On Neurovirology October 25–28, 2016, Toronto, Ontario, Canada

J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 DOI 10.1007/s13365-016-0478-8

Abstracts from the 14th International Symposium on NeuroVirology October 25–28, 2016, Toronto, Ontario, Canada

Published online: 27 September 2016 # Journal of NeuroVirology, Inc. 2016

P1 observed inhibition, similarities and differences between Comparative Modeling of Human Endogenous HIV and HERV-K protease were examined. There is no Retrovirus-K Protease Based on HIV Protease Predicts crystal structure of the HERV-K protease available, hence Efficacy of Protease Inhibitors comparative modeling using the sequence alignment with HIV protease was performed. A homology model was Rachel Abrams1, Richa Tyagi1, Wenxue Li1, Mario Bianchet2, generated and refined using the Prime program in the Avindra Nath1 Schrodinger Suites Software package. This model was (corresponding author: [email protected]) used to compare the active site of HIV protease with that of HERV-K. It can be seen from the model that while the 1National Institute of Neurological Disorders and Stroke, overall structures of the proteases appear quite similar, National Institutes of Health; 2Department of Neurology, changes in the active pocket caused by the few amino Johns Hopkins School of Medicine aciddifferencesmaybesufficienttoexplaintheobserved reduction in antiviral activity. Over the course of human evolution, retroviruses have infected cells of the germ line, allowing the genome of the virus to be passed down from parent to offspring. P2 These human endogenous retroviruses (HERVs) make up Role of ATF6b in HIV-Associated Neurocognitive about 8 % of the genome; however, in most cases multiple Disorders mutations have made them inactive. One of the most recently incorporated (HERV-K); however, has been impli- Cagla Akay Espinoza, Ping Lin cated in the development of amyotrophic lateral sclerosis (corresponding author: [email protected]) (ALS). Increased expression of HERV-K viral transcripts was observed in the brains of ALS patients and the ex- University of Pennsylvania, School of Dental Medicine, pression of HERV-K envelope causes damage to motor Department of Pathology neurons in vivo. While there are no treatment options for patients with ALS, in some patients with HIV infec- The underlying mechanism of cognitive impairment and tion that also display an ALS-like syndrome, antiretroviral brain injury in patients with HIV-associated drugs can reverse the ALS symptoms. Both HIV and neurocognitive disorder (HAND) on suppressive antiretro- HERV-K utilize an aspartic acid protease to process the viral therapy are not completely understood. However, viral polyprotein to its active components. To determine if synaptic injury, neuronal dysfunction, and damage in HIV protease inhibitors could inhibit HERV-K protease, these patients are partially driven by immune activation HIVproteaseinhibitorsweretestedinaninvitroHERV- and chronic inflammation in response to soluble factors K infection model. The compounds tested had a moderate released by HIV-infected and/or activated macrophages as protective effect; however, were less effective than against well as a low level of HIV replication in central nervous HIV infection. To investigate the reason for the level of system (CNS) reservoirs. A majority of these mediators as S2 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 well as many of the comorbid conditions were shown to Cell populations were stained with antibodies, run on a induce a ubiquitous cellular response, unfolded protein BD LSRII flow cytometer, and analyzed with FlowJo response (UPR) in vitro and in vivo. We have previously 7.6.5. Results: In rats infected by aerosol, infiltration of shown UPR activation in post-mortem tissue from HAND inflammatory cells into the CNS began at 4 dpi and patients in vivo. One of the three master UPR initiator consisted of neutrophils, macrophages, and lymphocytes. proteins, ATF6b, upon cleavage by site-1 and site-2 pro- There was also evidence of microglia activation. Rats in- teases (S1P and S2P), translocates to the nucleus for tran- fected subcutaneously do not develop overt disease yet scriptional induction of ER resident chaperones, apoptotic still had a detectable infiltrate that was intermediate com- genes, and secretory pathway regulatory genes. We hy- pared to uninfected rats. Neutrophils were a major com- pothesized that activation of the ATF6 pathway of the ponent in aerosol but not subcutaneously infected rats. UPR contributed to neuronal damage and death observed Conclusions: In addition to extensive viral replication in in HAND. We found that infection of primary human the brain, leukocyte infiltration and microglia activation monocyte-derived macrophages (MDMs) with HIV led are a significant component of disease in the Lewis rat to the nuclear translocation of the cleaved, thus active, model of RVFV. The relative contributions of viral cyto- ATF6b (N-ATF6b), which could be blocked by S1P inhi- pathology versus immunopathology remain to be deter- bition. We also observed that blocking nuclear accumula- mined, but this study represent the first attempt to char- tion of N-ATF6b in macrophages led to attenuation of acterize the leukocytic component. death of primary rat cortical neuroglial cultures exposed to supernatants from HIV-infected MDMs. Finally, we found nuclear N-ATF6b accumulation in neurons exposed P4 to HIV-infected MDM supernatants or excitotoxic stimu- Selection of gRNAs to target the HIV-1 quasispecies lus. These findings suggest that altered function of ATF6b with CRISPR/cas9 in several cell types within the CNS might be contributing to neuronal dysfunction and damage in patients with Alexander Allen1, Michael Nonnemacher1,William HAND. Dampier1,2, Matthew Desimone2, Vanessa Pirrone1,Katie Kercher1, Shendra Passic1, Jean Williams1, Brian Wigdahl1 (corresponding author: [email protected]) P3 Rift Valley Fever virus-induced encephalitis: 1Department of Microbiology and Immunology, Institute for characterization of leukocytes in the CNS Molecular Medicine and Infectious Disease, Drexel University College of Medicine; 2School of Biomedical Joseph Albe, Michael Kujawa, Tiffany Thompson, Engineering and Health Systems, Drexel University Amy L. Hartman (corresponding author: [email protected]) HIV-1 viral persistence during long-term antiretroviral therapy is a major hurdle to a cure. Genomic editing techniques, Department of Infectious Disease and Microbiology, Center like the CRISPR/Cas9 system, hold promise to permanently for Vaccine Research, University of Pittsburgh excise integrated virus from a host cell. Targets are defined by a 20 nucleotide guide RNA (gRNA) complementary to the Background: Rift Valley Fever Virus (RVFV) is a vector- desired genomic region. However, due to the rapid mutation borne infection endemic to the Horne of Africa; However rate intrinsic to HIV-1 replication, the virus in patients exists recent outbreaks in the Arabian Peninsula have expanded as a collection of distinct genomic variants, termed its potential range. People can develop encephalitis as a quasispecies. Presented here is a methodology for designing result of RVFV infection. Our lab uses an immunocompe- gRNA sequences to cleave a spectrum of HIV-1 quasispecies tent Lewis rat model to study neuropathogenesis of present within individual patients across a population of HIV- RVFV. Lewis rats develop uniformly lethal encephalitis 1-infected patients. PBMC genomic DNA was isolated from within 7–8 days after aerosol exposure. Rats infected sub- patients in the Drexel Medicine CNS AIDS Research and cutaneously do not develop disease. The goal of this study Eradication Study (CARES) Cohort as well as from brain is to characterize the phenotypes, timing, and extent of and spleen tissue from the National NeuroAIDS Tissue immune cell infiltrate into the CNS of RVFV-infected Consortium (NNTC) and the long terminal repeat (LTR) of Lewis rats. Methods: Lewis rats were infected with the HIV-1 quasispecies was sampled using Next Generation RVFV ZH501 by aerosol and subcutaneous routes, then Sequencing (NGS). gRNAs were computationally selected by serially sacrificed between days 3 and 6. Rat immune examining their binding potential across a random training set cells were isolated from brains via percoll gradients. of CARES patient samples. A package of 4 or 10 gRNAs were J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S3 selected based on the training set which in silico cleaved the target cells were shown to be lysed by HTLV-1 Tax-specific entire detectable quasispecies of the remaining CARES sam- cytotoxic T lymphocytes (CTL). Additionally, initial results ples and of the majority of the NNTC samples with an average indicate that Tax can be introduced into exosomes with trans- number of cleavages within each sample group of 3.4+/−1.7 fection of Tax plasmids into uninfected cells, and this process or 5.4+/−3 times, respectively. The package was further tested was unaltered with Tax mutation. Cumulatively, these find- in silico against a national sampling of subtype B North ings show that there are HTLV-1 proteins present in exosomes American LTRs from the Los Alamos National Database found in virus-free CSF of HAM/TSP patients and that HAM/ and was shown to cleave all sequences. Currently, we are TSP PBMCs can excrete these exosomes in culture. focused on cloning these packages of gRNAs to initiate func- Furthermore, exosomes containing HTLV-1 Tax can sensitize tional studies to confirm the in silico studies performed to target cells for HTLV-1 specific lysis by CTLs. This suggests date. These studies represent a step towards understanding that exosomes may play a role in perpetuating the activated the complex task of using excision therapy to target HIV-1 immune response that contributes to HAM/TSP. quasispecies in the infected patient population.

P6 P5 Extracellular vesicles are amyloid carriers in HIV-infected Functional consequences of HTLV-1 viral antigens brains detected in exosomes from HAM/TSP patients Ibolya E. Andríçs1, Ana Leda1, Marta Garcia-Contreras 2, Monique Anderson1, Yoshimi Enose-Akahata1, Maria Chiara Luc Bertrand1,MinseonPark1, Marta Skowronska1, Monaco-Kushner1, Ashley Vellucci1, Yuetsu Tanaka2, Ben Michal Toborek1 Lepene3, Fatah Kashanchi4, Steven Jacobson5 (corresponding author: [email protected]) (corresponding author: [email protected]) 1Department of Biochemistry and Molecular Biology, 1National Institutes of Health; 2University of the Ryukyus University of Miami School of Medicine; 2Diabetes Graduate School of Medicine; 3Ceres Nanosciences; Research Institute, University of Miami School of Medicine 4George Mason University Laboratory of Molecular Virology; 5National Institutes of Health HIV-infected brains are characterized by increased amyloid beta (Abeta) deposition. It is believed that the blood–brain Human T-cell lymphotropic virus Type I (HTLV-1) is a retro- barrier (BBB) is critical for Abeta homeostasis and contrib- virus that currently infects an estimated 10–20 million people utes to Abeta accumulation in the brain. Extracellular vesi- worldwide. The vast majority of those infected will remain cles (ECV) gained recently a lot of attention as potentially asymptomatic, but about 0.1–4 % of patients will develop playing a significant role in Abeta pathology. In addition, HTLV-1 associated myelopathy/tropical spastic paraparesis HIV-1 hijacks the exosomal pathway for budding and re- (HAM/TSP). HAM/TSP is a neuroinflammatory disease that lease. Therefore, we investigated the involvement of BBB- has been shown to be immunopathologically mediated. derived ECV in the HIV-1-induced Abeta pathology in the Recent data suggests that viral antigens can be acquired in brain. Our results indicate that HIV-1 increases ECV release the absence of viral infection through the transport of from brain endothelial cells and elevate their Abeta cargo as exosomes. Therefore, the presence of HTLV-1 products in compared to controls. Interestingly, brain endothelial cell- the exosomes isolated from HAM/TSP patient cerebrospinal derived ECV transferred Abeta to astrocytes and pericytes. fluid (CSF) was investigated. Using a novel nanotrap technol- Infusion of brain endothelial ECV carrying fluorescent ogy which utilizes hydrogel particles that trap exosomes, Abeta into the internal carotid artery of mice resulted in exosomes that were positive for HTLV-1 Tax by Western blot Abeta fluorescence associated with brain microvessels and could be detected from HAM/TSP patient CSF but not from also in the brain parenchyma. These data suggest that ECV control HTLV-1 seronegative multiple sclerosis (MS) patients. carrying Abeta can be successfully transferred across the Additionally, HAM/TSP PBMCs were shown to secrete BBB into the brain. Based on these observations, we con- exosomes containing HTLV-1 Tax in ex vivo cultures. To clude that HIV-1 facilitates shedding of brain endothelial understand the source of these exosomes, PBMCs were sorted ECV carrying Abeta, a process that may increase Abeta and found to have increased exosome production by activated exposure of cells of neurovascular unit, such as astrocytes CD4 + CD25+ and CD8 + CD25+ T cells. Importantly, to de- and pericytes. This mechanism may contribute to increased termine if these exosomes are functionally producing tax pro- amyloid deposition in HIV-infected brain. Supported by teins, exosomes isolated from HAM/TSP PBMCs were ap- MH098891, MH072567, DA039576, DA027569, plied to recipient target B cells. These exosome-sensitized HL126559, and by the Miami CFAR MH063022. S4 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89

P7 between the evolution of Vpr and gp120 in HIV-1- HIV-1 Vpr sequence variants associate specifically infected patients, demonstrate the existence of HIV-1 tro- with co-linear genetic determinants of viral co-receptor pism beyond the envelope, and suggest associations be- phenotype tween Vpr variants and R5 viral genotypes in the brain.

Gregory Antell1 , William Dampier2 ,Benjamas Aiamkitsumrit2, Michael Nonnemacher2, Vanessa Pirrone2, P8 Wen Zhong2, Katherine Kercher2, Shendra Passic2,Jean Divergent efficacies of antiretroviral therapy in blood Williams2, Yucheng Liu2, Tony James2, Jeffrey Jacobson3, and brain: HIV-1 suppression is dependent on drug Zsofia Szep4,BrianWigdahl2,FredKrebs2 and cell type. (corresponding author: [email protected]) Eugene Asahchop1,WingChan1, William Branton1,Gail 1School of Biomedical Engineering, Science, and Health Rauw2, Manmeet Mamik1, Lothar Resch3,JohnGill1,Glen Systems, Drexel University; 2Department of Microbiology Baker2, Christopher Power4 and Immunology, Institute for Molecular Medicine and (corresponding author: [email protected]) Infectious Disease, Drexel University College of Medicine; 3Department of Medicine, Section of Infectious Disease, 1Department of Medicine, University of Alberta; 2Department Lewis Katz School of Medicine, Temple University; of Psychiatry, University of Alberta; 3Department of 4Division of Infectious Diseases and HIV Medicine, Pathology, University of Calgary; 4Department of Medicine Department of Medicine, Drexel University College of and Psychiatry, University of Alberta Medicine Background: Perivascular macrophages and microglia are Viral protein R (Vpr) is a 14 kDa HIV-1 accessory protein the principal productive reservoir for active HIV-1 repli- that functions in HIV-1 replication and pathogenesis. Vpr cation in the brain. In patients receiving suppressive com- is also a likely contributor to the neuropathogenesis that bination antiretroviral therapy (cART), HIV-1 replication underlies deficits collectively referred to as HIV- in the brain reservoir is assumed to be latent or minimally associated neurocognitive disorders (HAND). HIV-1 in- productive although the susceptibility of this reservoir to fection of the brain is predominated by infected macro- suppression by cART is uncertain. We investigated the phages and microglial cells that harbor viruses character- efficacies and concentrations of established antiretroviral ized by an R5 (CCR5-utilizing) co-receptor genotype. In drugs (ARVs) in HIV-infected microglia and macrophages studies that have paralleled our identification of sequence together with evaluating the in vivo brain HIV-1 reservoir signatures in HIV-1 Tat and the long terminal repeat during suppressive cART. Methods: HIV-1 RNA and (LTR) that associate with co-receptor utilization, we have DNA levels in brain tissues from HIV-infected patients demonstrated associations betweenVprvariationinHIV- (n = 2) were quantified by digital droplet PCR within 1-infected patients and co-receptor usage. Co-linear HIV- 12 h of last ARV dosage. The efficacies of individual 1 Vpr and Env-V3 amino acid sequences were collected ARVs in HIV-infected primary microglia, bone marrow- from the LANL HIV-1 sequence database, as well as from derived macrophages and peripheral blood mononuclear well-suppressed patients enrolled in the Drexel/Temple cells (PBMCs) were compared. Tissue and cellular con- Medicine CNS AIDS Research and Eradication Study centrations of ARVs were measured by liquid (CARES) Cohort. Vpr sequences were grouped as X4 chromatography-mass spectrometry (LC-MS) in human (CXCR4-utilizing) or R5 according to genotypic classifi- myeloid cells and mouse brain. Results: In treated patients cation of the co-linear Env-V3 sequence. The amino acid with prolonged (>4 years) undetectable plasma viral RNA diversity of each Vpr population was assessed, as was the levels, HIV-1 RNA and DNA were detected in multiple Jensen-Shannon divergence between groups, in order to brain specimens with associated neuroinflammatory re- identify sequence signatures associated with co-receptor sponses. Zidovudine, etravirine and darunavir treatment utilization. Multiple amino acid alphabets were utilized of HIV-infected microglia exhibited significantly higher in order to better assess the impact of amino acid substi- EC50 values than those observed in PBMCs. tutions with similar physiochemical properties. Positions Intracellular and extracellular ARV levels in human mye- 35–45 and the C-terminus of Vpr consistently displayed loid cells were similar and unaffected by HIV-1 infection. statistically significant divergence across multiple alpha- In vivo concentrations of darunavir and raltegravir in mice bets. Specifically, positions 37 and 41 displayed a consen- varied depending on brain region and were >1.0 log lower susaminoacidswitchbetweenR5andX4sequencepop- than matched plasma levels at 1 h and undetectable at 8 h ulations. Overall, the results suggest a functional link post-treatment. Conclusions: ARVs displayed differential J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S5 concentrations and antiviral effects depending on the in- the HIV genome into overlapping fragments of 1000 (+/−200) dividual ARV and infected cell type (or tissue), which was base pairs allowing PCR amplification of each fragment. Next substantiated by the presence of persistent viral DNA and generation sequencing is then used to acquire sequence data RNA in brain tissue from patients with undetectable plas- with sufficient depth to identify genetic variants making up ma viral RNA. These findings underscore considerations less than 1 % of the quasispecies in the sampled genome, of assessing ARV selection for different viral reservoirs as allowing for design of potential CRISPR-cas9-based cure part of efforts to eradicate HIV-1. strategies.

P9 P10 Identification of HIV-1 quasispecies for targeted excision Human neural progenitor cells (hNPCs) are productively of latent infection using next generation sequencing infected by R5-tropic HIV-1: Morphine interactions on infection and function involve Cdk5 signaling Andrew Atkins1, William Dampier1, Katherine Kercher1, Shendra Passic1, Wen Zhong1, Jean Williams1,Sergey Joyce Balinang1,KurtHauser2, Pamela Knapp1 Balashov1, Joshua Mell1, Joshua Earl1, Vanessa Pirrone1, (corresponding author: [email protected]) Michael Nonnemacher1,ZsofiaSzep2, Jeffrey Jacobson3, Brian Wigdahl1 1Department of Anatomy and Neurobiology, Virginia (corresponding author: [email protected]) Commonwealth University; 2Department of Pharmacology and Toxicology, Virginia Commonwealth University 1Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease, Drexel Opiates augment HIV-induced neuropathogenesis in the CNS University College of Medicine; 2Division of Infectious through both direct and indirect mechanisms that disrupt glial Disease, Department of Medicine, Drexel University and neuronal function. All CNS macroglia and neurons derive College of Medicine; 3Department of Medicine, Lewis Katz from neural progenitor cells (NPCs) during development, and School of Medicine, Temple University NPCs in the adult brain contribute to repair processes. Since disruptions in NPC function are known to impact CNS popu- Although ART drastically improves the prognosis for HIV-1- lations and brain function in a number of disease/injury con- infected patients, latent infection due to integrated proviral ditions, we examined whether HIV ± opiate exposure affected DNA persists in some tissues (e.g. peripheral blood, lymphoid the maturation and fate of human NPCs. As hNPC infection tissue, and the brain). There is currently no effective strategy by HIV has occasionally been reported, we also reexamined for the removal of persistent proviral DNA from infected pa- this question, and parsed between effects due directly to hNPC tients and the end result of ART interruption is a resurgence of infection by HIV, or to hNPC dysfunction caused by the in- systemic viral load. However, recent success has been fective milieu. Multiple approaches confirmed the infection of achieved in the removal of HIV-1 from infected cell lines as hNPCs by R5 tropic HIVBaL, and demonstrated that active well as primary cells from infected patients and the results of infection could be sequentially transferred to naíve hNPCs. these experiments suggest a new approach to the targeted Exposure to supernatant from HIVBaL-infected cells removal of latent infection. Bioinformatic analysis of deep (HIVsup) reduced hNPC proliferation and led to premature sequencing data can potentially be used to design gRNAs differentiation into astrocytes and neurons. Morphine co- which target persistent HIV DNA using the CRISPR-Cas9 exposure prolonged hNPC infection and exacerbated func- system. In order to pursue this strategy, it is necessary to col- tional effects of HIVsup. Neither purified virions nor UV- lect sequence data from patients with ART regulated viral inactivated HIVsup altered proliferation, indicating that this loads. Furthermore, it is critically important to track genetic effect did not require infection. Gene array analysis and RT- variation over time in HIV-1-infected patients as the genetic qPCR with immunoblot validation suggested that Cdk5 sig- sequence of latent HIV infection is not static. To address this naling was involved in HIV-morphine interactions. siRNA- complex problem, we have utilized patient samples from the mediated knockdown of Cdk5 expression reversed premature Drexel Medicine CNS AIDS Research and Eradication Study MAP2 differentiation, but also increased hNPC death. (CARES) Cohort. The CARES Cohort collects longitudinal Pharmacological inhibition of Cdk5 activity with samples from patients allowing observation of the progress of roscovitine also reversed MAP2 differentiation without HIV-1 mutation. Presented here are the strategies that we have inducing hNPC death. However, roscovitine also inhibits developed to address the challenge of whole-genome se- Cdk2/Cdk4 activity, which may explain its anti- quencing of latent HIV infection in ART-regulated patients. proliferative effects on hNPCs independent of treatments. We have determined that an effective strategy is to subdivide Overall, dysregulation of hNPC functions by the S6 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 infectious environment may create cell population imbal- 1National Center for Biodefense and Infectious Disease - ances that contribute to CNS deficits in both adult and George Mason University; 2Ceres Nanosciences Inc., pediatric patients. Additionally, infected hNPCs may pass Manassas, VA; 3Florida International University, Miami, FL virus to their progeny, and serve as an unappreciated viral reservoir. The recent epidemic of opiate/heroin abuse HIV-1 infection results in a chronic illness since long-term highlights the clinical importance of HIV and opiate HAART can lower viral titers to an undetectable level. interactions. However, discontinuation of therapy rapidly increases virus burden. Moreover, patients under HAART frequently develop various metabolic disorders, neurocognitive abnormalities, and cardiovascular diseases. We have previ- P11 ously shown that exosomes containing trans-activating re- cAMP signaling enhances HIV-1 long terminal repeat sponse (TAR) element RNA enhance susceptibility of un- (LTR)-directed transcription and viral replication differentiated naíve cells to HIV-1 infection. Up to a mil- in human bone marrow progenitor cells lion copies of TAR RNA per microliter were also detected in the serum from HIV-1 infected humanized mice suggest- Anupam Banerjee, Luna Li, Vanessa Pirrone, Fred Krebs, ing that TAR RNA may be stable in vivo. We recently have Brian Wigdahl, Michael Nonnemacher found another viral non-coding RNA that we termed TAR- (corresponding author: [email protected]) gag which does not code for a protein but is present in the exosomes. Incubation of exosomes from HIV-1 infected Department of Microbiology and Immunology, Institute for cells with primary macrophages resulted in a dramatic in- Molecular Medicine and Infectious Disease, Drexel crease of pro-inflammatory cytokines, IL-6 and TNF-beta, University College of Medicine indicating that exosomes containing TAR RNA could play a direct role in control of cytokine gene expression. CD34+ hematopoietic progenitor cells have been shown Furthermore, the single stranded 5’ or 3’ processed stem to be susceptible to HIV-1 infection, possibly due to a low RNA binding to TLRs activates the NF-kappaB pathway level expression of CXCR4, a coreceptor for HIV-1 entry. and regulates cytokine expression. In our most recent data, Given these observations, we have explored the impact of we find that the number of exosomes from infected cells is forskolin on cell surface expression of CXCR4 in a cell increased when cells are treated with cART. This directly line model (TF-1). The elevation of intracellular cAMP by indicates that HIV viral release and exosome release have forskolin through adenyl cyclase resulted in transcription- overlapping biogenesis pathways, including the ESCRT al upregulation of CXCR4 with a concomitant increase in pathway. We will also discuss exosomes from other replication of the CXCR4-utilizing HIV-1 strain IIIB. neuro-tropic RNA viral infections, including HTLV-1, Transient expression analyses also demonstrated an in- Ebola, RVFV, and Zika, and how they behave differently crease in CXCR4-, CCR5, and CXCR4-/CCR5-utilizing than exosomes from HIV infected cells. Collectively, our HIV-1 (LAI, YU2, and 89.6, respectively) promoter activ- results imply that exosomes in viral infection control path- ity. Studies also implicated the protein kinase A pathway ogenesis and targeting these particles may be a method to and the downstream transcription factor CREB-1 in inter- lower overall viral burden in infected hosts. facing with cAMP response elements located in the CXCR4 and viral promoter. These observations suggest the cAMP signaling pathway may serve as a regulator of CXCR4 levels and concomitantly of HIV-1 replication in P13 bone marrow progenitor cells. Role of DNA damage response pathways in the activation of polyomavirus JC

P12 Anna Bellizzi1,2,MartynK.White2, Gabriele Ibba2, Kamel Exosomes from donor and recipient cells: Role Khalili2, Hassen S. Wollebo2 of exosomes in HIV latency in cART treated cells. (corresponding author: [email protected])

Robert Barclay1, Catherine DeMarino1,AngelaSchwab1, 1Department of Public Health and Infectious Diseases, Michelle Pleet1,GavinSampey1, Sergey Iordanskiy1,Ramin Institute Pasteur, Cenci-Bolognetti Foundation, Sapienza Hakami1, Benjamin Lepene2, Nazira El-Hage3, Fatah University,RomeItaly;2Center for Neurovirology, Kashanchi1 Department of Neuroscience, Lewis Katz School of (corresponding author: [email protected]) Medicine at Temple University, Philadelphia PA J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S7

Infection of glial cells by the human neurotropic polyomavi- oligodendrocytes, which produce myelin in the brain, with rus JC (JCV), the causative agent of progressive multifocal human neurotropic polyomavirus JC (JCV). PML lesions are leukoencephalopathy (PML) rapidly inflicts damage to cellu- areas of demyelination containing oligodendrocytes with viral lar DNA, which in turn evokes the DNA damage response nuclear inclusion bodies and bizarre astrocytes, which are also (DDR). We previously reported that JCV rapidly induces ex- productively infected by JCV. Although JCV was isolated pression of the DNA repair factor Rad51 and Rad51 activates over 40 years ago and has been extensively studied since, the JCV early promoter in co-operation with the transcription there is still no effective therapy for PML. Recently, a novel factor NF-kappaB p65. Thus Rad51 induction by JCV infec- genome-editing method was developed based on clustered tion serves as a positive feedback mechanism for viral activa- regularly interspaced short palindromic repeat (CRISPR) sys- tion early in JCV infection. It is known that the DDR may also tems. The CRISPR system uses a nuclease, CRISPR- stimulate NF-kappaB activity, a phenomenon known as associated (Cas9), that complexes with small RNAs as nucleus to cytoplasm or “inside-out” NF-kappaB signal- guides (gRNAs) to cleave DNA in a sequence-specific ing. This pathway is initiated by Ataxia telangiectasia manner upstream of the protospacer adaptor motif mutated (ATM) protein, a serine/threonine kinase that is (PAM) in any genomic location. Here, we use recruited and activated by DNA double-strand breaks and CRISPER/Cas9 system as a potential tool for JCV elimi- subsequently involves a series of post-translational modi- nation by designing guide RNAs (gRNAs) targeting re- fications of NF-B essential modulator (NEMO), also gions within the early gene encoding viral protein, T-an- known as inhibitor of NF-kappaB kinase subunit gamma tigen. Our results indicated that Cas9/gRNAs targeted to (IKK-gamma), which activates NF-kappaB. Here we the JCV T-antigen gene effectively delete target DNA se- show that JCV infection causes phosphorylation and acti- quence, reduce T-antigen expression and late promoter vation of ATM while a specific small molecule inhibitor activity and inhibit JCV DNA replication. No off-target of ATM, KU-55933, inhibits JCV replication. Analysis of effects of the JCV-specific CRISPR/Cas9 editing were the effect of JCV infection on the subcellular distribution detected using the SURVEYOR assay. These data reveal of NEMO by cell fractionation and immunocytochemistry the promise of CRISPR/Cas9 as a tool for the elimination showed a redistribution of NEMO from the cytoplasm to of the JCV genome and a potential as a cure for PML. the nucleus. Co-expression of JCV large T-antigen and FLAG-tagged NEMO in cells showed the occurrence of the sumoylation of NEMO in the presence of T-antigen, P15 while co-expression of ATM and FLAG-NEMO demon- Inhibition of HSV-1 replication by a gene editing strategy strated physical association between ATM and NEMO. Taken together, we propose a model where JCV infection Anna Bellizzi1,3,PamelaC.Roehm2,3,MasoudShekarabi3, induces both the expression level of Rad51 protein and Hassen S. Wollebo3, Gabriele Ibba3, Martyn K. White3, nucleus to cytoplasm NF-kappaB signaling, which then Lifan He3, Julian Salkind3, Kamel Khalili3 act together to enhance viral gene expression. (corresponding author: [email protected])

1Department of Public Health and Infectious Diseases, P14 Institute Pasteur, Cenci-Bolognetti Foundation, Sapienza CRISPR/Cas9 system as an agent for eradication University, Rome Italy; 2Department of Otolaryngology/ of polyomavirus JC infection Head and Neck Surgery - Department of Neurosurgery, Lewis Katz School of Medicine at Temple University, Anna Bellizzi1,2, Gabriele Ibba2, Rafal Kaminski2,MartynK. Philadelphia PA; 3Center for Neurovirology, Department of White2, Wenhui Hu2, Jennifer Gordon2, Kamel Khalili2, Neuroscience, Lewis Katz School of Medicine at Temple Hassen S. Wollebo2 University, Philadelphia PA (corresponding author: [email protected]) HSV-1 induced illness affects more than 85 % of adults world- 1Department of Public Health and Infectious Diseases, wide and there is no permanent curative therapy. We used Institute Pasteur, Cenci-Bolognetti Foundation, Sapienza RNA-guided CRISPR/Cas9 gene editing to specifically target University, Rome Italy; 2Center for Neurovirology, DNA sequences of the HSV-1 genome for deletion that span Department of Neuroscience, Lewis Katz School of the region directing expression of ICP0, a key viral protein Medicine at Temple University, Philadelphia, PA that stimulates HSV-1 gene expression and replication. We found that CRISPR/Cas9 introduced InDel mutations into ex- Progressive multifocal leukoencephalopathy (PML) is a fatal on 2 of the ICP0 gene profoundly reduced HSV-1 infectivity demyelinating disease of the CNS caused by lytic infection of in permissive human cell culture models and protected S8 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 permissive cells against HSV-1 infection. CRISPR/Cas9 cells. Functional studies showed that both Tat.B and mediated targeting ICP0 prevented HSV-1-induced disin- Tat.AG decreased the expression of endothelial tight junc- tegration of promonocytic leukemia (PML) nuclear bod- tion proteins claudin-5 and ZO-1, and decreased brain ies, an intracellular event critical to productive HSV-1 trans-endothelial electric resistance (TEER), but greater infection that is initiated by interaction of the ICP0 N- reduction in TEER was observed with Tat.B compared terminus with PML. Combined treatment of cells with to Tat.AG. Overall, our data showed increased BBB in- CRISPR targeting ICP0 plus the immediate early viral flammation and BBB disruption with Tat.B, compared to proteins, ICP4 or ICP27, completely abrogated HSV-1 Tat.G. This may suggests these two HIV-1 subtypes dif- infection. We conclude that RNA-guided CRISPR/Cas9 ferentially affect the BBB and the CNS. Our future studies can be used to develop a novel, specific and efficacious will validate these findings by analyzing the direct effects therapeutic and prophylactic platform for targeted viral of these viral strains on the BBB and the CNS. genomic ablation to treat HSV-1 diseases.

P17 P16 Dysregulation of epigenetic control of memory in HIV Mechanisms associated with differential inflammatory induced mild cognitive disease in mice and disease relief effects of Tat proteins derived from HIV-1 subtypes-B by valproic acid treatment and recombinant CRF02_AG on human brain microvascular endothelial cells Alejandra Borjabad1, Wang Hongyin1, Wei Chao1, Boe-Hyun Kim1, Kelschenbach Jennifer1, Chao-Jiang Gu1, Jacinta BIJU BHARGAVAN1, GEORGETTE KANMOGNE1 Murray1, Susan Morgello1, Mary Jane Potash1,DavidJ (corresponding author: [email protected]) Vo ls ky 1 (corresponding author: [email protected]) 1Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center 1Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, USA Previous studies showed that HIV-1 Tat induce blood–brain barrier (BBB) dysfunction and is involved in HIV-associated HIV infected individuals on suppressive antiretroviral therapy neurocognitive disorders (HAND). These studies were per- develop chronic mild neurocognitive impairments (mild NCI) formed mostly with Tat from HIV-1 subtype-B (Tat.B), that may affect everyday activities but do not progress to HIV whereas 70 % of the 37 millions HIV-infected subjects are dementia. The pathobiology of mild NCI is unclear and there in Sub-Saharan Africa and are infected with non-B subtypes, is no treatment, but one feature indicative of disease mecha- including the recombinant HIV-1 CRF02_AG, prevalent in nism is an apparent discordance between broad neuronal dys- West-Central Africa. The effects of this recombinant virus function and limited neuronal death. We investigated NCI on the BBB and HAND are not known. In the current study, biology and treatment in experimentally induced disease in we analyzed the effects of Tat.B and Tat from HIV-1 immunocompetent mice infected with chimeric HIV, CRF02_AG (Tat.AG) on primary human brain microvascular EcoHIV. We first show in behavioral tests that infected ani- endothelial cells (HBMEC), the major component of the mals develop chronic NCI involving at least two cognitive BBB. Exposure of HBMEC to Tat.B increased the expression domains, memory and executive function. The resemblance and secretion of the proinflammatory cytokine IL6 by 9-fold of murine disease to human mild NCI was indicated by low (p < 0.001) and increased IL6 mRNA by 113-fold (p <0.001), HIV burdens, mild neuroinflammation, and diffuse dendritic whereas Tat.AG increased IL6 expression by 2.7 to 3.8-fold, pruning in multiple brain regions but absence of overt neuro- and increased IL6 mRNA by 35.7-fold (p <0.001)compared pathology and cellular apoptosis. We then applied systems to controls. Mechanistic studies showed that Tat.B induced biology approach to gain an insight into disease pathobiology. BBB inflammation through the IRAK1/4,MKK/JNK path- Comparative analysis of brain gene expression profiles from ways, in an AP1- and NFkB-independent manner, whereas cognitively impaired mice and patients with HIV cognitive Tat.AG effects occurred via the MKK/JNK/AP1/NFkB disease revealed common downregulation of pathways con- pathways. We further showed that Tat-induced effects trolling neuronal signal transmission and memory. were associated with activation of c-jun(serine-63) and Assessment of epigenetic control of these functions by chro- SAPK/JNK(Thr183/Tyr185), demonstrated increased ex- matin immunoprecipitation and sequencing indicated high pression of transcription factors associated with these correlation between histone 3 hypermethylation at lysine pathways (Jun, STAT1, RELB, CEBPA) with higher 9 m3 and transcriptional suppression of genes associated with levels in Tat.B-treated cells compared Tat.AG-treated synaptodendritic functions. Treatment of cognitively impaired J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S9 mice with valproic acid significantly mitigated murine NCI, to −0.10, p = 0.007), and with lower SYP in hippocampus normalized selected synaptic gene expression, and reversed (B = −0.25, 95 % CI −0.42 to −0.03, p = 0.003) but not histone hypermethylation on their promoters. The results sug- putamen or frontal cortex (p > 0.05). Higher plasma gest dysregulation of epigenetic control of memory in HIV sCD163wasassociatedwithhigherHLA-DRinputamen induced mild cognitive dysfunction and indicate that the dis- (B = 0.17, 95 % CI 0.08 to 0.26, p < 0.001) and when aver- ease is treatable. aged across three brain regions (B = 0.11, 95 % CI 0.05 to 0.17, p < 0.001). Cerebrospinal fluid sCD163 was not associated with any histological measure (p >0.05). P18 Conclusion: Higher plasma sCD163 in life is associated Plasma soluble CD163 is associated with postmortem with greater synaptodendritic damage (SYP, MAP2) and brain pathology in human immunodeficiency virus microglial activation (HLA-DR) in cortical and subcortical infection. brain regions. Plasma sCD163 warrants additional study as a biomarker of neurologic damage in HIV infection. Alex Bryant1, David Moore1, Tricia Burdo2, Jessica Lakritz3, Ben Gouaux1, Virawudh Soontornniyomkij4, Cristian Achim4, Eliezer Masliah5, Igor Grant4,AndrewLevine6, P19 Ronald Ellis5 Extracellular vesicle mediated shuttling of miR-9 mediates (corresponding author: [email protected]) synaptodendritic alterations in HAND

1HIV Neurobehavioral Research Program, San Diego, CA; Shilpa Buch, Lu Yang, YeonHee Kook, Yu Cai, Guoku Hu 2Department of Neuroscience, Temple University, Lewis (corresponding author: [email protected]) Katz School of Medicine, Philadelphia, PA; 3Department of Biology, Boston College, Chestnut Hill, MA; 4Department of Department of Pharmacology and Experimental Psychiatry, University of California San Diego, La Jolla, CA; Neuroscience, University of Nebraska Medical Center 5Department of Neurosciences, University of California San Diego, La Jolla, CA; 6Department of Neurology, University of Reversible synaptodendritic injury and underlying neuroin- California Los Angeles, Los Angeles, CA flammation are important phenotypes and correlates of HIV- associated neurocognitive disorders (HAND). Similar to Background: Plasma soluble CD163 (sCD163), shed by HIV+ subjects on antiretroviral therapy (ART), SIV-infected monocytes and macrophages, correlates with rhesus macaques on ART have also been shown to exhibit loss neurocognitive impairment in human immunodeficiency of synaptophysin, increased glial activation and dysregulation virus (HIV) infection and is a potential biomarker for neu- of various signature microRNAs (miRs). MiR-mediated reg- rologic damage in HIV. We sought to determine the asso- ulation of disease pathogenesis represents an evolving area of ciation between plasma or cerebrospinal fluid sCD163 and research that has ramifications for identification of potential postmortem brain pathology. Methods: We measured therapeutic targets for various neurodegenerative disorders, sCD163 levels in antemortem plasma (n = 54) and cerebro- for which, currently there exists no cure. Parallel to the ad- spinal fluid (n = 32) samples from 74 HIV+ participants vances made in miRNA research has been the advent of the (median 5 months before death) who donated their brains field of extracellular vesicles (EVs). EVs represent an impor- to research at autopsy. We quantified markers of tant mode of intercellular communication by serving as con- synaptodendritic damage (microtubule-associated protein duits for transfer of membrane and cytosolic proteins and 2 [MAP2], synaptophysin [SYP]), microgliosis (HLA- RNA (including miRs) between cells. Despite effective sup- DR, ionized calcium binding adaptor molecule 1), pression of virus replication in the presence of ART, viral astrocytosis (glial fibrillary acidic protein) and impaired proteins such as HIV Tat have been found to be present in protein clearance (beta-amyloid) in frontal cortex, hippo- tissues such as the CNS and the lymph nodes, likely contrib- campus, putamen, and internal capsule. Multivariable uting to ongoing neuroinflammation. In the current study we least-squares regression was used to evaluate the associa- demonstrate that exposure of astrocytes to HIV Tat resulted in tion between plasma or cerebrospinal fluid sCD163 and increased induction/release of miR-9 in the EVs. MiR-9- histological measures, accounting for medical comorbidi- enriched EVs, were in turn, taken up by the neurons resulting ties and correcting for multiple comparisons. Results: in significant synaptodendritic injury. Furthermore, we also Higher plasma sCD163 was associated with lower MAP2 observed downregulated expression of miR-9 targets - in frontal cortex (B = −0.23, 95 % CI −0.41 to −0.06, PDGF-CC, NLGN2 and MCPIP1 (key players in regulating p = 0.007), putamen (B = 0.32, 95 % CI −0.52 to −0.12, the normal synaptic functioning in neurons) in SIV+/HIV+ p = 0.004), and hippocampus (B = −0.23, 95 % CI −0.35 brains compared with the uninfected controls. Validation of S10 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 these findings in rat primary neurons infected with lentivirus potential of cathepsin B antibody was confirmed by immu- expressing miR-9 demonstrated decreased expression of the nofluorescence, demonstrating increased neuronal MAP2 respective targets. Moreover, we also observed reduced ex- staining and recovered morphology. Then, we examined pression of these targets and concomitant synaprodendritic the presence of cathepsin B in exosomes released by unin- injury in the neurons exposed to EVs isolated from condi- fected and HIV-infected MDM at 12 days post-infection. tioned media collected from HIV Tat-treated astrocytes. Western blots revealed that exosomes isolated from MDM Taken together, our findings implicate that HIV-Tat mediated infected in vitro contain cathepsin B in both pro-peptide alteration of synaptodendritic injury is regulated by exosomal and mature forms, and express CD63, an exosome marker. miR-9 via its targets (PDGF-CC/MCPIP1/NLGN2). Our results suggest that cathepsin B might be secreted in exosomes by HIV-infected macrophages, proposing a novel mechanism by which cathepsin B triggers neuronal dys- P20 function and opening doors for new studies in search for Neurotoxic lysosomal protease cathepsin B is present approaches to decrease the neuronal dysfunction and sub- in macrophage-derived exosomes and internalized sequent development of HAND. by neurons

Yisel Cantres-Rosario1, Karla Negron Roure2, Marines P21 Plaud1, Loyda Melendez3 A novel deep sequencing platform (ViroFind) reveals (corresponding author: [email protected]) a complex JC virus population in the brain of PML patients 1Department of Microbiology and Medical Zoology, School of Medicine, University of Puerto Rico-Medical Sciences Spyros Chalkias1,JoshuaGorham1, Erica Mazaika1,Michael Campus; 2Department of Biology, University of Puerto Rico Parfenov1,XinDang1, Steve DePalma1, David McKean1, - Bayamon Campus; 3Department of Microbiology and Jonathan Seidman1,IgorKoralnik2 Medical Zoology, School of Medicine & Translational (corresponding author: [email protected]) Proteomics Center, University of Puerto Rico-Medical Sciences Campus 1Harvard Medical School; 2Rush University Medical Center

Despite the efficacy of antiretroviral therapy, mild forms of Background: Deep sequencing enables the unbiased detection HIV-associated neurocognitive disorders (HAND) remain of viruses in clinical samples but it is limited by low cost- prevalent. HIV-infected macrophages infiltrate into the effectiveness. We developed a novel target-enrichment deep- brain secreting viral and cellular proteins that trigger neu- sequencing platform (ViroFind) for the cost-effective se- ronal dysfunction and death. One of the monocyte-derived quencing of viral genomes and we investigated the composi- macrophages (MDM) secreted proteins is cathepsin B, a tion of viral populations in the brain of 5 PML patients and in lysosomal protease that triggers neuronal apoptosis 18 controls with no known neurological disease. Methods: in vitro, and co-localizes with beta-amyloid peptides in ViroFind comprises 165,433 probes, complementary to the brain tissue from HAND and Alzheimer’spatients.Pre- entire genomes of 386 DNA and RNAviruses known to infect treatment of macrophage-conditioned media (MCM) with humans. The ViroFind probes are used in a hybridization re- anti-cathepsin B antibodies or inhibitors reduces neuronal action with nucleic acid libraries from clinical samples to en- apoptosis and beta-amyloid peptides in vitro. We have rich only viral sequences. The output of this reaction is se- demonstrated that recombinant active cathepsin B added quenced via an Illumina platform and the sequencing data are in MCM is internalized by neurons, and the levels of inter- aligned against a database of all viral genomes. Mapped se- nalization are proportional to the levels of HIV infection. quences are used to analyze viral genomes and identify viral Therefore, we hypothesize that targeting cathepsin B in variants. Results: Compared to direct deep sequencing, MCM represents a viable approach to elucidate its mecha- ViroFind enriched the JC virus (JCV) sequences detected nism of neuronal dysfunction. To test this hypothesis, we per PML brain sample, ranging from 584 viral sequences exposed SK-N-SH neuroblastoma cells to histidine-tagged (each of 75 bp length) per 1,000,280 total sequences up to cathepsin B in culture media alone or in presence of anti- 375,653 viral sequences per 430,842 total sequences. The cathepsin B antibody, and localized the histidine tag in enrichment of JCV sequences attributable to ViroFind ranged neurons by intracellular flow cytometry. Histidine-tagged from 33 to 127-fold. Each JCV nucleotide was sequenced at cathepsin B was internalized by neurons (52.0 %), a mech- least 10 times (coverage 10X). We identified 24 viral capsid anism that was partially reduced by the pre-treatment with protein VP1 variants and 12 of these variants were associated anti-cathepsin B antibody (34.9 %). The neuroprotective with amino acid substitutions. Mutation D66H, likely related J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S11 to VP1 conformational changes that could impact tissue tro- which binds different region of CD46 (SCR1-2 domains). pism, was observed in 4 of 5 (80 %) PML brain samples. These results suggested that induction of HERV-W is spe- Lastly, we detected low frequency JCV sequences in 10 of cific for HHV-6A infection in neural and immune cells and 18 (56 %) control brains. Conclusion: Multiple JCV variants that engagement of CD46-SCR3 domain might be required constitute highly complex viral populations in the brain of for the induction of HERV-W. The reactivation of HERV-W- PML patients. JCV genome fragments can also be detected ENV was shown to induce inflammatory responses through in the brain of up to 56 % of individuals with no known TLR4. Therefore, these results expand the spectrum of neurological disease, which suggests that JCV is present at a HHV-6 effects and of its receptor CD46 in the modulation latent stage in the human brain. of the immune response and support the hypothesis that cross-talk between HHV-6A and HERV as well as complement activation and consequent CD46 activation may par- P22 ticipate in the pathogenesis of the neuroinflammation. HHV-6A infection induces the expression of Human Endogenous RetroViral elements (HERV-W-Env) in human cells: link to the neuroinflammation P23 Neuro-inflammation modulates Fcgamma receptor Benjamin Charvet1, Josephine Reynaud1, Gilda Raguenez2, expression on microglial cells following viral brain Hei-Lanne Dougier2, Julie Medina3, Herve Perron3,Patrice infection Marche2, Branka Horvat1 (corresponding author: [email protected]) Priyanka Chauhan, Shuxian Hu, Wen Sheng, Sujata Prasad, James Lokensgard 1International Centre for Infectiology Research, INSERM (corresponding author: [email protected]) U1111, CNRS UMR5308, Ecole Normale Superieure de Lyon, University of Lyon 1, 69365 Lyon, France; 2INSERM Department of Medicine, University of Minnesota Medical U823, Universite J Fourier, IAPC, 38041 Grenoble, France; School 3GeNeuro Innovation, Lyon, France Fcgamma receptors (FcgammaRs) for IgG couple innate and Herpesvirus infections have been associated with the induc- adaptive immunity through their ability to activate effector tion of human endogenous retroviruses (HERV), suggesting cells by antigen-antibody complexes. Brain-resident they could synergistically participate in the establishment of microglial cells, which are pivotal to pathogen detection different pathologies in humans. Among the HERV-W fam- and initiation of innate neuro-immune responses, co- ily, the envelope protein (HERV-W-ENV) seems to be in- express activating (FcgammaRI, FcgammaRIII, and volved in the pathogenesis of multiple sclerosis (MS). We FcgammaRIV) as well as inhibitory Fcgamma Receptor have analyzed here the interaction between HHV-6A and (FcgammaRIIB). Thus, the threshold for cellular activation HERV-W-ENV and have particularly focused on the is determined by the relative ratios of these opposing transactivator role of CD46 molecule, acting as the receptor FcgammaRs. In this study, we investigated the relative ex- for several viruses, including HHV-6A and measles. We in- pression of both activating and inhibitory FcgammaRs on fected several primary and immortalized human cell lines, microglia using an in vivo model of chronic neuro- including T lymphocytes, monocytes, glioblastoma and neu- inflammation following murine cytomegalovirus (MCMV)- roblastoma cells with HHV-6A (GS). The mRNA expression induced encephalitis. Flow cytometric analysis of microglial of ENV from different HERVs (syncytin-1, MSRV and cells obtained from infected brain tissue demonstrated that HERV-K18) was determined by quantitative RT-PCR and the activating FcgammaRs were expressed maximally at 5 d results were confirmed by immunofluorescence. The in- post infection (dpi), while the inhibitory receptor volvement of CD46 in HERV-W-ENV activation was ana- (FcgammaRIIB) remained highly elevated during the chron- lyzed using several CD46 ligands, including UV-inactivated ic phase of infection (i.e., 30 and 60 dpi). The highly in- HHV-6A, measles virus and anti-CD46 antibody and recom- duced expression of activating FcgammaRIV during the binant C3b complement fragment, which binds naturally acute phase of infection was also noteworthy. While, CD46. We showed that HERV-W-ENV over-expression in FcgammaRI was found to be constitutively expressed, there HHV-6A-infected human cells at transcript and protein level. was minimal expression of FcgammaRIII. Subsequently, Furthermore, the stimulation of HHV-6A receptor CD46, when the relative expression of FcgammaRs was analyzed using UV-inactivated HHV-6A, anti-CD46-SCR3 domain within the brains of mice post MCMV-infection by semi- Ab or C3b fragment of complement also increases the pro- quantitative RT-PCR (semi-qRT-PCR), we observed maxi- duction of HERV-W-ENV, in contrast to measles virus, mal expression of FcgammaRIV during the acute phase S12 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 commensurate with our flow cytometric studies. the ability of researchers to design gRNA that can cleave Furthermore, in vitro analysis of cultured primary murine HIV-1 while avoiding off-target effects. Future studies will microglial cells using flow cytometry and semi-qRT-PCR be performed to validate the off-target predictions using demonstrated maximal expression of FcgammaRIV and biomedical research laboratory techniques including FcgammaRIIB following stimulation with either IFN- GUIDE-seq. gamma or IL-4, respectively. We also demonstrated the role of IFN-gamma and IL-4 in polarizing microglia towards M1 or M2 phenotype, respectively. Thus, we conclude that acute P25 neuro-inflammation following viral infection increases ex- Altered mitochondrial biogenesis in brains during HAND: pression of activating FcgammaRs to promote pathogen Another result of Tat-mediated neurotoxicity? clearance through increased effector cell activation. In contrast, preferential expression of the inhibitory receptor during Hamza Coban1, Jerel A. Fields1, Edward Rockenstein2, Sarah chronic infection may provide a protective mechanism to Gough1, Ilse Flores1, Eliezer Masliah2, Paula Desplats2, prevent hyper-immune responses and subsequent bystander Cristian L. Achim1 brain damage. (corresponding author: [email protected])

1Department of Psychiatry, University of California San P24 Diego; 2Department of Neuroscience, University of Computational detection of off-target effects California San Diego of CRISPR/Cas9-associated gRNAs Background: Despite combined antiretroviral therapy Cheng-Han (James) Chung, Michael Nonnemacher, Brian (cART), HIV-associated neurocognitive disorders Wigdahl, William Dampier (HAND) remain a significant problem for the 1.4 million (corresponding author: [email protected]) people living with HIV in the USA. The causes of HAND are probably a diverse set of initiating factors, such as Department of Microbiology and Immunology, Institute for low-level viral expression, drugs of abuse, cART neuro- Molecular Medicine and Infectious Disease, Drexel toxicity, a combination of these factors and others, which University College of Medicine set in to action a cascade of inflammatory events that culminate in the neurodegeneration associated with Despite antiretroviral therapy, HIV-1 infection remains a HAND. Multiple recent publications implicate alterations life-long clinical problem due to reservoirs harboring provi- in mitochondrial dynamics in neurons as a mechanism of ral DNA in a latent/persistent form. Recently, gene-editing neurotoxicity in some HAND patients. Methods: In the strategies utilizing the CRISPR/Cas9 system have been de- current study, we asked whether alterations in mitochon- veloped to eradicate the HIV-1 genome from infected cells. drial biogenesis activities might also contribute to the These results offer a new avenue toward the elimination of pathogenesis of HAND in the cART era. To this end, we HIV-1 to cure HIV/AIDS. However, due to the promiscuous analyzed expression levels and distribution patterns of nature of the gRNA targeting, there is concern over off- mitochondrial biogenesis related genes and proteins in target binding sites that may cause unwanted DNA damage the frontal cortex of 37 well-studied cART-era HAND within the human genome that need to be assessed. This is donors from the NNTC cohorts. Results: We found sig- complicated by the non-linear nature of the binding penal- nificant increases in the master regulator of mitochondrial ties associated with gRNA recognition. With the availability biogenesis, peroxisome proliferator-activated receptor of unbiased identification of double stranded breaks enabled gamma coactivator (PGC)-1alpha, in brain tissues of by sequencing (GUIDE-seq), the off-target cleavage sites HAND donors; mRNA levels also indicated altered can be precisely observed. In order to measure the off- PGC-1alpha expression. Immunohistochemichal analyses target effect, we have developed a new database containing of PGC-1alpha show a distinct pattern in the brains of all potential cleavage sites within the entire human genome HAND donors compared to HIV- control. Importantly, along with all known single nucleotide polymorphisms in we found similar alterations brains of in inducible Tat tg dbSNP. Tree construction occurs on the order of mice compared to their non-tg counterparts. Discussion: O(N) ~ Nlog(N) time where N is the size of the database In combination with recent reports, our data suggest that and tree search occurs in O(m) ~ m where m is the length alterations at both ends of the mitochondrial biogenesis- of the query. In practice, the entire human genome and dynamics axis may contribute to HAND, and Tat may be dbSNP were indexed within 72 h and a query takes less an important player in some patients. As mitochondrial than 10 ms per gRNA. This database will greatly increase dysfunction is implicated in many neurodegenerative J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S13 disorders, further elucidation of these mechanisms may demonstrated. We are currently exploring the functions of provide a promising therapeutic target. these regions by mutagenesis. This study has been made possible by grants awarded to M. Safak from NIH (1R01NS090949-01A1) and Temple University Drug P26 Discovery Initiative (161398). Three Dimensional NMR Structure of the Human Polyomavirus JC Agnoprotein Revealed That the NH2- and COOH-termini of the Protein Adopt Intrinsically P27 Unstructured Conformation While the Center Portion Effects of cocaine and Tat on astrocyte-derived cholesterol Forms an Alpha-helix supplied to neurons

Pascale Coric1, A. Sami Saribas2, Jason Saredy2,Martyn Bianca Cotto1, William Yen1, Timothy Luongo2,Prasun White2, Serge Bouaziz1, Mahmut Safak2 Datta1, Satish Deshmane1,JohnElrod2, Dianne Langford1 (corresponding author: [email protected]) (corresponding author: [email protected])

1Universite Paris Descartes, Sorbonne Paris Cite, Laboratoire 1Department of Neuroscience, Center for Neurovirology; de Cristallographie et RMN Biologiques, UMR 8015 CNRS, 2Department of Pharmacology, Center for Translational 4av.del’Observatoire, Paris, France; 2Department of Medicine, Lewis Katz School of Medicine at Temple Neuroscience, Center for NeuroVirology, Laboratory of University, Philadelphia, PA USA Molecular Neurovirology, Lewis Katz School of Medicine at Temple University, Philadelphia, USA The human brain requires a balance of cholesterol homeostasis to meet the high metabolic requirements of neurons JC virus (JCV) causes a fatal demyelinating disease of the and maintain myelin integrity. Neurons rely on cholesterol central nervous system (CNS), known as progressive multi- synthesized by astrocytes for synaptogenesis and normal focal leukoencephalopathy (PML), in a subset of the patients functioning. In the brain, cholesterol synthesis and clear- with underlying immunosuppression, including lymphoma, ance are tightly regulated to maintain cholesterol levels. leukemia and AIDS due to the lytic infection of the glial cells, Liver X receptors (LXRs) are the master regulators of including oligodendrocytes, by the virus. The incidence of cholesterol homeostasis in the CNS. LXR activation leads PML dramatically increased in the era of the AIDS epidemic to the transcription of target genes involved in cholesterol and this disease has also been steadily increasing among au- trafficking and efflux, including apolipoprotein E (ApoE). toimmune disorder patients, including multiple sclerosis and The disturbance of LXR signaling in the brain can lead to Crohn’s disease, who are treated with antibody-based drugs. significant dysfunction in cholesterol homeostasis and dis- JCV establishes a sub-clinical persistant infection in the body ruptions have been implicated in several neurological dis- during early childhood, but upon reactivation, it causes the eases. Likewise, HIV infection and cocaine use are asso- neurological manifestations of PML. JCV encodes a limited ciated with myelin loss and synaptodendritic damage sug- number of structural and regulatory proteins. Among those, gesting that dysregulation in CNS cholesterol metabolism agnoprotein has been shown to play essential regulatory roles may be involved in the progression of neurological impair- during the viral replication cycle where JCV was demonstrat- ment. Therefore, given the importance of astrocytes in ed to be unable to sustain its productive life cycle in the LXR-mediated cholesterol regulation and its role in pro- absence of agnoprotein expression. Previous studies also viding metabolic support to other CNS cells, we hypothe- demonstrated that agnoprotein forms highly stable dimers sized that exposure of astrocytes to cocaine and Tat would and oligomers through its Leu/Ile/Phe-rich domain. lead to disruptions in LXR signaling and the expression of Subsequent NMR structural studies using a partial LXR regulated genes. Alterations in these pathways may agnoprotein peptide revealed that the amino acids from in turn decrease the bioavailability of cholesterol from Lys23 to Phe39, containing Leu/Ile/Phe-rich domain, adopt astrocytes to neurons and oligodendrocytes and promote a alpha-helical conformation. Here we report the complete 3D cellular dysfunction. We found that in astrocytes, exposure structure of the full-length peptide of agnoprotein by NMR. to cocaine and Tat significantly decreased LXRbeta ex- This 3D structure confirmed the localization of the previously pression. In addition, we discovered that LXR target genes reported alpha-helical domain of the protein and further re- including ApoE were also significantly reduced upon treat- vealed the presence of another shorter alpha-helix located ment with cocaine and Tat. In response, the ABC1a trans- between residues Leu6 and Lys13. The remaining regions porter responsible for transporting ApoE bound cholesterol of the agnoprotein exhibit an intrinsically unstructured con- out of the cell is increased significantly. Our findings formation, the significance of which has yet to be demonstrate the combined effects of cocaine and Tat on S14 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 the overall bioavailability of cholesterol provided by astro- P29 cytes to neurons and identify a novel role for LXR agonist Agnoprotein interferes with the neuroimmune response as a therapeutic intervention in the severity and progres- to JC Virus sion of HIV infection and/or cocaine abusing individuals. Michael Craigie, Stephanie Cicalese, Martina Donadoni, Ilker Sariyer (corresponding author: [email protected]) P28 Microglial activation and cocaine addiction Department of Neuroscience, Center for Neurovirology, LewisKatzSchoolofMedicineatTempleUniversity, Bianca Cotto1, Christina Chambers2, Hongbo Li2, William Philadelphia, PA USA Yen1, Ronald Tuma2, Sara Jane Ward2, Dianne Langford1 (corresponding author: [email protected]) JC virus is a human neurotropic polyomavirus and the 1Department of Neuroscience, Center for Neurovirology; etiologic agent of progressive multifocal 2Department of Pharmacology, Center for Substance Abuse leukoencephalopathy, a demyelinating disease of gray Research, Lewis Katz School of Medicine at Temple and white matter. PML is primarily observed in immu- University, Philadelphia, PA USA nocompromised patients, including patients with AIDS and those undergoing immunomodulatory therapies. Recent research shows that glial cells are intimately During JCV infection, the virus encodes multiple viral involved in synaptic plasticity, and that drugs of abuse proteins including the regulatory agnoprotein. We have affect glial activity. Specifically, microglia contribute to recently discovered that agnoprotein is secreted from synaptic plasticity via direct interactions with dendritic infected cells into the extracellular matrix. Also, previ- spines, synaptic pruning, and regulation of hippocampal ous data demonstrated that JCV infection is suppressed neurogenesis. We have combined a series of in vivo, by treatment with conditioned media from peripheral ex vivo, and in vitro experiments to test the hypothesis blood mononuclear cells, suggesting that the immune that cocaine exposure can lead to significant alterations system and immunological soluble factors play a major in microglial activation, enhancing cocaine-induced role in the regulation of JCV. Here, we have investi- neuroplasticity. In turn, these cascades contribute to pe- gated the possible role of extracellular agnoprotein in ripheral immune cell invasion, thereby priming the the neuroimmune response to JCV in glial cells. neuroimmune axis for a cycle of neuroinflammation in Following the cellular expression of agnoprotein, the context of cocaine addiction. We found that cocaine agnoprotein is released by glial cells and is able to self-administration in the rat model increases microglial be internalized by both astrocytes and microglia activation in reward regions of the brain, and increases in vitro. This internalization of agnoprotein was found expression of the transcription factor and synaptic plas- to impact the profile of cytokines released by both cell ticity marker MeCP2. We then determined in vitro that types. Moreover, the treatment of astrocytes with me- microglia express MeCP2 and that this expression india containing agnoprotein resulted in a significant re- creases significantly following cocaine exposure and induction in GM-CSF secretion. Subsequent reporter ducing release of BDNF, suggesting that cocaine’sef- gene analysis demonstrated that agnoprotein is capable fects on MeCP2 expression may participate in cocaine- of suppressing the transcription of GM-CSF, implicat- induced neuroplasticity. In addition, we observed that ing a possible mechanism for the reduction of released chronic cocaine administration increases cerebrovascular GM-CSF levels. Likewise, the treatment of a human leukocyte rolling and adhesion and subsequent BBB monocyte cell line, U937, with agnoprotein resulted weakening that persists during withdrawal, setting up in decreased macrophage differentiation, including de- the likelihood for a persistent dysregulation of creased attachment and decreased phagocytic ability. neuroimmune signaling that may mirror the cycle of Similarly, treatment of peripheral blood mononuclear cocaine addiction. These findings identify novel cells with agnoprotein resulted in decreased overall mi- neuroimmune targets such as activated microglia for gration across an in vitro blood brain barrier model. treating psychostimulant abuse, for which no approved These findings have suggested that extracellular medications exist. Our findings also illustrate mecha- agnoprotein is able to modulate aspects of the immune nisms in the cocaine abuse paradigm that are likely response to JCV, primarily through the suppression of important in CNS disease progression in HIV-infected GM-CSF release and subsequent impact on monocyte/ patients who use cocaine. macrophage function. J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S15

P30 P31 HIV-1 genetic variation resulting in the development Defining Vpr sequence variants and associated of new quasispecies continues to be encountered mechanisms that enhance HIV CNS pathogenesis in the peripheral blood of well-suppressed patients and disease

William Dampier1, Michael Nonnemacher1, Joshua Mell1, William Dampier1, Gregory Antell2, Michael Cruz3, Joshua Earl1, Vanessa Pirrone1, Benjamas Aiamkitsumrit1, Benjamas Aiamkitsumrit1, Michael Nonnemacher1, Zsofia Wen Zhong1, Katherine Kercher1, Shendra Passic1,Jean Szep4, Jeffrey Jacobson5, Vanessa Pirrone1, Wen Zhong1, Williams1, Zsofia Szep2, Jeffrey Jacobson3,BrianWigdahl1 Katherine Kercher1, Shendra Passic1, Jean Williams1, Tony (corresponding author: [email protected]) James1,KathrynDevlin6, Tania Giovannetti7, David Libon8, Garth Ehrlich1,BrianWigdahl1,FredKrebs1 1Department of Microbiology and Immunology, Institute for (corresponding author: [email protected]) Molecular Medicine and Infectious Disease; 2Department of Medicine, Division of Infectious Diseases and HIV Medicine, 1Department of Microbiology and Immunology, Institute for Drexel University College of Medicine; 3Department of Molecular Medicine and Infectious Disease, Drexel Medicine, Section of Infectious Disease, Lewis Katz School University College of Medicine; 2School of Biomedical of Medicine, Temple University Engineering, Science, and Health Systems, Drexel University; 3Interdisciplinary Health Sciences (IHS) As a result of antiretroviral therapeutic strategies, human Program, Graduate School of Biomedical Sciences and immunodeficiency virus type 1 (HIV-1) infection has be- Professional Studies, Drexel University College of come a long-term clinically manageable chronic disease Medicine; 4Department of Medicine, Division of Infectious for many infected individuals. However, despite this prog- Diseases and HIV Medicine, Drexel University College of ress in therapeutic control, including undetectable viral Medicine; 5Department of Medicine, Section of Infectious loads and CD4+ T-cell counts in the normal range, viral Disease, Lewis Katz School of Medicine, Temple mutations continue to accumulate in the peripheral blood University; 6 Department of Neuroscience and compartment over time, indicating either low level reacti- Comprehensive NeuroAIDS Center, Lewis Katz School of vation and/or replication. Using patients from the Drexel Medicine, Temple University; 7Department of Psychology, Medicine CNS AIDS Research and Eradication Study Temple University; 8Department of Geriatrics and (CARES) Cohort, whom have been sampled longitudinal- Gerontology, New Jersey Institute for Successful Aging, ly for more than 7 years, genetic change was modeled School of Osteopathic Medicine, Rowan University against the dominant integrated proviral quasispecies with respect to selection pressures such as therapeutic interven- Studies of HIV-associated neurocognitive disorders tions, AIDS-defining illnesses, and other factors. (HAND) have identified viral protein R (Vpr) as a Phylogenetic methods based on the sequences of the neuropathogenic factor. Extracellular Vpr, which has been LTR and tat exon 1 of the HIV-1 proviral DNA detected in the sera and cerebrospinal fluids of HIV-1- quasispecies were used to obtain an estimate of an aver- infected patients, causes adverse changes in brain resident age mutation rate of 5.3 nucleotides (nt)/kilobasepair (kb)/ cells, including macrophages, astrocytes, and neurons. Our year (yr) prior to initiation of antiretroviral therapy recent studies of Vpr have turned to investigating connec- (ART). Following ART the baseline mutation rate was tions between naturally occurring Vpr sequence variation reduced to an average of 1.02 nt/kb/year. The post-ART and neuropathogenesis in HIV-1-infected patients from the baseline rate of genetic change, however, appears to be Drexel Medicine CNS AIDS Research and Eradication unique for each patient. These studies represent our initial Study (CARES) Cohort. Analyses of peripheral blood- steps in quantifying rates of genetic change among HIV-1 derived Vpr sequences from Cohort patients revealed three quasispecies using longitudinally sampled sequences from amino acids in Vpr (positions 37, 41, and 55 in the 96 aa patients at different stages of disease both before and after protein) associated with significant differences in neuro- initiation of combination ART. Notably, while long-term psychological impairment (niVpr variants) measured by ART reduced the estimated mutation rates in the vast ma- comprehensive neuropsychological testing. 37-I and 41-S jority of patients studied, there was still measurable HIV-1 were linked to reductions in diagnosed neuropathogenesis mutation even in patients with no detectable virus by stan- (with respect to the average Global Deficit Score or GDS), dard quantitative assays. Determining the factors that af- while 41-N and 55-A were associated with increased fect HIV-1 mutation rates in the peripheral blood may lead neuropathogenesis and higher GDS. The effects of niVpr to elucidation of the mechanisms associated with changes variants appear to be cumulative, since the combined ef- in HIV-1 disease severity. fect sizes for Vpr sequences with two or three niVpr S16 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 variants preliminarily correlate with changes in average creation of 3D animations to visualize spike progression in patient GDS. Deep sequencing demonstrated the appear- individual electrodes over time, burst identification, spike ance of niVpr variants in HIV-1-infected patient blood, and burst firing rates, and periodicity detection. We found spleen, and brain samples, and indicated sequence com- that exosomes released from astrocytes in response to ATP partmentalization as well as variations in relative preva- stimulation produced a dose-dependent increase in dendritic lence. The pathogenic effects of Vpr variation on complexity, increased neural network connectivity, and peri- immunopathogenesis and neuropathogenesis were sug- odic synchronous network burst activity. In contrast, gested, respectively, by higher CD4 T-cell counts in pa- exosomes shed from astrocytes in response to HIV-MDS tients with the 41-N variant (relative to cell counts in reduced dendritic complexity. These data suggest that EVs patients with the 41-S variant) and greater reductions in shed from astrocytes strengthen neural network connectivity astrocyte glutathione pools subsequent to transient expres- and in the setting of HIV infection EVs may deconstruct sion of the 41-N Vpr variant (relative to the 41-S variant). neural networks possibly by modifying the composition of EV cargo.

P32 Astrocyte-shed extracellular vesicles regulate synchronous P33 network burst activity Follicular DCs within deep cerebral lymph nodes are potential reservoir of HIV-CNS infection Raha Dastgheyb1, Saja Khuder1, Joelle Dorskind1,Sheng Wang1, Amrita Datta Chaudhuri1, Seung-Wan Yoo1, Rajnish S. Dave1,ArtinderP.Nehra1, Roshell R. Muir2, Norman Haughey2 Rashida Ginwala1, Claire Deleage3,DivyaSagar1,Brian (corresponding author: [email protected]) Wigdahl1, Zafar K. Khan1, Elias Haddad4,JacobD.Estes3, Francois Villinger5,AftabAnsari5, Siddappa Byrareddy6, 1Department of Neurology, Johns Hopkins University School Pooja Jain1 of Medicine; 2Department of Neurology, Johns Hopkins (corresponding author: [email protected]) University School of Medicine. Department of Psychiatry, Johns Hopkins University School of Medicine 1Department of Microbiology and Immunology, and the Institute for Molecular Medicine and Infectious Disease, Synaptic damage and disruption of neuronal networks are Drexel University College of Medicine, Philadelphia, PA; prominent features of HIV Associated Neurocognitive 2Division of Infectious Disease and HIV Medicine, Disorders (HAND). The underlying mechanisms for these Department of Medicine, Drexel University College of pathological alterations in synaptodendritic networks are Medicine, Philadelphia, PA; 3AIDS and Cancer Virus largely unknown, but are thought to involve chronic glial Program, Frederick National Laboratory for Cancer activation and inflammation. Based on evidence that Research, Leidos Biomedical Research, Inc., Frederick, MD; astroglia to neuron communication regulates the formation 4Division of Infectious Disease and HIV Medicine, and strength of synaptic connections, we hypothesized that Department of Medicine, Drexel University College of HIV infection disrupts neural network activity by modifying Medicine, Philadelphia, PA; 5Department of Pathology & the quality of astrocyte to neuron communication. Laboratory Medicine, School of Medicine and Emory Extracellular vesicles (EVs) are emerging as a mechanism Vaccine Center, Emory University, Atlanta, GA; by which astroglia regulate neuronal communication through 6Department of Pharmacology and Experimental the delivery of protein, RNA and lipid cargo that regulate Neuroscience, University of Nebraska Medical Center, the activity of target cells. In this study we isolated Omaha, NE exosomes shed from astrocytes in response to ATP (a trophic stimulus), or media from HIV-infected macrophages Human immunodeficiency Virus (HIV) neuroinvasion leads (HIV-MDM; HIV and EV depleted), and exposed primary to neurodegeneration and HIV-associated neurocognitive dis- rodent neurons to a dose response of 5–100 EVs/cell. The order. In the central nervous system (CNS) circulating dendrit- effects of EVs on dendritic length, branching, and complex- ic cells (cDCs) encounter HIV virions or viral proteins and ity were determined using structured fluorescent imaging, migrate along the rostral migratory system to the cervical or and neural connectivity was determined using multichannel cerebral lymph nodes (CxLNs). Subsequently, HIV accumu- electrode arrays. Spike detection was accomplished using lates in the germinal centers (GCs) and there it appears bound filtered voltage measurements with a threshold for detection to follicular dendritic cells (FDCs). In this study, we sought to of 5 standard deviations above baseline. A MATLAB based dissect the mechanisms underlying harboring of HIVon FDCs code was developed to extract spike times that allow for the and their transmission to CD4+ T follicular helper (TFH) cells J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S17 in CxLNs of chronically SIV-infected rhesus macaques duration) from non-signals (no illumination) during the (RMs). Chronic SIV infection was established in all RMs. duration task. Both HIV-1 Tg and control females were CD35+ FDCs in CxLNs were identified to harbor SIV. unable to discriminate signals presented at shorter dura- Furthermore, FcRs (FcgammaRIIB) immune complexes co- tions than their male counterparts. Collectively, these localized with the FDCs and B-cells, suggesting SIV presen- findings suggest that the factor of biological sex has a tation by FDCs to B-cells. Significantly, B-cell interaction significant effect on the development of NCI for HIV-1 with TFH cells resulted in the latter getting infected. Hence, patients. Funded by NIH grants DA013137, HD043680, FDCs in CxLN not only retained SIV, but they also infected and MH106392. TFH cells. Based on these observations, we infer a novel role for FDCs in viral entrapment and creation of persistent CNS reservoir. Hence, our study highlights the role of FDCs in P35 accumulation and transmission of HIV with implications for IFN-Gamma Inhibits JC Virus Replication in Glial Cells HIV egress from the CNS. by Suppressing T-Antigen Expression.

Francesca Isabella De Simone, Rahsan Sariyer, Shadan P34 Yarandi, Michael Craigie, Jennifer Gordon, Ilker Sariyer Sex Matters: Differential Deficits in Sustained Attention (corresponding author: [email protected]) in the Male and Female HIV-1 Transgenic (Tg) Rat Temple University Lewis Katz School of Medicine Iris K. Dayton, Kristen A. McLaurin, Rosemarie M. Booze, Charles F. Mactutus Patients undergoing immune modulatory therapies for the (corresponding author: [email protected]) treatment of autoimmune diseases such as multiple sclerosis, and individuals with an impaired-immune system, most nota- Department of Psychology, University of South Carolina bly AIDS patients, are in the high risk group of developing progressive multifocal leukoencephalopathy (PML), an often Neurocognitive impairment (NCI) is a prevalent condition lethal disease of the brain characterized by lytic infection of of HIV-1 infected individuals because of the remarkable oligodendrocytes in the central nervous system (CNS) with JC success of combined antiretroviral therapy. Recent studies virus (JCV). Here, we investigated the impact of soluble im- suggest an effect of gender on the domains of speed of mune mediators secreted by activated PBMCs on viral repli- information processing, verbal fluency, learning, and cation and gene expression by cell culture models and molec- memory. Although it has been reported that the risk for ular virology techniques. Our data revealed that viral gene developing NCI for HIV-1 men and women differs, the expression and viral replication were suppressed by soluble examination of biological sex differences in NCI, inde- immune mediators. Further studies demonstrated that soluble pendent of societal factors, has not been previously con- immune mediators secreted by activated PBMCs inhibit viral ducted. Deficits in executive function are a distinctive replication induced by T-antigen, the major viral regulatory characteristic of NCI. Presently, we examined the poten- protein, by suppressing its expression in glial cells. This un- tial sex differences in sustained attention deficits, as a expected suppression of T-antigen was mainly associated with core component of executive function using the HIV-1 the suppression of translational initiation. Cytokine/ Tg rat. Male Fischer HIV-1 Tg (n = 35) and F344/N con- chemokine array studies using conditioned media from acti- trol rats (n = 35), and intact female Fischer HIV-1 Tg vated PBMCs revealed several candidate cytokines with pos- (n = 35) and F344/N control rats (n = 33) were trained sible roles in this regulation. Among them, only IFN-gamma (shaping) and tested (vigilance and manipulation of stim- showed a robust inhibition of T-antigen expression. While ulus duration) in a signal detection task. There were potential roles for IFN-beta, and to a lesser extent IFN-alpha marked sex differences in initial training of the signal have been described for JCV, IFN-gamma has not been pre- detection task, acquisition of the vigilance task, and per- viously implicated. Further analysis of IFN-gamma signaling formance on the duration task. Females acquired the sig- pathway revealed a novel role of Jak1 signaling in control of nal detection task at a much slower (4X) rate than males. viral T-antigen expression. Furthermore, IFN-gamma sup- Females met criterion of five consecutive days or seven pressed JCV replication and viral propagation in primary hu- non-consecutive days at 70 % or higher for the vigilance man fetal glial cells, and showed a strong anti-JCV activity. task at a significantly slower rate than males; there was a Our results suggest a novel role for IFN-gamma in the regu- 14-day lag before any female met the acquisition criteri- lation of JCV gene expression via downregulation of the ma- on. The factor of biological sex altered the ability to dis- jor viral regulatory protein, T-antigen, and provide a new av- criminate signals (illumination for 100, 500, or 1000 msec enue of research to understand molecular mechanisms for S18 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 downregulation of viral reactivation that may lead to develop- play a crucial role in neurotoxicity associated with al- ment of novel strategies for the treatment of PML. cohol consumption.

P36 P37 Alcohol-Mediated Alternative Splicing of Mcl-1 The role of infiltrating macrophages and IL-6 production pre-mRNA is Involved in Ethanol Induced Neurotoxicity on seizure development after viral infection

Francesca Isabella De Simone1, Rahsan Sariyer1, Sulie L. Ana Beatriz DePaula-Silva, Tyler Hanak, Daniel Doty, Jordan Chang2,IlkerSariyer1 Sim, Jane Libbey, Robert Fujinami (corresponding author: [email protected]) (corresponding author: [email protected])

1Department of Neuroscience, Center for NeuroVirology, University of Utah, School of Medice, Department of Lewis Katz School of Medicine at Temple University; Pathology 2Institute of NeuroImmune Pharmacology, Seton Hall University Epilepsy is a chronic neurological disorder characterized by recurrent seizures. Seizures, which are often associ- Heavy and chronic ethanol exposure can cause signifi- ated with viral encephalitis, occur in response to imbal- cant structural and functional damage to the adult brain. ances between excitatory and inhibitory inputs within The developing nervous system is even more vulnerable the brain. Patients with viral encephalitis are 16 times to ethanol exposure. Prenatal exposure of ethanol during more likely to develop epilepsy. It is estimated that pregnancy can lead to fetal alcohol spectrum disorders around 65 million people worldwide suffer from epilep- (FASD), characterized by malformation of the nervous sy. Although treatments to prevent seizures are avail- system and mental retardation. The most devastating able, they are mainly anticonvulsants and around 30 % consequence of ethanol exposure is the neurotoxicity of the patients do not respond to the medication and associated with the depletion of neurons. It is crucial numerous side effects related to the drugs have been to elucidate mechanisms of neuroapoptosis in order to reported. In order to develop new immunomodulatory develop effective therapeutic approaches to overcome treatments that could lead to an eventual cure for sei- ethanol-induced neuropathologies. Regulation of splice zures/epilepsy, a better understanding of the seizure de- variants in the brain can modulate protein functions, velopment mechanism is required. We have developed which may ultimately affect behaviors associated with an experimental model of virus-induced seizures/epilep- alcohol dependence and ethanol-mediated neurotoxicity. sy. In our model, C57BL/6 mice are infected intra- Limited number of studies has shown that pre-mRNA cranially (i.c) with the Daniels strain (DAV) of splicing patterns of genes are potentially altered and Theiler’s murine encephalomyelitis virus (TMEV). involved in behavior changes associated with alcohol- Between3and10dayspostinfection(d.p.i),around ism. Since alcohol consumption is associated with neu- 50 % of the infected mice will develop spontaneous rotoxicity, it is possible that altered splicing of survival acute seizures and approximately 50 % of the mice that and pro-survival factors during the development of al- have experienced spontaneous acute seizures will devel- coholism may contribute to the neurotoxicity. Our re- op epilepsy. Because seizures begin to develop at 3 sults suggest that ethanol exposure can lead to pre- d.p.i, we believe it happens as a consequence of the mRNA missplicing of Mcl-1, a pro-survival member of activation of the innate immune response. We found that the Bcl-2 family, by downregulating the expression seizures are correlated with an increase in myeloid and levels of serine/arginine rich splicing factor 1 (SRSF1). lymphoid cells infiltrating into the brain. We also found The pre-mRNA of Mcl-1 can be alternatively spliced to that the pro-inflammatory cytokines IL-6 and TNF alpha remove exon 2, which produces shortened form of Mcl- were elevated at 3 d.p.i. IL-6 knockout mice infected 1, named Mcl-1S. While the longer gene product Mcl- with DAV experienced significant fewer seizures. 1 L enhances cell survival, the alternatively spliced Because infiltrating macrophages are the main producers shorter gene product Mcl-1S promotes apoptosis. Our of IL-6, we depleted macrophages from mice and we preliminary data has indicated that ethanol exposure to infected them with DAV. We did not observed seizures neurons leads to a decrease in the ratio of Mcl-1 L/Mcl- in animals lacking macrophages, suggesting macro- 1S by favoring pro-apoptotic Mcl-1S splicing over anti- phages are playing a central role in the development apoptotic Mcl-1 L isoform suggesting that Mcl-1S may of seizures after viral infection. J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S19

P38 (corresponding author: [email protected]) Macrophage OXPHOS regulation by HIV-1 Vpr and cocaine 1Section of Infection of the Nervous System; National Institute of Neurological Disease and Stroke (NINDS); NIH; Satish Deshmane, Shohreh Amini, Prasun Datta 2Bioinformatics Section of Information Technology Program, (corresponding author: [email protected]) NINDS, NIH; 3Neural Differentiation Unit, NINDS, NIH; 4 Community Networks Program, Section of Neuroscience, Comprehensive NeuroAIDS center, LKSOM Neurophysiology and Biophysics, NINDS, NIH; 5National at Temple University Cancer Institute, NIH; 6Section of Infection of the Nervous System, NINDS, NIH HIV-1 infected macrophages play a significant role in the pathogenesis of AIDS. HIV-1 viral protein R (Vpr) not Human Endogenous Retroviruses (HERVs) comprise approx- only facilitates HIV-1 infection but also contribute to imately 8 % of the human genome and play a critical, highly long-lived persistence in macrophages. Our previous stud- regulated spatiotemporal role in cellular differentiation during ies using SILAC-based proteomic analysis showed that the development. We hypothesize that HERVs play a critical role expression of critical metabolic enzymes in the glycolytic in neuronal differentiation, and their dysregulation results in pathway and tricarboxylic acid (TCA) cycle were altered in neurodevelopmental tumors. Atypical Teratoid Rhabdoid response to Vpr expression in macrophages. Mitochondria Tumor (AT/RT) is a rare type of pediatric brain cancer. The are key players in the generation and regulation of cellular tumor cells have an expression profile similar to embryonic bioenergetics, producing the majority of adenosine triphos- stem cells (ESCs) and induced pluripotent stem cells (iPSCs). phate molecules by the oxidative phosphorylation system We characterized the cell line by immunostaining and found (OXPHOS). We therefore assessed the effects of Vpr alone the cells strongly express Pax6, a neuro-ectodermal marker, and Vpr in combination with cocaine on the relative pro- and Oct4, an iPSC marker while having low expression of tein expression of OXPHOS mitochondrial complexes in Beta tubulin, a neuronal marker, and Nestin, a neuronal stem macrophages. U937 differentiated macrophages were cell marker. Interestingly, when the AT/RT cell line was grown transduced with adenoviral vector harboring Vpr gene. as an adherent line using matrigel and incubated in differenti- Transduced cultures were either left untreated or treated ation media, the cells began to differentiate into neuronal pre- with 5‘microM of cocaine for 1 and 3 days. Cell-lysates cursors and neurons with increased expression of both MAP2 were prepared and electron transport complexes were de- and Nestin proteins. We analyzed each of the cell types and tected using an antibody mix that detects key component determined the stage of differentiation using RT-PCR and proteins from each complex (CI-NDUFB8, CII-SDHB, qPCR; furthermore, we compared the AT/RT derived neuro- CIII-UQCRC2, CIV-MTCO1 and CV-APT5A). Complex nal cells with neurons generated from iPSCs. Similar to III, IV and V levels stayed constant however, complex I iPSCs, the AT/RT cells expressed HERV-K; however, and II were found to be negatively affected by Vpr. HERV-K expression decreased dramatically as the cells dif- Complex-I was found to be downregulated by 32 % on ferentiated into neuronal precursors and finally neurons. day 1 and day 3 post-transduction with Vpr. Complex-II When we blocked HERV-K expression using siRNA in was found to be downregulated by 33 and 71 % on day 1 iPSCs we observed neuronal differentiation as determined and day 3. With cocaine, a transient upregulation of com- by RT-PCR, qPCR, and immunostaining. Our data leads us plex I and II was found on day 1. However, both com- to conclude that HERV elements likely play a key role in plexes I and II were found to be downregulated by 70 % neuronal differentiation; furthermore, it raises the possibility and 32 % respectively on day 3 post-transduction. that manipulation of HERV expression could be utilized as a Together, our data show that Vpr and cocaine reprogram treatment modality for neurodevelopmental tumors. macrophage OXPHOS. Funded by NIDA.

P39 P40 Critical Role of Human Endogenous Retroviruses Use of a lip scarification mouse model to study (HERVs) in Neurodevelopment and Neurodevelopmental the pathology, immunology, and virology associated Tumors. HSV-1 Infection of the periphery and corresponding trigeminal ganglia Tara Doucet-O’Hare1, Kory Johnson2, Tongguang Wang3, Marie Medynets3,WinsonHo4, Zhengping Zhuang5, Kevin Egan, Alex Allen, Brian Wigdahl, Stephen Jennings Avindra Nath6 (corresponding author: [email protected]) S20 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89

Department of Microbiology and Immunology, Institute for through dynamic neuroglia crosstalk. Susceptibility to HIV-1 Molecular Medicine and Infectious Disease, Drexel infection and subsequent latency in astrocytes contribute to University College of Medicine neuronal damage that culminates into HIV-1 Associated Neurocognitive Disorders (HAND). The neurotoxic viral Herpes simplex virus type 1 (HSV-1) replicates in epithelial protein HIV-1 Transactivator of Transcription (Tat), is re- cells of mucosal surfaces before establishing a lifelong latent leased in the brain even in successful cART cases. Tat is infection within the trigeminal ganglion. Although there is reported to be produced in HIV-1 infected astrocytes. an established ocular infection model in the laboratory Recently, we have demonstrated that astrocytes mediate in- mouse which reproduces primary infection and latency ob- direct neuronal death by releasing excess ATP through acti- served in human, the majority of primary human infections vation of purinergic receptor system and experimentally occur within the lip and latency is established within a dif- proved that enhanced ATP levels induce astrocytes to secrete ferent branch of the trigeminal ganglion. Based on this, the more MCP-1(CCL2), that is critical for monocyte aided viral lip scarification model has been used to study the genetic trafficking into CNS. Using well characterized model system loci of HSV-1 resistance in the laboratory mouse, however of human primary astrocytes and neurons, we further probed the basic kinetics of and the pathologic and immunologic into the molecular mechanism for enhanced ATP release in responses to viral infection have not been fully defined. Tat affected astrocytes. It was observed that HIV-1 Tat mod- Using the McKrae strain of HSV-1, we have proceeded to ulates the miRNA machinery in astrocytes to perturb the define the kinetics of viral infection in the lip. The lower lip levels of voltage dependent anion channel-1 (VDAC1), a of 3-month-old mice were scarified and inoculated with channel present in the outer mitochondrial membrane and HSV-1 and tissue was collected at 7 time points up to day plasma membrane which regulates extracellular ATP release. 60 post infection for detection of infectious virus and We report a novel molecular cascade of miRNA-mediated responding immune cells. High virus titers were detected ATP release through regulation of VDAC1 and provide ev- in the lip at early time points that resolved after 8 days of idence that HIV-1 Tat dysregulates miR- 320a that in turn exposure. Lip pathology peaked 5 days post-infection and perturbs astrocytic ATP release. Down-regulation of resolved 15 days post-infection with residual lymphocytes VDAC1 either with miR-320a mimic or VDAC1 siRNA present. CD45+ cells including CD4+ and CD8+ T lympho- in HIV-1 Tat affected astroglia, could rescue the neurons cytes infiltrated the TG and associated with neurons. CD8+ from glia-mediated indirect death. We corroborated these and CD4+ T lymphocytes were retained in the trigeminal in vitro findings using HIV-1 patients suffering from mild ganglia and were present at 30 and 60 days post-infection. cognitive disorder (MCI) and age-matched control; we ob- Analysis of genome copies revealed that genome copies served dysregulated miR-320a- VDAC1 axis in frontal cor- peaked at 8 days post-infection in the lip. In the trigeminal tex of HIV-MCI patients as compared to control subjects. ganglia genome copies established a steady level at 5 days Our findings reveal a novel upstream therapeutic target that post-infection. These results are the first descriptions of could be employed to abolish the astroglia-mediated neuro- HSV-1 kinetics and pathology in the mouse lip. This model toxicity in HIV neuropathogenesis. will be useful in studying the virologic and immunologic aspects associated with HSV-1 infection of the lower lip where most human infections occur. P42 Expression of mitochondrial biogenesis and innate inflammation genes in brains of HIV+ donors stratified P41 by mtDNA haplogroups Novel molecular pathway of perturbation of ATP release in astrocytes contributes to neuronal death in HAND Jerel Fields, Hamza Coban, Sarah Gough, Ilse Flores, Eliezer Masliah, Paula Desplats, Cristian Achim Mahar Fatima1, Bharat Prajapati2, Kanza Saleem2, Chitra (corresponding author: [email protected]) Singal2, Pankaj Seth2 (corresponding author: [email protected]) University of California, San Diego

1Cellular and Molecular Neuroscience, National Brain Despite combined antiretroviral therapy (cART), HIV- Research Centre, Manesar, India; 2Cellular and Molecular associated neurocognitive disorders (HAND) remain a signif- Neuroscience, National Brain Research Centre, Manesar icant problem for 1.4 million people living with HIV in the USA, and minority populations are disproportionately affect- Astroglia are indispensable component of the tripartite synap- ed. The causes of HAND are multifactorial and may include se that regulate neuronal functions, health and survival low-level viral expression, drugs of abuse, cART J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S21 neurotoxicity, a combination of these and others, which initi- a Tat-mediated HIV LTR transactivation assay with which ate a cascade of inflammatory events that culminate in neuro- we can identify new antiviral agents that target Tat itself. degeneration. Recent studies suggest mitochondrial DNA Using the Tet-On 3G regulated transcription system, we (haplogroups), and hence mitochondrial function. A re- have successfully developed and optimized a stable, induc- cent study suggests that Hispanics identified as carrying ible HeLa cell line that: a) is fast-growing and easy to haplogroup B mtDNA may be less likely to develop maintain in culture, b) exhibits minimal background Tat HAND. Here we sought to investigate mitochondrial bio- expression in the absence of induction, c) enables Tat ex- genesis and inflammatory gene expression in HAND do- pression and LTR transactivation to be monitored by a nors from different haplogroups. In the frontal cortex of fluorescent tag and a luminescent reporter, respectively, d) 60 (32 Hispanic) well-studied cART-era HAND donors, demonstrates reliable and consistent GFP fluorescence and we found significant increases in the master regulator of Luciferase luminescence data reproducibility and e) dis- mitochondrial biogenesis, peroxisome proliferator- plays minimal LTR activation in the absence of Tat stimu- activated receptor gamma coactivator (PGC)-1alpha, in lation. Using this cell line in high throughput screening brain tissues of HAND donors; mRNA levels also indi- assays in 384 well plate format, we’ve screened more than cated altered PGC-1alpha expression, with more signifi- 2000 compounds from the Spectrum Collection to identify cant increases in Hispanic donors. Immunohistochemichal therapeutic antagonists to Tat-TAR binding and subsequent analyses show a distinct pattern in the brains of HAND LTR transactivation. Subsequent studies will confirm the donors compared to HIV- control and HIV+ Normal, and activity of the “hit” compounds, characterize them in con- between haplogroups. Importantly, we found similar alter- centration dependent analyses and determine their direct ations in complement pathway protein expression, with binding interactions with HIV Tat, in order to provide a the highest gene expression levels detected in Hispanics. novel therapeutic for HIV infection. In combination with recent reports, these data suggest that alterations at both ends of the mitochondrial biogenesis- dynamics axis may contribute to HAND. In Hispanic do- P44 nors, other than those of haplogroup B, altered comple- Whole body mapping shows a relatively small pool ment pathway protein expression may increase the risk of of latent HIV in the CNS not strongly related to pools developing HAND. As mitochondrial dysfunction is im- in other body compartments plicated in many neurodegenerative disorders, further elucidation of these mechanisms may provide a therapeutic Benjamin Gelman1,JoshuaLisinicchia1,PatelVipulkumar1, target to improve disparities in HAND outcomes. Lalita Singh1,JaniceEndsley1,DennisKolson2 (corresponding author: [email protected])

P43 1Department of Pathology, University of Texas Medical Development of an inducible cell line screening Branch; 2Department of Neurology, University of for therapeutic antagonists to HIV Tat protein Pennsylvania

Nicholas Geiger1, Wenxue Li1,RichaTyagi1, Muzna The effectiveness of therapies to eradicate latent HIV DNA Bachani2, Nasir Malik2, Joseph Steiner2, Avindra Nath3 must be assessed primarily by sampling peripheral blood (corresponding author: [email protected]) mononuclear cells (PBMCs). The relationship between changes in the size of the latent pool of HIV DNA in 1Section of Infections of the Nervous System, NINDS/NIH, PBMCs versus that of the deep body compartments such Bethesda, MD; 2Translational Neuroscience Center, NINDS/ as the central nervous system (CNS) is unknown. A whole NIH, Bethesda, MD, USA; 3SINS and Translational body map of HIV DNA concentration was produced in 16 Neuroscience Center, NINDS/NIH, Bethesda, MD, USA HIV infected people; half of the decedents were virally suppressed. 17 tissue types were assayed for HIV RNA and The Tat protein of HIV is a small, highly basic, regulatory DNA using PCR and the two-step alu-gag assay for integrat- protein that is the first protein to be produced during viral ed HIV DNA. Correction for latent HIV DNA within blood replication. Tat plays a critical role in driving viral replica- pools transiting through the tissues was estimated with he- tion by transactivation of the Long Terminal Repeat (LTR) moglobin assays. The largest HIV DNA pool size in the promoter region of the virus, and has long been implicated body was in the gastrointestinal (GI) tract which contained in the pathophysiology of HIV-Associated Neurocognitive 52 % of the total. Latent HIV in PBMCs had substantial Disorders (HAND). Although Tat has been extensively predictive power pertaining to latent pool sizes in many deep studied, it remains an elusive target. We have established body compartments including the gut, but notably not in S22 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89

CNS. The CNS pool of HIV DNA contained less than 1 % sIR secreted from cells exposed to the HIV-1 Tat or TNF- of the whole body total and was more concentrated in white alpha when compared to untreated. Incubation of cells with matter, which contained 70 % of the CNS pool. Gray matter a combination of HIV-1 Tat and TNF-alpha also increased contained 30 % of the pool and was about half as concen- significantly (p < 0.0001) sIR secretion, however this combi- trated. In the liver active hepatitis was significantly asso- nation did not enhanced the effects observed with either pro- ciated with a high latent pool size. In the spleen high tein individually. R5070 was able to partially reverse the in- latent pool size was associated with low expression of creased sIR secretion induced by either HIV-1 Tat or TNF- candidate biomarker mRNAs including CXCL16, alpha, however the drug completely abolished the receptor CXCL13 and CCL1. Whole body mapping shows that secretion induced by the combination of both proteins. the latent pool of HIV DNA in PBMCs varies proportion- Conclusion: Our data suggests that TNF-alpha and ately with latent pools in many deep body compartments, HIV-1 Tat regulate the secretion of sIR from neuronal but not with the relatively small pool sequestered within cells and that the effects of Tat on sIR is dependent on the CNS. The size of the HIV DNA pool in PBMCs also TNF-alpha receptor activation. Further studies will inves- was not significantly related to the degree of viral sup- tigate if the effects of Tat on sIR secretion are mediated pression in patients with less than 100,000 copies/ml by neuronal TNF-alpha being released to the extracellu- blood plasma. lar medium. Supported by R01NS099036, R21MH095524, U54MD007587, G12RR003051, G12MD007600. P45 HIV-1 Tat Induces Neuronal Insulin Receptor Secretion Through the TNF-Alpha Receptor P46 Microglial gene expression is altered in HIV infection, Yamil Gerena1, Raissa Mení©ndez-Delmestre2, Andrea even in the absence of detectable virus in brain Delgado-Nieves1, Anibal Gonzíçlez-Escalante1,Luis Colí_n-Cruz1, Valerie Marshall-Freites1,PaolaRivera- Stephen D. Ginsberg1, Melissa J. Alldred1,SatyaM. Morales1, Avindra Nath3, Valerie Wojna4 Gunnam2, Tracy Fischer2 (corresponding author: [email protected]) (corresponding author: [email protected])

1Department of Pharmacology, University of Puerto Rico- 1Center for Dementia Research, Nathan Kline Institute, Medical Sciences Campus; 2NeuroAIDS Research Program, Orangeburg, NY; Departments of Psychiatry and Physiol. & University of Puerto Rico-Medical Sciences Campus; 3NIH, Neurosci., New York University Langone Medical Center, National Institute of Neurological Disorders and Stroke; New York, NY; 2Temple University School of Medicine, 4Department of Internal Medicine, Neurology Division, Department of Neuroscience, Philadelphia, PA University of Puerto Rico-Medical Sciences Campus We have previously reported substantial accumulation of Background: Previously we found that high levels of soluble CD163+/CD16+ macrophages and microglia in brains of pa- insulin receptor (sIR) in the cerebrospinal fluid (CSF) of HIV- tients with HIV encephalitis (HIVE), a neuropathological cor- seropositive women were associated with presence and sever- relate of the most severe form of HIV-associated ity of cognitive impairment. However, the possibility that neurocognitive disorders (HAND), HIV-associated dementia components present in the CSF of these patients may influ- (HIV-D). More recently, we found neurocognitively impaired ence sIR secretion from neuronal cells remains totally unclear. HIV+ individuals without encephalitis (HIV/noE) or detect- In this study we investigated if HIV-1 Tat protein and TNF- able virus production in the brain also have increased CD163+ alpha influence the secretion of sIR from human neuronal and CD16+ brain macrophage’s and microglia. Accordingly, cells and then analyzed the effects of R5070, a TNF-alpha we hypothesize that microglial activation is a common mech- receptor blocker, on HIV-1 Tat or TNF-induced neuronal sIR anism between lesser and more severe HIV-associated neuro- secretion. Methods: Human neuronal cells (SH-SY5Y; degenerative processes, regardless of virus production in the 5 × 106 cells) were exposed to HIV-1 Tat (100nM), TNF- brain. To begin to explore this premise, we investigated gene alpha (5 pg/ml), or a combination of both proteins for 24 h. expression changes of select classes of transcripts in paren- Culture medium was collected and sIR levels were quantified chymal macrophage’s/microglia from archival brain tissue of by ELISA. The secretion of sIR was also measured in medium patients with HIVE, HIV/noE, and age-matched seronegative of cells exposed to either TNF-alpha, HIV-1 Tat, or a combi- (HIV-) controls. Microarray analyses were performed on laser nation of both in the presence of R7050 (10-8 M) for 24 h. capture microdissected parenchymal CD163+, CD16+ or Results: A significant increase (p < 0.0001) was observed in CD68+ macrophage’s/microglia, using terminal continuation J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S23

(TC) RNA amplification and a custom-designed array plat- engraftment of human CD34+ hematopoietic stem cells. form. Here, we report pronounced differences in gene expres- Flow cytometry and histological analyses revealed recon- sion between HIV- and HIV infection with and without en- stitution of mice with human immune cells, including cephalitis. Altered expression of several classes of transcripts, macrophages, dendritic cells, T cells and B cells. including genes associated with cell death, inflammatory Proviral load (PVL) was determined in the peripheral markers, and protein kinases, are observed in the context of blood, spleen, and other organs of Rag1 and BLT mice HIV infection. Several transcripts suggesting macrophage’s/ by droplet digital PCR. Within blood, PVL and viral pro- microglia activation are up regulated in cells recovered from tein Tax was detected as early as 2 weeks post-infection HIV/noE brain; however, many of these are decreased in (wpi) with continued expression until 16 wpi. Both PVL HIVE. This may suggest impaired function of macro- and Tax expression was considerably higher in the adult phage’s/microglia, resulting from prolonged neuroinflamma- Rag1 mice as compared to the neonates with the latter tion. These data indicate the utility of profiling macrophage’s/ showing less than 20 % PVL in the peripheral blood, microglia in HIV infection to identify potential factors for brain, and liver. Thus far, several members of the CD28:B7 targeted treatment modalities. In addition, our work provides family of immunoglobulin superfamily co-signaling mole- important insights into the diverse roles microglia play in cules that include PD-1 and its ligand PDL-1 have been asso- maintaining brain homeostasis, as well as alterations in ciated with T-cell dysfunctions in HTLV-infected patients. We microglial function that likely contribute to neuronal injury thus assessed and found over expression of PD-1, TIM-3, and cognitive impairment. TIGIT and 2B4 on CD4 and CD8 T cells in HTLV-1 infected samples from both hu-mice and human subjects. Moreover, lymphocytic infiltration with concomitant Tax expression and P47 resulting myelin disruption was observed in the spinal cord HTLV-1 infection and neuropathogenesis in the context and brain of the infected mice. This data represents the first of Rag1−/−γc−/− (RAG1) mice attempt to establish HTLV-1 neuropathogenesis in the context of RAG1 and BLT mice. Rashida Ginwala1,PaigeCharlins2, Ramesh Akkina2, Breanna Caruso3, Ronak Loonawat1, Steven Jacobson3, Sreesha Nambiar1, Glen M. Chew4, Lishomwa C. Ndhlovu4, P48 Pooja Jain1, Zafar K. Khan1 Nutraceutical Apigenin regulates DC function (corresponding author: [email protected]) in a RelB-dependent manner during neuroinflammation

1Department of Microbiology and Immunology, and the Rashida Ginwala1, Emily McTish1, Nikil Revuri1, Chander Institute for Molecular Medicine and Infectious Disease, Raman2, Narendra Singh3, Mitzi Nagarkatti3,Prakash Drexel University College of Medicine, Philadelphia, PA; Nagarkatti3,DivyaSagar1, Pooja Jain1, Zafar K. Khan1 2Department of Microbiology, Immunology and Pathology, (corresponding author: [email protected]) Colorado State University, Fort Collins, CO;3Viral Immunology Section, Neuroimmunology Branch, National 1Department of Microbiology and Immunology, and the Institutes of Health, Bethesda, MD; 4Department of Tropical Institute for Molecular Medicine and Infectious Disease, Medicine, Medical Microbiology and Pharmacology, John A. Drexel University College of Medicine, Philadelphia, PA; Burns School of Medicine, University of Hawaii, Honolulu, 2Division of Clinical Immunology and Rheumatology HI University of Alabama School of Medicine, Birmingham, AB; 3Department of Pathology, Microbiology and HTLV-1-associated myelopathy/tropical spastic Immunology University of South Carolina, Columbia, SC paraparesis (HAM/TSP) is a disabling chronic inflammatory disease of the central nervous system (CNS) with Apigenin, a natural flavonoid, found in several plants is similarities to multiple sclerosis. To date, the lack of a known to have anti-oxidant and anti-inflammatory properties suitable small animal model has hindered our quest to indicated by its use for centuries as a medicinal approach to understand the immuno- and neuropathogenesis of treat inflammatory disorders. However, there is a considerable HTLV-1 in an in vivo system. Additionally, the host im- dearth of information regarding its effect on immune cells, mune response that plays a critical role in the outcome of especially dendritic cells (DC) that maintain the critical bal- HTLV-1 infection could be better tested in the context of ance between an immunogenic and tolerogenic immune re- humanized (hu) mice. Thus, we employ here neonatal and sponse, in an immunospecialized location like the central ner- adult Balb/c-Rag1−/−gammac−/− or Rag1 as well as Bone vous system (CNS). Thus we looked at the anti-inflammatory marrow-Liver-Thymic (BLT) mouse models for properties of Apigenin in restoration of immune cell function, S24 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 especially DCs. In vitro upon LPS stimulation, Apigenin the half-life of ARVs and enhance drug delivery to viral res- inhibited IL-6 and TNF-alpha secretion and the cell surface ervoirs. This is achieved by co-administration of long-acting expression of alpha4 integrin, CD86, CLEC12A and MHC II nanoformulated ARVs (nanoARV) with URMC-099, a mixed molecules on murine DCs. Further, Apigenin treatment ame- lineage kinase-3 inhibitor. URMC-099 was shown to be neu- liorated severity of disease progression and relapse after onset roprotective and anti-inflammatory in a rodent model of HIV- of experimental autoimmune encephalomyelitis (EAE) in associated neurocognitive disorders (HAND). We now show C57BL/6 and SJL mouse models of multiple sclerosis immu- that URMC-099 activates autophagy through nuclear nized with MOG35-55 and PLP139-151 respectively. translocation of transcription factor EB (TFEB) resulting Apigenin treated EAE mice showed decreased expression of in improved antiretroviral activities and extended drug de- alpha4 integrin and CLEC12A on splenic DCs and an in- pots in human monocyte derived macrophages (MDMs). creased retention of DCs and macrophages in the periphery Induction of autophagy also leads to retention of compared to untreated EAE mice. This correlated with de- nanoARV, containing the protease inhibitor atazanavir, in creased immune cell infiltration and reduced demyelination the autophagosomes of MDMs. This occurs in parallel with in the CNS. Mechanistically, we observed Apigenin treat- an up to 4-fold improvement in mitochondrial activities ment reduces cytoplasmic RelB expression in presence of and cell vitality compared to nanoARV alone. In rodents, LPS in human peripheral blood DCs, which is central to URMC-099 induced transcriptional activation of autopha- DC maturation, its antigen presentation capabilities and gy led to a 50-fold increase in the half-life of the viral DC-mediated T cell activation. Concomitantly, IFN- integrase inhibitor dolutegravir. Activation of autophagy gamma a downstream target of RelB was also reduced lead to decrease in HIV-1 induced IL-1beta secretion in upon Apigenin treatment in these cells. RelB also plays infected MDMs and humanized mice. Such improved a role in mitochondrial bioenergetics during inflammation, anti-inflammatory and antiretroviral responses paralleled which explains the metabolic shift away from glycolysis what was shown previously in HIV-1 infected humanized that we observed upon Apigenin treatment in the inflamed mice. We conclude that pharmacologic induction of au- DCs. These results indicate a protective role of Apigenin tophagy provides a novel means to further extend the ac- against neurodegenerative effects resulting from the entry tion of long acting nanoformulated antiretroviral therapy. of DC stimulated pathogenic T cells into the CNS thus implicating a potential therapy for neuroinflammatory disease. P50 Cognitive Change Trajectories in Virally Suppressed HIV-Infected Individuals Suggest High Prevalence P49 of Ongoing Disease Activity Autophagy Facilitates Nanoformulated antiretroviral Drug Depots for Sustained Release and Enhanced Chloe Gott1,ThomasGates2, Nadene Dermody3,Bruce Anti-Retroviral Activity Brew4, Lucette Cysique5 (corresponding author: [email protected]) Divya Prakash Gnanadhas1, Prasanta Dash1,BradySillman1, Zhiyi Lin1,AdityaBade1, Harris Gelbard2, JoEllyn 1Psychology Department, Macquarie University, Sydney, McMillan1, Benson Edagwa1, Howard Gendelman1, Santhi NSW, Australia; 2Centre for Applied Medical Research, St Gorantla1 Vincent’s Hospital Sydney, NSW, Australia; 3Neuroscience (corresponding author: [email protected]) Research Australia (NeuRA), Sydney, NSW, Australia; 4Centre for Applied Medical Research, St Vincent’sHospital 1Department of Pharmacology and Experimental & UNSW Australia, Sydney, NSW, Australia; 5Neuroscience Neuroscience, College of Medicine, University of Nebraska Research Australia (NeuRA), & UNSW Australia, Sydney, Medical Center, Omaha, Nebraska; 2Center for Neural NSW, Australia Development and Disease, University of Rochester Medical Center, Rochester, NY Background: In persons with stable, treated, and virally- suppressed HIV infection, cognitive decline rate and profile Long-acting anti-HIV products can substantively change the is not established. As such this study aims to quantify the rate standard of care for HIV/AIDS. To this end, antiretroviral of cognitive decline and define clinically meaningful cogni- drugs (ARVs) that can be administered once a month or longer tive trajectories in a cohort of virally-suppressed HIV+ per- and the ability to transform short acting hydrophilic drugs to sons. Methods: Ninety-six HIV+ (97 % virally undetectable; extended release prodrugs are positive steps forward towards median current cART duration: 24 months; median HIV du- realizing this goal. We now report yet another means to extend ration: 19 years, mean age: 56 years) and 44 demographically J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S25 comparable HIV- participants underwent standard neuropsy- has previously been implicated in HAND via animal and chological testing assessing 7 cognitive domains at baseline in vitro studies, and in a large gene expression study in and 18-months follow-up. We determined clinically relevant humans. Therefore, the KEAP1/nrf2 pathway is a candidate cognitive trajectories based on a history of HAND, baseline target for HAND treatment/prevention. Here we assess the HAND status and cognitive decline using norms for change safety and efficacy of Bardoxolone, a compound that in- corrected for practice effect. The definition of cognitive de- duces nrf2 nuclear translocation, stimulating expression of cline was based on a continuous global change score (GCS) several protective genes. Methods: Several experiments were and dichotomous definition of clinically meaningful cognitive conducted in two independent labs: 1) To assess neuropro- decline (decline/stable; 90 % confidence interval, 1-tailed tective effects, monocyte-derived macrophages (MDM) were around normative GCS). Results: Relative to HIV- controls pre-treated for 24-h prior to infection with Bardoxolone, (4.5 %), 14 % of HIV+ participants declined (p =.11);more- Tempol (0.5 or 5microM), or vehicle and the following mea- over the HIV+ group scored significantly lower on the GCS sures were evaluated several times over 15 days: HIV re- (p = .03) and showed greater decline in processing speed verse transcriptase (RT) activity in supernatants, heme- (p = .02) and mental flexibility/inhibition (p = .02). Baseline oxygenase-1 (HO-1) expression in lysates, and microtubule HAND status significantly predicted cognitive decline at associated protein-2 (MAP2) expression in primary rodent follow up (p = .005). We determined 7 clinically relevant neurons exposed to culture supernatants 2) HIV-infected U1 cognitive trajectories which in order of prevalence were: 1) monocytes were stimulated using LPS (1microg/ml) to de- Always neurocognitively-normal (39 %), 2) Stable base- termine if pretreatment with Bardoxolone (5 or 50nM) line impairment (35 %), 3) Sustained long-term impair- inhibited viral replication. Control cells were compared to ment since historical HAND, (9 %) 4) Baseline impairment cells pre-treated with either Tempol or Bardoxolone over- and decline (7 %), 5) Historical HAND fully recovered night. Drug efficacy was assessed by measuring levels of (3 %), 6) Incident decline at 18 months (3 %) 7) Always p24 gag expression. Results: 1) Bardoloxone pre-treatment progressing (3 %). There was no relationship between reduced HIV replication in MDM, demonstrated by reduced these trajectories, and traditional HIV disease biomarkers HIV RT activity in the culture supernatants. Bardoxolone (nadir, current, standard difference baseline and follow-up enhanced expression of HO-1 in HIV-infected and non- CD4+ T cell counts; study period undetectable/detectable; infected MDM. Bardoxolone treatment of HIV-infected baseline HIV duration, and baseline cART duration). MDM dramatically reduced the neurotoxicity of cell culture Conclusions: When determining clinically relevant trajec- supernatants, demonstrated by maintenance of MAP2 levels tories, we found that more than half of virally-suppressed in the neuron cultures as compared to vehicle and Tempol. HIV+ individuals (57 %) have ongoing disease activity 2) Bardoxolone pre-treatment decreased the amount of p24 that is not related to traditional HIV biomarkers. gag expression following stimulation by LPS. Conclusion: This series of preclinical studies lends support for the safety and efficacy of Bardoxolone in the prevention and/or treat- P51 ment of HAND. Preclinical studies of Bardoxolone for the prevention and treatment of HIV-associated neurocognitive disorders

Analise Gruenewald1, Irene Kim2,DennisKolson1,Dimitrios P52 Vatakis2,AndrewLevine3 Macrophage-tropic Simian Immunodeficiency Virus (corresponding author: [email protected]) Molecular Clone causes NeuroAIDS in Rhesus Macaques

1Department of Neuroscience, University of Pennsylvania; Sanjeev Gumber1, Shawna Woollard2,PraveenKumar 2Department of Medicine, Division of Hematology- Amancha3,Po-JenYen4, Dana Gabuzda4,Francois Oncology, UCLA AIDS Institute, David Geffen School of Villinger3, Siddappa Byrareddy2 Medicine at the University of California Los Angeles; (corresponding author: [email protected]) 3Department of Neurology, David Geffen School of Medicine at the University of California Los Angeles 1Division of Pathology, Yerkes National Primate Research Center, Emory University, Atlanta, GA 30329; 2Department Background: HIV-associated neurocognitive disorders of Pharmacology and Experimental Neuroscience, University (HAND) are the clinical manifestation of inflammation and of Nebraska Medical Center, Omaha, NE 68198; 3New Iberia oxidative stress in the CNS. Nrf-2 is a transcription factor Research Center, University of Louisiana at Lafayette, New that promotes expression of a wide variety of antioxidant Iberia, LA 70560; 4Department of Cancer Immunology and and anti-inflammatory factors. The KEAP1/nrf-2 pathway Virology, Dana-Farber Cancer Institute, Boston, MA, USA S26 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89

Macrophages are a major target of HIV/SIV and are relatively nervous system (CNS) lead to neuronal dysfunction. resistant to the cytopathic effects of infection. These cells play However, how HIV infection leads to accelerated trafficking an important role in disease pathogenesis and serve as viral of macrophages into the CNS is largely unknown. Previously, reservoirs in the central nervous system (CNS). Previously, a we demonstrated that during infection, HIV triggers chemo- unique early SIVmac251 Env variant, deSIV147, was isolated tactic signaling through gp120 binding to CXCR4, which ac- from blood of a rhesus macaque with rapid disease progression tivates cofilin and Arp2/3, two of the major regulators of actin and SIV-associated encephalitis (SIV-E). A replication- dynamics. Given that cell migration are driven by actin dy- competent SIV251 molecular clone encoding deSIV147 Env namics, we investigated whether R5 virus binding to macro- was shown to be highly fusogenic, replicate efficiently in phages may alter their chemotactic responses and mobility. rhesus macaque PBMC and macrophages, and induce multi- Here, we report the modulation of both the cofilin and nucleated giant cell formation during macrophage infection Arp2/3 signaling pathways in macrophages by R5 viruses. in vitro. Furthermore, deSIV147 Env shared close sequence In addition, we also probed the magnitude of R5 gp120- homology to sequences in the brains of infected animals with mediated signal transduction in primary macrophages, neurological disease. Here, we show that molecularly cloned using a systematic pathway mapping technology, the re- SIV251 virus expressing deSIV147 Env is neurotropic verse phage phospho-protein micro-array (RPPA), we in- in vivo. The deSIV147 virus infected rhesus macaques fol- terrogated over 200 phospho-proteins for their involve- lowing intravenous or intrarectal exposure, causing sys- ment in R5 gp120 signaling. We identified four major temic infection with plasma viral loads ranging from pathways, the actin signaling network (cofilin, LIMK1, 10^5 to 10^7 copies/ml. Furthermore, immunohistological PTEN, VASP, FAK, Pyk2), the PI3K-Akt signaling net- evidence of macrophage infection was present in brain, work (Akt), the MAPK signaling network (c-Raf, lymph nodes, spleen, colon, lung, and liver, and CD4+ T ERK1/2), and the Jak/Stat signaling network (Jak1, cells in gut were depleted during acute infection in 3/3 Stat3), that are selectively activated. We future demon- animals tested. Next, deSIV147 virus was inoculated into strated that targeting some of these signaling networks macaques depleted of CD4+ T cells, creating selection effectively block R5 viral infection of primary macro- pressure for the virus to infect macrophages in the CNS. phages, Our studies suggest that novel therapeutics can All animals were infected, with plasma and CSF viral loads be developed based on targeting R5 HIV-mediated signal- of 10^5 to 10^8 and 10^3 to 10^7 copies/ml, respectively, ing pathways. These novel approaches may prevent the and evidence of SIVp27-positive macrophages in brain. establishment of viral reservoirs in the CNS. Furthermore, accumulation of perivascular macrophages, formation of multinucleated giant cells, microgliosis, and multifocal neuronal injury in gray and white matter of the P54 brain was detected in 1 of 3 animals tested. These findings Nef, an auxiliary protein produced by HIV-1 inhibits suggest the neurotropic deSIV147 clone will be useful to autophagy in neonatal cardiac myocytes study the role of macrophages in HIV/SIV-associated neurocognitive disorders, gain new insights into the CNS Manish Gupta1, Rafal Kaminski1, Brian Mullen1, Jennifer as a viral reservoir, and test strategies for eradication Gordon1, Joseph Cheung2, Arthur Feldman2, Muniswamy Madesh3, Kamel Khalili3 (corresponding author: [email protected]) P53 System mapping of CCR5 signaling in HIV 1Department of Neuroscience, Center for Neurovirology, neuropathogenesis and viral infection of primary Lewis Katz School of Medicine at Temple University; macrophages 2Department of Medicine, Lewis Katz School of Medicine at Temple University; 3Center for Translational Medicine, Jig Guo, Yuntao Wu Department of Medical Genetics and Molecular (corresponding author: [email protected]) Biochemistry, Lewis Katz School of Medicine at Temple University NCBID, George Mason University Introduction: Patients with HIV-1 infection develop heart fail- The CCR5-utilizing viruses are the predominant species dur- ure with reduced ejection fraction (HFrEF); however, the ing the course of HIV infection. The R5 viruses infect causative mechanism is unclear because cardiac dysfunction monocytes/macrophages that are considered as major media- often occurs in HIV infected individuals in the absence of tors of HIV-mediated neuropathogenesis; the activation and myocarditis. Hypothesis: We tested the hypothesis that the trafficking of infected/uninfected macrophages into the central expression of the HIV-1 auxiliary protein Nef inhibits J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S27 autophagy leading to diminished protein quality control and with altered fMRI response in the ACC (Jiang et al., enhanced cell death in cardiac myocytes. Methods and result: CROI 2016), providing further evidence suggesting cin- Using primary cardiomyocytes isolated from neonatal rat ven- gulate might be affected in HIV. Here using a modified tricle we tested the role of Nef protein in cardiomyocyte au- voxel-based morphometry (VBM) technique that is sensi- tophagy and cell survival. Our data show that Nef protein tive to local changes in gray matter (GM) volume, we causes inhibition of cardiomyocyte autophagy at the terminal examined the impact of aging and HIV on the volume of steps and induces accumulation of p62 and ubiquitin positive cingulate cortex. Forty-two adults (43–63 years old, 27 aggregates at the perinuclear space. Mechanistically, we found HIV+) without major psychiatric disorders and other con- that Nef protein interacts with the autophagy maturation fac- founding health problems participated in this study. All tors, Belin1 and Rab7 and forms aggregate at the perinuclear HIV+ subjects were on antiretroviral therapy, and did space. In addition, Nef dysregulated mitochondrial turnover as not have a clinical diagnosis of cognitive impairments. evidenced by accumulation of Tom20, increased generation of At a whole brain level, there is no difference between reactive oxygen species and decreased mitochondrial mem- the HIV-infected and HIV-uninfected groups. However, brane potential. Interestingly, pharmacological drug the volume of cingulate cortex was reduced in the HIV+ rapamycin-mediated induction of autophagy can neutralizes group than the age-matched uninfected controls (after the Nef induced cardiomyocyte’s abnormalities. Conclusion: controlling for age). In addition, a negative correlation Nef reversibly inhibits cardiomyocyte autophagy through in- between cingulate volume and age was observed. hibition of autophagy maturation factors and may therefore Furthermore, a strong correlation between the reduced serve as a therapeutic target in patients with HIV-associated functional connectivity and the cingulate volume was ob- HFrEF. served. These results provide further evidence suggesting that the cingulate cortex might be one of brain regions affected by HIV early on, and the additive impairments P55 due to aging and HIV might underlie the increased risk Reduced cingulate structural and functional integrity of cognitive impairments in HIV+ older adults. in HIV and aging

Shiva Hassanzadeh1, Manya Magnus2, Matthew Dawson3, P56 Allison Budzinski4, Rebecca Barasky2,CuiweiWang5, Informatic Interrogation of CSF Proteomic Profiles Princy Kumar5, Mary Young5, David Moore3, Ronald Ellis4, from HIV-Infected Subjects Implicates Acute Phase Xiong Jiang1 and Complement Systems in Shifting Cognitive Status (corresponding author: [email protected]) Norman Haughey1, Ceereena Ubaida-Mohien1,Benjamin 1Department of Neuroscience, Georgetown University Lamberty2, Alex Dickens1, Michelle Mielke3,Thomas Medical Center; 2Department of Epidemiology and Marcotte4, Scott Letendre4, Donald Franklin4, Ned Sacktor1, Biostatistics, George Washington University; 3Department of Igor Grant4, Pawal Cibrowski2, Ravi Tharakan1,Justin Psychiatry, University of California, San Diego; 4Department McArthur1,HowardFox2 of Neuroscience, University of California, San Diego; (corresponding author: [email protected]) 5Department of Medicine, Georgetown University Medical Center; 1Johns Hopkins; 2University of Nebraska; 3Mayo Clinic; Cingulate cortex is one of less understood brain regions, 4UCSD but has been implied in many cognitive functions, including executive function. A recent study with a task- The prevalence of HIV-Associated Neurocognitive Disorders switching paradigm revealed that HIV+ older adults adapt (HAND) has not changed considerably in the last two de- less quickly to changing task demands than HIV- cades. Potent antiretroviral therapy (ART) has shifted the se- uninfected controls, and the behavioral impairments cor- verity of HAND to milder phenotypes, but excess morbidity related with reduced brain activation in the anterior cin- and mortality continue to be associated with HAND. Changes gulate cortex (ACC) (Jiang et al., AIDS Care 2016), sug- in numerous markers of immune function, inflammation and gesting ACC dysfunction might underlie reduced execu- cellular stress have been repeatedly associated with HAND, tive function in HIV. In addition, using resting-state func- but the underlying systems that drive these changes have not tional connectivity MRI (rs-fcMRI) technique, we have been identified. In this study we conducted an untargeted pro- foundthatbothHIVandagingledtoanisolatedcingu- teomic analysis of CSF samples from the Central Nervous late, and the reduced functional connectivity correlated System HIV Anti-Retroviral Therapy Effects Research S28 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89

(CHARTER) and the HIV Neurobehavioral Research Center release as shown by quantification of HIV reverse transcrip- (HNRC) studies. Subject selection was based on a case review tase (RT) in culture supernatant. Surprisingly, overexpression of ~3,500 clinical visits from 430 study participants. Subjects of TDP-43 in astrocytes strongly inhibited viral transcription, had CSF available from two consecutive study visits with suggesting that the role of TDP-43 on HIV replication is cell- complete clinical and neuropsychological data available and type specific and may be dependent on TDP-43 post-transla- were on stable ART for at least 3 months before the first visit tional modifications or availability of cellular co-factors. and for the duration of the observation period. Participants Furthermore, TDP-43-mediated enhancement of HIV did were demographically matched, and were similar in duration not require previously identified binding sites on the HIV of HIV infection, HCV serostatus, nadir CD4+ T-cell count, LTR, as mutation of these regions had no effect on HIV and plasma and CSF HIV RNA. Based on changes in replication. Similarly, deletion of NFkB elements from neurocognitive (NC) performance, participants were cate- HIV LTR had no impact on the TDP-43 effect, indicating gorized as: Stably normal (n = 25), Improving (n =20), that TDP-43 association with p65 was not involved. Worsening (n =22)andStablyImpaired(n =22).Toen- In vitro binding assays and RNA immunoprecipitation sure the stability of these categories of NC performance, (RIP) revealed that TDP-43 bound to multiple HIV tran- NC data from a third visit was used to validate the trajec- scripts and identified UG-rich sequences in the 5’ untrans- tory of change in cognitive status. An untargeted proteo- lated region (UTR) of HIV mRNA as the putative binding mic analysis was conducted using CSF from the two in- region. These findings strongly suggest that TDP-43 plays dex visits. Systems informatics was used to interrogate the a regulatory role in HIV infection, its effects are cell type proteomic content of CSF. The patterns of change in CSF specific and that it may exert this effect via altered pro- protein content implicated the induction of acute phase cessing or stability of HIV transcripts. and complement systems as important regulators of NC status. Worsening NC performance was preceded by induction of acute phase and complement systems, while P58 improving NC performance was preceded by downregu- Detection of HIV-1 Tat protein and TAR RNA lation of these systems. in cerebrospinal fluid from patients on antiretroviral therapy: targeting persistent Tat production using antisense oligonucleotides P57 Modulation of HIV replication in lymphocytes, Lisa Henderson1, Tory Johnson2, Robert Barclay3,Richa macrophages and astrocytes by association of HIV mRNA Tyagi1, Muzna Bachani4, Ned Sacktor2, Scott Letendre5, with TAR DNA binding protein 43 Joseph Steiner4, Fatah Kashanchi6, Avindra Nath1 (corresponding author: [email protected]) Lisa Henderson, Wenxue Li, Richa Tyagi, Avindra Nath (corresponding author: [email protected]) 1Section of Infections of the Nervous System, National Institute of Neurological Disorders and Stroke; 2Department Section of Infections of the Nervous System, National of Neurology, Johns Hopkins University School of Medicine; Institute of Neurological Disorders and Stroke 3National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University; Human immunodeficiency virus (HIV) colonizes the brain 4Translational Neuroscience Center, National Institute of early in infection, where it infects macrophages, microglia Neurological Disorders and Stroke; 5University of California and astrocytes. These cells are long-lived and resistant to cy- San Diego; 6Laboratory of Molecular Virology, School of topathic effects of HIVand can thus harbor virus for extended Systems Biology, George Mason University periods of time. It is vital to gain a greater understanding of the mechanism(s) that regulate HIV expression in these cells. Background: Antiretroviral therapy (ART) has greatly dimin- TAR DNA binding protein 43 (TDP-43) plays critical roles ished the mortality associated with HIV-1 infection and limit- in mRNA splicing and stability. TDP-43 can also bind directly ed its transmission. However, HIV remains a life-long infec- to target promoters, including the HIV long terminal repeat tion with associated health concerns including persistent in- (LTR), to modulate transcription. TDP-43 also binds to NFkB flammation and neurocognitive impairment. HIV-1 Tat is a subunit p65, to function as co-activator for NFkB genes and neurotoxic and pro-inflammatory viral protein not targeted regulates splicing of interleukin-6 and interleukin-10 mRNA. by current therapies. Methods: Tat levels in cerebrospinal fluid We found that overexpression of TDP-43 enhanced HIV tran- (CSF) from patients on ART (n = 57) or collected from pa- script levels in monocyte-derived macrophages (MDM) and tients pre- and post-ART initiation (n =5)wereevaluatedby lymphocytes as measured by qRT-PCR and enhanced viral ELISA. Tat and viral RNA were also measured in exosomes J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S29 isolated from patient CSF by ultracentrifugation using western only been detected in cerebrospinal fluid (CSF) from one blot and qRT-PCR, respectively. We designed DNA antisense AFM patient to date. The lack of consistent CSF viral isolation oligonucleotides (ASO) complementary to exon 1 of tat or nucleic acid amplification, and the fact that EV-D68 strains mRNA or an intronic sequence not present in the mature tat were not generally considered neurotropic, made the causal transcript but retained in env. ASO efficacy was evaluated by role of EV-D68 in AFM uncertain. Here we show that a co-transfection of HeLa or primary human astrocytes with representative EV-D68 strain (US/MO/18947) isolated HIV and ASO followed by Tat ELISA or product enhanced from a respiratory sample during the 2014 outbreak in- reverse transcriptase (PERT) assay to measure viral release. duces paralytic disease in neonatal mice following intrace- Results: Tat protein was detected in 42 % (24/57) of pa- rebral, intramuscular, intraperitoneal, and intranasal inocu- tient CSF samples. CSF Tat levels increased in 4 out of 5 lation that shares key features with the human disease. In individuals after initiation of therapy, indicating that Tat contrast, the 1962 prototype strain, Fermon, does not pro- was not diminished by ART. Similarly, exosomes isolated duce disease in mice. In-depth investigation of disease from CSF contained Tat protein (3/10) and/or TAR RNA pathogenesis of US/MO/14-18947 revealed infectious vi- (4/10), with 2 out of 10 samples positive for both Tat and rus, viral RNA corresponding to the full genome, and viral TAR. ASO directed against the first exon of Tat effective- particles in spinal cords of paralyzed mice. Paralysis was ly reduced Tat protein levels but did not inhibit productive associated with infection and subsequent loss of motor viral replication in astrocytes. In contrast, ASO that neurons in the corresponding spinal cord anterior horn as targeted the intronic sequence repressed Tat translation assessed by immunohistochemistry. On-going investiga- and reduced viral release. Conclusions: These findings tion of other 2014 EV-D68 strains has shown that many, confirm that both Tat and viral RNA continue to be pro- but not all, genetically distinct isolates have the ability to duced in individuals otherwise controlled on ART, and induce paralytic disease in mice. These studies establish highlight a need for new therapies that target Tat. Tat- the neuronotropic and neurovirulent potential of EV-D68 directed antisense are effective in reducing Tat protein as and provide a novel experimental model to further study well as inhibiting total viral replication and provide new EV-D68-induced disease. In addition, preliminary investi- avenues for therapeutic intervention. gations of disease modifying treatments, such as passive antibody transfer, show promise that this model can be used to develop EV-D68 specific therapies. P59 A mouse model of enterovirus D68-induced paralysis P60 Alison M. Hixon1,GuixiaYu2, J. Smith Leser3, Shigeo Yagi 4, Role of endolysosome de-acidification in antiretroviral Penny Clarke3, Charles Y. Chiu2,KennethL.Tyler5 drug-induced amyloidogenesis (corresponding author: [email protected]) Liang Hui, Leo Lakpa, Jonathan Geiger, Xuesong Chen 1Medical Scientist Training Program, Neuroscience Program, (corresponding author: [email protected]) University of Colorado; 2Department of Laboratory Medicine and Medicine, Division of Infectious Diseases, Abbott Viral School of Medicine & Health Science Department of Diagnostics and Discovery Center, University of California, Biomedical Sciences, University of North Dakota San Fransicso; 3Department of Neurology, University of Colorado; 4California Department of Public Health; 5Denver Combined antiretroviral therapeutic (ART) strategies have ef- VA Medical Center; Department of Neurology, Department of fectively increased the long-term survival of HIV-1 infected Microbiology and Immunology, Department of Medicine, individuals. However, along with increased longevity comes University of Colorado age-related neurological disorders including a very high prevalence of HIV-1 associated neurocognitive disorders (HAND) Enterovirus D68 (EV-D68) is a unique enterovirus that shares including clinical manifestations and pathological features of characteristics with rhinoviruses, including respiratory trans- Alzheimer’s disease (AD). However, the pathogenesis of mission. Although previously rare, the prevalence of EV-D68 HAND remains unclear, and little is known about how AD- respiratory disease has increased in the last decade. In 2014, like pathology is developed as a result of HIV-1 infection and/ the US saw an unusual increase in acute flaccid myelitis or long-term use of ART drugs. We postulate that neuronal (AFM) cases in children coincident with a nationwide out- endolysosome de-acidification leads to the co-pathogenesis of break of EV-D68 respiratory disease, and up to 50 % of HAND and AD. Here, we used rat primary cultured neurons AFM patients with available samples had EV-D68 RNA de- to test the effects of 14 separate ART drugs on endolysosome tected by RT-PCR in their respiratory secretions. EV-D68 has pH and amyloid beta (Abeta) protein generation. The 14 ART S30 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 drugs tested included (1) the nucleoside reverse-transcriptase stress on HIV-1 transcription and viability of infected cells. inhibitors zidovudine, abacavir, lamivudine, deoxythymidine Treatment of both T cells and monocytes with therapeutic IR and emtricitabine, (2) the nucleotide reverse-transcriptase doses led to dramatic increase of HIV-1 transcription, as evi- inhibitors tenofovir and disoproxil, (3) the non-nucleoside denced by the presence of Pol II and reduction of HDAC1 and reverse-transcriptase inhibitors efavirenz and nevirapine, methyl transferase SUV39H1 on the HIV-1 promoter using (4) the protease inhibitors ritonavir, nelfinavir and ChIP assay. Increased HIV-1 replication after IR correlated darunavir, (5) the integrase inhibitors dolutegravir and with higher cell death as compared with uninfected cells. elvitegravir, and (6) the booster (liver enzyme inhibitor) Importantly, the level of phosphorylated Ser46 in p53, cobicistat. The ART drugs that de-acidified endolysosome responsible for apoptosis induction, was markedly higher pH also increased Abeta levels, whereas those ART drugs in HIV-1 infected cells following IR treatment. Exposure that acidified endolysosome pH decreased Abeta levels. of HIV-1 infected humanized mice treated with Furthermore, an agent that acidifies endolysosomes antiretrovirals, which did not display viral RNA in the (MLSA-1) blocked ART drug-induced increases in plasma and PBMCs, to IR resulted in a significant in- Abeta levels. Collectively, our data suggest endolysosome crease of HIV-1 RNA in plasma, lung, and brain tissues de-acidification plays an important role in ART drug- of these animals. IR-induced cellular stress facilitates en- induced amyloidogenesis. (This work was supported by hanced apoptotic death of infected cells, possibly via p53. P30GM103329, and R01MH100972, and Collectively, these data point to the use of combination of R01MH105329.) low to moderate dose of IR with HIV-1 transcription activators as a potential application for the “Shock and Kill” strategy for latently infected cells. P61 A new approach to “Shock and Kill” for various organs including CNS: Therapeutic doses of irradiation activate P62 viral transcription and induce apoptosis in HIV-1 infected Use of novel Nano-particles to deliver specific HIV cells. activator Tat and proteins that kill infected cells

Sergey Iordanskiy1, Gavin Sampey1, Rachel Van Duyne2, Sergey Iordanskiy1,BenjaminLepene2, Fatah Kashanchi1 Caitlin Woodson1, Kelsi Fry1,MohammedSaifuddin1, Fabio (corresponding author: [email protected]) Romerio3, Fatah Kashanchi4 (corresponding author: [email protected]) 1National Center for Biodefense and Infectious Disease - George Mason University; 2Ceres Nanosciences Inc., 1National Center for Biodefense and Infectious Disease - Manassas, VA George Mason University; 2Center for Cancer Research, National Cancer Institute, National Institues of Health; HIV-1 infection results in a chronic illness since long-term 3Deparment of Medicine, Uvinersity of Maryland School of HAART can lower viral titers to an undetectable level. Medicine; 4National Center for Biodefense and Infectious However, discontinuation of therapy rapidly increases virus Disease - George Mason University burden. Moreover, patients under HAART frequently develop various metabolic disorders, neurocognitive abnormalities, The highly active antiretroviral therapy reduces HIV-1 RNA and cardiovascular diseases. Our understanding of both the in plasma to undetectable levels. However, the virus continues degree of treatment adherence needed to maintain long-term to persist in the long-lived resting CD4+ Tcells, macrophages, virological suppression and the pathophysiology of some of and astrocytes, which form a viral reservoir in infected indi- the major adverse events associated with HIV infection and viduals. Selective activation of viral transcription is critical therapy has improved. Anti-retroviral drugs targeting HIV since the host immune response, in combination with antire- encoded enzymes have slowed the death rate from AIDS troviral therapy, may eradicate the virus following reactivation but, to date, no effective vaccines have been developed and (“Shock and Kill”). X-ray irradiation (IR), a well-defined there is no practical cure. Thus, the selective activation of the stress signal that is widely used for many therapeutic pur- latently HIV-infected cells resulting in the replication of pro- poses, has been shown to be capable of activating HIV-1 tran- viral genome is critical, since the virus itself, as well as the scription, progeny virion formation, and eventual apoptosis of host immune response in combination with antiretroviral infected cells. Using chronically HIV-1 infected T drugs (“Shock and Kill” strategy), may eliminate the virus lymphoblastoid and monocytic cell lines, primary resting following reactivation. Additionally, elicitation of the treat- CD4+ T cells, and humanized mice infected with dual-tropic ment that can directly eliminate latently HIV-infected cells HIV-1 89.6, we examined the effect of IR-induced cellular within the pool of resting T cells is also important for effective J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S31 depletion of the viral reservoirs. A direct means of achiev- strengthened the correlation (r =0.44, p =0.038). The 18 ing this is to first specifically activate the latent virus (75.0 %) participants who had CSF collected after 11:15 am (i.e., with Tat) and then kill infected cells based on dis- had higher HIV RNA in CSF (Cohen’sd=0.83,p =0.053). play of specific antigens such as the HIV envelope gly- Conclusions: Circadian variation in two CSF constituents coprotein (Env) on the external surface of productive is consistent with experimental observations of the infected cells, where it can be recognized by a specific glymphatics system. While the observed differences may binding molecule such as an antibody. The Env-targeting not be clinically meaningful, they are consistent with moiety can be linked to various types of cytotoxic glymphatic-mediated surges in CSF flow and suggest that agents, yielding novel molecules that selectively kill research studies should account for the CSF collection HIV-infected cells. We will show data on how this ap- time in analyses. proach can be used to target the infected cells in vivo using specific set of nanoparticles. Our approach is novel and unique in the sense that it can specifically activate P64 HIV transcription and specifically destroy infected cells Myocyte enhancer factor (MEF)-2 plays essential roles in vitro and in vivo. in T-cell transformation associated with HTLV-1 infection by stabilizing complex between Tax and CREB

P63 Pooja Jain1, Alfonso Lavorgna2, Mohit Sehgal1,Linlin Time of Day May Influence Concentrations of Albumin Gao3,DivyaSagar1, Rashida Ginwala1,EdwardHarhaj2, and HIV RNA in Cerebrospinal Fluid (CSF) Zafar Khan1 (corresponding author: [email protected]) Jennifer Iudicello1, Scott Letendre1, J. Allen McCutchan1, Qing Ma2,IgorGrant1, Ronald J. Ellis1 1Department of Microbiology and Immunology, and the (corresponding author: [email protected]) Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA; 1University of California, San Diego; 2University at Buffalo 2Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Background: Recent research shows that glial aquaporin Baltimore, MD;3Graduate Program in Cancer Biology, channels generate large water fluxes through interstitial and Sylvester Comprehensive Cancer Center, The University of CSF during sleep, flushing solutes from brain tissue into Miami, Miller School of Medicine, Miami, FL glymphatic and venous channels. We tested the effect of this circadian glymphatic flushing on levels of albumin and HIV The exact molecular mechanisms regarding HTLV-1 Tax-me- RNA in CSF, hypothesizing that levels would be lower in diated viral gene expression and CD4 T-cell transformation samples collected earlier in the day. Methods: Albumin and have yet to be fully delineated. Herein, utilizing virus- HIV RNA were measured in CSF and blood in 165 HIV+ infected primary CD4+ T cells and the virus-producing cell adults who were taking antiretroviral therapy (ART) and line, MT-2, we describe the involvement and regulation of who enrolled in the CHARTER project. Concentrations were Myocyte enhancer factor-2 (specifically MEF-2A) during compared to the time of collection of either CSF or blood the course of HTLV-1 infection and associated disease syn- using bivariate and multivariate regression. Recursive drome. Inhibition of MEF-2 expression by shRNA and its partitioning was used to identify potentially informative activity by HDAC9 led to reduced viral replication and T- threshold values in the time-of-day. Results: CSF was collect- cell transformation in correlation with a heightened expression ed between 9:00 am and 5:20 pm (median 1:10 pm). Later of MEF-2 in ATL patients. Mechanistically, MEF-2 was re- collection times tended to correlate with higher albumin con- cruited to the viral promoter (LTR, long terminal repeat) in the centrations (r =0.15,p = 0.057). The 35 (21.2 %) participants context of chromatin, and constituted Tax/CREB transcrip- who had CSF collected after 2:50 pm had higher albumin tional complex via direct binding to the HTLV-1 LTR. concentrations (mean 21.8 vs. 17.4 mg/dL, Cohen’sd=0.60, Furthermore, an increase in MEF-2 expression was observed p = 0.019). Multivariate analysis indicated that higher CSF upon infection in an extent similar to CREB (known Tax- albumin was associated with both serum albumin and CSF interacting transcription factor), and HATs (p300, CBP, and collection time after 2:50 pm (Model R2 = 0.106, p/CAF). Confocal imaging confirmed MEF-2 co-localization p = 0.0001). Among the 24 participants with HIV RNA in with Tax and these proteins were also shown to interact by co- CSF > 50 c/mL, CSF collection time did not correlate with immunoprecipitation. MEF-2 stabilization of Tax/CREB HIV RNA (p = 0.41). Excluding 2 outliers that fell near the complex was confirmed by a novel promoter-binding assay limit of quantification and farthest from the regression line that highlighted the involvement of NFAT (nuclear factor of S32 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 activated T cells) in this process via Tax-mediated activation neuroprotective by limiting crucial steps in mature mono- of calcineurin (a calcium-dependent serine-threonine phos- cyte transmigration in response to CCL2 in the context of phatase). MEF-2-integrated signaling pathways (PI3K/Akt, HIV neuropathogenesis and opioid abuse. We are testing NF-kappaB, MAPK, JAK/STAT, and TGF-beta) were also this hypothesis using the ECOHIV mice model that reca- activated during HTLV-1 infection of primary CD4+ T cells, pitulates HIV infection and cognitive impairments ob- possibly regulating MEF-2 activity. served in HIV infected people on cART.

P65 Effects of Buprenorphine on CCL2 mediated P66 CD14+CD16+ Monocyte Migration in the context Inducible zinc finger nuclease (ZFN) by HIV Tat to excise of NeuroAIDS HIV-1 from host genome for eradication of AIDS

Matias Jaureguiberry-Bravo1, Loreto Carvallo 2, Chaojiang Haiyan Ji1,XiyingQu2, Won-Bin Young2, Huanzhang Zhu1 Gu2, Jennifer Kelschenbach2,DavidVolsky2,JoanW.Berman1 (corresponding author: [email protected]) (corresponding author: matias.jaureguiberry@ phd.einstein.yu.edu) 1Key Laboratory of Medical Molecular Virology of Ministry of Education/Health, School of Life Sciences, Fudan 1Department of Pathology, Albert Einstein College of University, Shanghai, China; 2Department of Radiology, Medicine; 2Department of Medicine, Division of Infectious University of Pittsburgh School of Medicine Diseases, Icahn School of Medicine at Mount Sinai Current combination antiretroviral therapy (cART) cannot HIV entry into the CNS mediates viral seeding, chronic clear the infected cells harboring HIV proviral DNA from neuroinflammation, and CNS damage that lead to HIV the HIV-infected patients. We previously demonstrated that associated neurocognitive disorders (HAND) in more than Zinc-finger nucleases (ZFNs) could specifically and effi- 50 % of infected people despite successful cART. HIV ciently excise HIV-1 proviral DNA from the latently infected enters the CNS by transmigration of infected monocytes human T cells by targeting the long terminal repeats (LTRs), across the blood brain barrier (BBB). A mature subpopu- a novel and alternative antiretroviral strategy for eradicating lation of CD14+CD16+ monocytes is increased in number HIV-1 infection. To prevent the unwanted off-target effects in infected people. Mature monocytes can be productively from constantly expressed ZFN, in this study, we engineered infected with HIV, and transmigrate preferentially across the expression of ZFN under the control of HIV LTR, by the BBB to CCL2, a chemokine elevated in the CNS of which ZFN expression can be activated by the HIV Tat infected people. Thus, CD14+CD16+ monocytes are key protein during or after HIV infection. Our results show that mediators of HAND. Drug abuse is a major risk factor for functional expression of ZFN induced by Tat excised the HIV infection and opioids have been shown to alter the integrated proviral DNA of HIV-NL4-3-GFP at the position progression and severity of HAND, in part by modulating of Ch16p13.3 in at least 30 % population of previously immune cell functions. Buprenorphine is an opiate deri- established cell line. For visualizing the induction of ZFN vate used to treat opioid dependency. It is a partial agonist by HIV infection, an internal ribosome entry site (IRES) was of MOR, and a full antagonist of KOR, opioid receptors. placed between ZFN and an optical imaging reporter gene However, its effects on CCL2 mediated CD14+CD16+ NanoLuc, a Deep-Sea shrimp luciferase, to become a LTR- monocyte migration and subsequent neuroinflammation ZFN-IRES-Nanoluc. The induced expression of NanoLuc have not been studied. We showed by FACS that and ZFN by HIV transduction was first demonstrated CD14+CD16+ monocytes express MOR and KOR, and in vitro using bioluminescence imaging and Western blotting that mature monocytes obtained from HIV infected people on the tested HuT-R5 or GHOST(3) X4/R5 cells. To dem- express higher surface levels of both receptors compared onstrate the proof of principle in vivo, this LTR-ZFN-IRES- to monocytes from uninfected people. We also demon- Nanoluc was delivered in vivo using Adeno-Associated viral strated that treatment of CD14+CD16+ monocytes with (AAV) vectors for visualizing the induction of ZFN in buprenorphine reduced CCL2-mediatedadhesiontobrain mouse models for HIV-1 infection. The spatial-temporal microvascular endothelial cells. Additionally we found biodistribution of the ZFN-treated cells along with HIV dis- that buprenorphine decreased mature monocyte chemotax- semination in the same mice are currently under evaluation is to CCL2, but that methadone, another opioid derivate using dual-reporter bioluminescence imaging. Taken togeth- used to treat heroin dependency, does not appear to affect er, positively regulated expression of ZFN in the presence of this process. Our findings suggest buprenorphine is HIV Tat may provide a safer and novel implementation of J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S33 the genome-editing technology, including Cas9/CRISPR, for P68 eradicating the HIV-1 proviral DNA from the infected host. Varicella zoster virus downregulates PD-L1 and MHC-1 in human brain vascular adventitial fibroblasts, perineurial cells and lung fibroblasts

P67 Dallas Jones, Anna Blackmon, C. Preston Neff, Brent Palmer, TZIP, a synthetic Pur-protein-based peptide, highlights Don Gilden, Hussain Badani, Maria Nagel a pathway linking CNS actions of Pur-alpha in HIV-1 (corresponding author: [email protected]) infection and in amyotrophic lateral sclerosis University of Colorado School of Medicine Edward M. Johnson1,EarlW.Godfrey2, Dianne C. Daniel1 (corresponding author: [email protected]) Varicella zoster virus (VZV) vasculopathy is a significant cause of intracerebral vasculopathy (stroke), giant 1Department of Microbiology and Molecular Biology, Eastern cell arteritis and granulomatous aortitis. All 3 forms of Virginia Medical School, Norfolk VA 23507; 2Department of VZV vasculopathy develop after virus reactivates from Anatomy, Eastern Virginia Medical School, Norfolk VA ganglia and spreads transaxonally to the adventitia of 23507 arteries where VZV induces persistent inflammation, which in turn leads to pathological vascular remodel- Amyotrophic lateral sclerosis (ALS, Lou Gehrig’sdisease) ing. The mechanism(s) by which inflammatory cells involves degeneration of neurons in the spinal cord and persist to cause vascular damage in VZV-infected ar- brain leading to loss of neuromuscular control. Effects of teries is unknown. However, downregulation of pro- HIV-1 infection on ALS have previously been noted but grammed death ligand 1 (PD-L1) on target cells causes not mechanistically dissected. We report here that the pa- persistent inflammation in some autoimmune diseases thology of ALS can be influenced by the HIV-1 protein, and may play a role in the VZV vasculopathies. Tat, through its interaction with cellular protein, Pur-alpha. Specifically, PD-L1 can be expressed on virtually all This influence is highlighted by a new synthetic peptide, nucleated cells and suppressestheimmunesystemby TZIP, which strongly modulates the action of Tat in HIV-1 interacting with the programmed cell death protein re- transcription as well as the interaction of Pur-alpha with ceptor 1 (PD-1) found exclusively on immune cells. polynucleotides essential for induction of a type of ALS. Inhibition of PD-L1 by autoantibodies is associated ALS is of mixed etiology, including a familial form called with disease progression in rheumatoid arthritis, and C9ORF72 (C9). This chromosome locus contains a repeat deletion of PD-L1 increases neuroinflammation in a of the hexanucleotide GGGGCC, which is greatly expand- murine model of stroke. Thus, we hypothesized that ed in the C9 form of ALS. GGGGCC is a Pur-alpha VZV infection of adventitial cells downregulates PD- binding element, both as ssDNA and + strand mRNA. L1 and contributes to persistent vascular inflammation. Pur-alpha binds a 24-mer of this sequence with a Kd of Adventitial cells (brain vascular adventitial fibroblasts nearly 10-8 M. TZIP was engineered to contain a generic (HBVAFs) and perineurial cells (HPNCs)), as well as Pur protein nucleic acid-binding motif and a transporter human fetal lung fibroblasts (HFLs), were mock- and sequence allowing exogenous cell entry. TZIP has a VZV-infected and analyzed at 24 and 72 h post-infec- cysteine-containing motif essential for Pur-alpha binding tion. Flow cytometry and immunofluorescence analyses to Tat. TZIP binds the C9 hexanucleotide repeat nearly showed decreased PD-L1 expression in VZV-infected 100-fold tighter than Pur-alpha and causes the repeat to BRAFs, HPNCs, and HFLs compared to mock- bind Pur-alpha in an altered 3-dimensional structure. In its infected cells. Quantitative RT-PCR analyses showed transcribed RNA form the C9 repeat is reportedly involved no change in PD-L1 transcript levels between mock- in the formation of cytoplasmic stress granules (SG) and and VZV-infected cells, indicating a post- atypically-translated dipeptide repeats, considered patholog- transcriptional mechanism for VZV-mediated inhibition ical features of ALS. We have found that ectopic Pur- of PD-L1 expression. Flow cytometry analyses showed alpha expression influences formation of the SG and ame- decreased major histocompatibility complex 1 (MHC-I) liorates certain other aspects of ALS in primary neurons. expression in both VZV-infected cells and adjacent un- TZIP also inhibits Tat-dependent transcription of HIV-1 at infected cells compared to mock-infected cells. Overall, <10-10 M. HIV-1 does not infect neurons, but Tat from reduced PD-L1 expression in VZV-infected adventitial infected glia can enter neurons exogenously. Elucidation of cells may contribute to persistent vascular inflamma- the Tat-Pur-alpha pathway may provide clues to treatment tion, while downregulation of MHC-1 may prevent vi- of non-AIDS-related ALS. ral clearance in the VZV vasculopathies. S34 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89

P69 individuals infected with HIV-1 CRF02_AG as compared HIV-1 Subtype G associated with higher impairment to those with subtype G. Further studies are required to of neurocognitive function compared to CRF02_AG characterize the mechanistic basis for the observed among treatment naíve patients in Nigeria differences.

Jibreel Jumare1, Nicaise Ndembi1, Samer El-Kamary2, Laurence Magder2, Laura Hungerford2, Tricia Burdo3, P70 Lindsay Eyzaguirre1, Anya Umlauf4, Mariana Cherner4, Elevated Serum S100B Levels are Associated With Alash’le Abimiku1, Man Charurat1, William Blattner1, Impaired Working Memory and Reduced Brain Gray Walter Royal III5 Matter Volumes in ART-Experienced Chronically (corresponding author: [email protected]) HIV-Infected Adults

1Division of Epidemiology, Institute of Human Virology, Kalpana Kallianpur, Brooks Mitchell, Cecilia Shikuma, University of Maryland Baltimore; 2Department of Lishomwa Ndhlovu Epidemiology and Public Health, University of Maryland (corresponding author: [email protected]) Baltimore; 3Boston College, Chestnut Hill, MA; 4HIV Neurobehavioral Research Program, University of California Hawaii Center for AIDS, John A. Burns School of Medicine, San Diego; 5Department of Neurology, University of University of Hawaii, Honolulu, HI Maryland School of Medicine, Baltimore Neuropsychological (NP) impairment and brain atrophy per- Introduction: HIV-1 subtype has been shown to be associat- sist in chronically HIV-infected patients virally suppressed on ed with disease progression. We compared cognitive func- antiretroviral therapy (ART). Produced by adipocytes, S100B tion between individuals infected with HIV-1 CRF02_AG protein in serum correlates with body mass index (BMI) in and subtype G in Nigeria. Methods: A total of 146 antire- healthy individuals. High circulating S100B concentration is troviral naïve participants with plasma HIV RNA ≥1000 also a marker of brain injury and of blood–brain barrier dis- copies/ml were selected at baseline for this study. ruption that may precede neuronal damage. We examined se- Genotypic analysis was performed by nested PCR of prote- rum S100B for cross-sectional associations with NP test per- ase and reverse transcriptase genes. Amplification products formance, regional brain volumes, and abdominal fat mass in were sequenced, assembled using Sequencer 5.4, and a subset of participants from the HIVAging with HIV Cohort- aligned with HIV-1 curated subtype references. Utilizing de- Cardiovascular Disease study. Serum S100B was assayed by mographically adjusted T scores obtained from a 7-domain ELISA. Composite, domain-specific NP z-scores were obtain- neuropsychological test battery, cognitive status was deter- ed, and T1-weighted magnetic resonance imaging at 3 T was mined by the global deficit score (GDS) approach, with a performed. Visceral, subcutaneous, and total abdominal fat GDS of ≥0.5 indicating cognitive impairment. Results: A content was assessed by computed tomography. Serum total of 76 (52.1 %) participants were infected with the cir- S100B data were available for 39 HIV+ adults on ART [me- culating recombinant form CRF02_AG, 48 (32.8 %) with dian age = 50 years; median CD4 count = 466 cells/mL; medi- subtype G, and 22 (15.1 %) with other HIV-1 strains (A, an BMI = 25.9; 35 male; 33 with plasma HIV RNA <50 C, D, A1G, CRF06_cpx, CRF43_02G). In a multivariable copies/mL]. Twenty-nine had undetectable S100B (<10 pg/ linear regression adjusting for plasma HIV RNA viral load, mL) and 10 had detectable S100B levels (median 287.4 pg/ CD4 count and depression score, global T score was higher mL). Detectability of serum S100B was associated with di- among CRF02_AG infected participants as compared to minished working memory (z-scores: −0.80 vs. 0.07, those with subtype G [Mean difference (MD): 2.3; P-value: p = 0.008) and psychomotor speed (z-scores: 0.17 vs. 0.38, 0.01]. Also, T scores were significantly higher among p = 0.11); lower CD4 count (390 vs. 502 cells/mL, p =0.04); CRF02_AG as compared to subtype G infected participants and higher frequencies of activated (CD38 + HLA-DR+) for the speed of information processing, executive function, CD8+ T-cells (26.1 % vs. 10.3 %, p = 0.008). High S100B and verbal fluency cognitive ability domains [MD: 4.4, 5.4, correlated with decreased volumes of brain regions involved 2.9; P-values: 0.002, 0.001, and 0.02 respectively]. in working memory and psychomotor speed; e.g., anterior Adjusting for viral load and CD4 count in a multivariable cingulate cortex (Spearman rho = −0.89, p = 0.001); total cor- logistic regression, the odds of cognitive impairment tical gray matter (rho= −0.72, p = 0.02); and caudate nucleus were2.4 times higher among subtype G as compared to (rho = −0.75, p = 0.01). S100B did not relate to BMI, waist-to- CRF02_AG infected participants (OR: 2.4 [95 % CI: 0.95, hip ratio, or abdominal fat mass. Elevated serum S100B in 6.1]; P-value: 0.065). Conclusion: We found significantly ART-treated chronic HIV infection appears to reflect an in- better cognitive performance among antiretroviral naíve creased release from brain tissue and may hold potential as a J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S35 biomarker of HIV-related brain atrophy and neurocognitive Li4, Rosemarie M. Booze4, Jennifer Gordon1, Wenhui Hu1, impairment. Furthermore, circulating S100B may help to clar- Kamel Khalili1 ify mechanisms underlying neurodegeneration in well- (corresponding author: [email protected]) controlled HIV disease. 1Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine at Temple University, P71 Philadelphia; 2Microbiology and Clinical Microbiology, Elimination of HIV-1 genomes from human T-lymphoid Department of Biomedical, Surgical and Dental Sciences, cells by CRISPR/Cas9 gene editing University of Milan, Milan; Department of Pharmacology and Experimental Neuroscience, University of Nebraska Rafal Kaminski1, Yilan Chen1, Tracy Fischer1,EllenTedaldi2, Medical Center, Durham Research Center, Omaha; Alessandro Napoli1, Yonggang Zhang1, Jonathan Karn3, 4Behavioral Neuroscience, Department of Psychology, Wenhui Hu1,KamelKhalili1 University of South Carolina, Columbia (corresponding author: [email protected]) A CRISPR/Cas9 gene editing strategy has been remarkable 1Department of Neuroscience/Center for Neurovirology, in excising segments of integrated HIV-1 DNA sequences Comprehensive NeuroAIDS Center, Lewis Katz School of from the genome of latently infected human cell lines and Medicine, Temple University, Philadephia; 2Department of by introducing InDel mutations, suppressing HIV-1 replica- Medicine, Temple HIV Program, Comprehensive tion in patient-derived CD4+ T-cells, ex vivo. Here, we NeuroAIDS Center, Lewis Katz School of Medicine, employed a short version of the Cas9 endonuclease, Temple University, Philadephia; 3Department of Molecular saCas9, together with a multiplex of guide RNAs (gRNAs) Biology and Microbiology, Case Western Reserve for targeting the viral DNA sequences within the 5’-LTR University, Cleveland and the Gag gene for removing critically important segments of the viral DNA in transgenic mice and rats encompassing We employed an RNA-guided CRISPR/Cas9 DNA the HIV-1 genome. Tail-vein injection of transgenic mice editing system to precisely remove the entire HIV-1 ge- with a recombinant Adeno-associated virus 9 (rAAV9) vec- nome spanning between 5’ and 3’ LTRs of integrated tor expressing saCas9 and the gRNAs, rAAV:saCas9/gRNA, HIV-1 proviral DNA copies from latently infected human resulted in the cleavage of integrated HIV-1 DNA and exci- CD4+ T-cells. Comprehensive assessment of whole- sion of a 978 bp DNA fragment spanning between the LTR genome sequencing of HIV-1 eradicated cells ruled out and Gag gene in the spleen, liver, heart, lung and kidney as any off-target effects by our CRISPR/Cas9 technology well as in the circulating lymphocytes. Retro-orbital inocu- that might compromise the integrity of the host genome lation of rAAV9:saCas9/gRNA in transgenic rats eliminated and further showed no effect on several cell health indices a targeted segment of viral DNA and substantially decreased including viability, cell cycle and apoptosis. Persistent co- the level of viral gene expression in circulating blood lym- expression of Cas9 and the specific targeting guide RNAs phocytes. The results from the proof-of-concept studies, for in HIV-1-eradicated T-cells protected them against new the first time, demonstrate the in vivo eradication of HIV-1 infection by HIV-1. Lentivirus-delivered CRISPR/Cas9 DNA by CRISPR/Cas9 on delivery by an rAAV9 vector in a significantly diminished HIV-1 replication in infected pri- range of cells and tissues that harbor integrated copies of mary CD4+ T-cell cultures and drastically reduced viral viral DNA. load in ex vivo culture of CD4+ T-cells obtained from HIV-1 infected patients. Thus, gene editing using CRISPR/Cas9 may provide a new therapeutic path for P73 eliminating HIV-1 DNA from CD4+ T-cells and potential- Negative feedback regulation of HIV-1 by gene editing ly serve as a novel and effective platform toward curing strategy AIDS. Rafal Kaminski1, Yilan Chen1, Julian Salkind1,Ramona Bella2, Won-bin Young3, Pasquale Ferrante2,Jonathan P72 Karn4,ThomasMalcolm5, Wenhui Hu1,KamelKhalili1 Excision of HIV-1 DNA by gene editing: (corresponding author: [email protected]) aproof-of-conceptinvivostudy 1Department of Neuroscience/Center for Neurovirology, Rafal Kaminski1,RamonaBella2, Chaoran Yin1, Jessica Comprehensive NeuroAIDS Center, Lewis Katz School of Otte1, Pasquale Ferrante2, Howard E. Gendelman3, Hailong Medicine, Temple University, Philadelphia; 2Microbiology S36 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 and Clinical Microbiology, Department of Biomedical, Inventory-II to assess the prevalence and severity of depres- Surgical and Dental Sciences, University of Milan, Milan; sive symptoms in 270 controls and HIV-infected 3Department of Radiology, University of Pittsburg School of Cameroonians. Univariable analysis showed a trend toward Medicine, Pittsburg; 4Department of Molecular Biology and higher risk of depressive symptoms among cases, compared Microbiology, Case Western Reserve University, Cleveland; to controls (p = 0.055), and among subjects >40 years-old, 5Excision Biotherapeutics, Inc. compared to subjects ≤40 years (p = 0.059); but multivariable analyses showed no effect of age and no significant The CRISPR/Cas9 gene editing method is comprised of the difference in the risk of depressive symptoms between cases guide RNA (gRNA) to target a specific DNA sequence for and controls. Analysis of depression severity showed that cleavage and the Cas9 endonuclease for introducing breaks in 33.73 % of cases had moderate-to-severe depressive symp- the double-stranded DNA identified by the gRNA. Co- toms, compared to 19.8 % of controls (p < 0.01). Both expression of both a multiplex of HIV-1-specific gRNAs univariable and multivariable regression analyses showed and Cas9 in cells results in the modification and/or excision significantly higher risk of depressive symptoms among fe- of the segment of viral DNA, leading to replication-defective males compared to males (p = 0.02); both among female virus. In this study, we have personalized the activity of controls (p = 0.04; 95 % CI: 0.02 to 1.04) and female cases CRISPR/Cas9 by placing the gene encoding Cas9 under the (p = 0.03; 95 % CI: 0.04 to 1.03), compared to male con- control of a minimal promoter of HIV-1 that is activated by the trols. Gender, ART, or the presence of opportunistic infec- HIV-1 Tat protein. We demonstrate that functional activation tions did not influence the risk of depression among cases. of CRISPR/Cas9 by Tat during the course of viral infection Lower (≤10 years) education was associated with increased excises the designated segment of the integrated viral DNA risk of depression among controls (p = 0.04), but education and consequently suppresses viral expression. This strategy did not influence the risk of depression among cases, and was also used in a latently infected CD4+ T-cell model after there was no association between education and depression treatment with a variety of HIV-1 stimulating agents including severity. This study shows increased presence of depressive PMA and TSA. Controlled expression of Cas9 by Tat offers a symptoms in both controls and HIV-infected Cameroonians. new strategy for safe implementation of the Cas9 technology Integrating care for mental disorders such as depression into for ablation of HIV-1 at a very early stage of HIV-1 replication primary health care and existing HIV/AIDS care programs during the course of the acute phase of infection and the reac- in Cameroon may enhance the wellbeing of the general pop- tivation of silent proviral DNA in latently infected cells. ulation and could lower HIV/AIDS burden.

P74 P75 Depression among HIV-infected and seronegative control Transgenic expression of HIV-1 in Tg26 mice accelerates subjects in Cameroon: effect of age, education, and gender atherosclerosis; and implications in vascular dementia

Georgette Kanmogne1, Fonsah Julius2, Fang Qiu1, Claude Alison Kearns, Shen Dai, Fengming Liu, Elizabeth Kiernan, Tagny2, Dora Mbanya2, Dora Njamnshi3, Callixte Kuate2, Xiao Peng, Jennifer Gordon, Xuebin Qin Roland Doh2, Anne Marie Kengne4, Mariana Cherner5, (corresponding author: [email protected]) Robert Heaton5, Alfred Njamnshi2 (corresponding author: [email protected]) Department of Neuroscience and Center for Neurovirology, Lewis Katz School of Medicine at Temple University 1University of Nebraska Medical Center; 2University of Yaounde-I; 3Yaounde Day Hospital; 4Yaounde Central HIV-1-associated comorbidities including vascular dementia Hospital; 5University of California San Diego; and cardiovascular disease are the most frequent cause of death among HIV-1+ patients in the HAART era. Extensive Depression is a leading cause of disease burden; it worsens clinical evidence suggests that HIV-1 accelerates develop- HIV/AIDS outcome and quality of life. Addressing this ment of atherosclerosis, an immune and inflammatory dis- problem requires accurate quantification of the added burden ease that is associated with vascular dementia and cardio- of depression to HIV/AIDS in a given population, and vascular diseases including brain vascular disorders and knowledge of the baseline depression prevalence in the gen- stroke. However, the mechanisms underlying HIV-1- eral seronegative population. There has been no previous associated atherosclerosis have not been fully elucidated study of depression in the general Cameroon population. experimentally and how atherosclerosis contributes to the The current study begins to fill that important gap in one development of vascular dementia, especially in the context Sub-Saharan African country. We use the Beck Depression of chronic HIV-1 infection, remains unclear. This is mainly J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S37 due to the lack of a suitable in vivo model. Here, we pathogenesis. Preliminary data using conventional mice in- present the characterization of a new mouse model for fected with a mouse-tropic HIV, EcoHIV, suggests that chron- HIV-1-associated atherosclerosis. By crossing Tg26 mice ic morphine administration prior to primary HIV infection (Tg26+/−) which contain a transgene encoding the HIV-1 accelerates HIV neuroinvasion and induction of genome with Apolipoprotein E (ApoE) deficient mice neurocognitive impairments (NCI) as assessed by radial arm (ApoE−/−) on a C57BL/6 J background, we introduced water maze (RAWM) testing. This finding was accompanied Tg26 mice to hyperlipidemia conditions (Tg26/ApoE−/−), by a significant increase in bran viral load, suggesting that a widely accepted method for inducing atherosclerosis in chronic morphine administration may facilitate viral transit mice. We demonstrate that 1) Tg26/ApoE−/− mice fed on into the brain. In addition, both NCI as well as the virological high fat diets for either 8 or 12 weeks developed more changes were reversed in the presence of mu opioid receptor severe atherosclerosis in both the aortic root and aorta than antagonist, naltrexone, indicating that these findings are opiate ApoE−/− mice, 2) Tg26/ApoE−/− mice highly express dependent. Based on these results we hypothesize that HIV-1 transcripts in hematopoietic tissues, which are the chronic morphine exposure facilitates induction of origin of inflammatory cells including macrophages and HAND pathogenesis at the early stage of HIV infection T-cells, 3) Tg26/ApoE−/− did not have significantly higher when brain reservoirs of virus are being established and levels of cholesterol or triglycerides than ApoE−/− mice, 4) molecular processes that pre-determine NCI are initiated. Tg26/ApoE−/− mice did not display signs of nephropathy, The importance of the proposed research is underscored 5) HIV-1 expression accelerated the production of foam by significant coincidence between illicit substance use cells, a hallmark for atherogenesis, and 6) HIV-1 expression and the progression of HIV infection to HAND. The ulti- increased the activation of the caspase-1, a signaling path- mate goal of this work is to maximize currently available way critical for inflammasome formation, in Tg26/ApoE therapeutics to help the individuals at risk for the devas- −/−‘s macrophages. Taken together, these results indicate tating neurocognitive complications associated with HIV- that Tg26/ApoE−/− mice provide a new mouse model for 1 infection, with a particular focus on drug abusing HIV understanding the pathogenesis of HIV-1-associated athero- populations, which are both prevalent and underserved. sclerosis. The implications of these findings on potential Supported by DA037611, MH104145, and DA017618. mechanisms underlying HIV-associated changes in the brain microvasculature will be discussed. P77 HIV-1 TAT protein expression enhances P76 methamphetamine behavioral sensitization and alters Chronic morphine administration exacerbates dopaminergic function in mice. EcoHIV-induced neurocognitive impairments in mice James P. Kesby1, Julia A. Najera2,BenedettoRomoli1,Yiding Jennifer Kelschenbach1, Alejandra Borjabad1, Boe-Hyun Fang2, Liana Basova2,AmandaBirmingham3, Maria Cecilia Kim1, Chao-Jiang Gu1, Hongxia He1, Wei Chao1, Ottavio G. Marcondes2,DavideDulcis1, Svetlana Semenova1 Arancio2,MaryJanePotash1, David Volsky1 (corresponding author: [email protected]) (corresponding author: [email protected]) 1Department of Psychiatry, School of Medicine, University of 1Department of Medicine Icahn School of Medicine at Mount California San Diego, La Jolla, CA; 2Department of Sinai; 2Department of Pathology and Cell Biology, Columbia Molecular and Cellular Neurosciences, The Scripps University Research Institute, La Jolla, CA; 3Center for Computational Biology and Bioinformatics, University of California San Mild HIV associated neurocognitive disorders (mild HAND) Diego, La Jolla, CA continue to occur at high frequency despite the success of combined antiretroviral therapies (cART) in alleviating immu- Methamphetamine abuse is common among humans with im- nological abnormalities of HIV infection and preventing or munodeficiency virus (HIV). The HIV-1 regulatory protein TAT delaying onset of AIDS. Recent data suggest that the molec- is one of several HIV-related proteins associated with HIV- ular processes underlying mild HAND are triggered without induced neurotoxicity. TAT-induced dysfunction of the overt symptoms early, possibly during primary/acute infection mesolimbic dopaminergic systems may result in impaired re- when absence of adaptive antiviral immunity facilitates virus ward processes and contribute to methamphetamine abuse. expansion, neuroinvasion, and establishment of stable virus Transgenic mice with doxycycline-induced TAT protein expres- reservoirs. Host and environmental factors, such as drugs of sion in the brain show neuropathology resembling brain abnor- abuse, may facilitate these processes and enhance HAND malities observed in humans affected by HIV disease. These S38 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 studies investigated the impact of TAT expression on the brain early following infection, pre-exposure prophylaxis methamphetamine-induced locomotor sensitization and underly- (PrEP) (tenofovir, emtricitabine) could prevent HIV-infection ing changes in dopamine function/receptors and adenosine re- but could also decrease virus entry into the brain. However, ceptors in mesolimbic brain areas. Mice were tested for locomo- efficacy drops when patients are non-compliant. We hypoth- tor activity in response to repeated methamphetamine injections esize that combining PrEP with a CCR5-inhibitor, such as and methamphetamine challenge after a 7-day abstinence period. maraviroc (MVC) could reduce HIV-infected monocyte Dopamine function in the nucleus accumbens (Acb) was deter- (MO) trafficking into the CNS. We present an in-vitro mined using high performance liquid chromatography. blood–brain-barrier (BBB) model to test our hypothesis. Expression of dopamine and adenosine A receptors (ADORA) Objective: Determine the level of HIV-infected MO in the in the Acb was assessed using RT-PCR. Microarrays with path- presence of PrEP compared to PrEP+MVC. Assess the level way analyses assessed dopamine and adenosine signaling in the of HIV-infected MO trafficking across the BBB with PrEP caudate putamen (CPu). Activity-dependent neurotransmitter compared to PrEP+MVC. Methods: Human brain astrocytes switching of reserve pool neurons to a dopaminergic pheno- (HBA) and human brain microvascular endothelial cells type in the ventral tegmental area (VTA) was determined using (HBMVEC) were cultured on opposite sides of a matrix- immunohistochemistry and stereology. TAT expression en- coated cell-culture insert. In-vitro BBB integrity was assessed hanced methamphetamine-induced sensitization. Levels of do- by trans-endothelial electronic resistance (TEER) and perme- pamine and associated metabolites in the Acb did not differ ability with 0.45 % Evan’s Blue Albumin (EBA). MO HIV- between TAT-expressing and control mice regardless of meth- infection will be assessed in the presence of 0, 0.02, 0.06, and amphetamine exposure. However, TAT expression alone de- 0.2 microM emtricitabine and 0, 0.01, 0.03, and 0.1 microM creased striatal dopamine (D1, D2, D4, D5) and ADORA1A tenofovir; and compared to infection with PrEP+MVC at receptor expression, while increasing ADORA2A receptors MVC concentrations of 0, 0.01, 0.05, and 0.2 microM. expression. Moreover, TAT expression combined with meth- Transmigration of MO across the BBB will then be mea- amphetamine exposure was associated with increased adeno- sured to assess MVC efficacy. Results: A BBB bilayer mod- sine A receptors (ADORA1A) expression and increased re- el was established. BBB integrity reached its highest on day cruitment of dopamine neurons in the VTA. Together these 8 of culture with TEER ranging from lowest 101 Ω /cm2 to findings suggest that TAT-induced enhancement of metham- highest 495 Ω/cm2 (4–24 replicates). BBB EBA permeabil- phetamine sensitization may involve altered dopamine and ity on days 6–8 measured mean of 5.3–6.0 % respectively adenosine receptors expression and enhanced recruitment of compared to cell-free controls 65–68 % (p =0.0001). BBB reserve pool neurons to a dopaminergic phenotype. TEER remained ≥60 Ω /cm2 (3–15 replicates) up to 6 days Dopamine-adenosine receptor interactions and reserve pool post-media change from growth to basal medium. neuronal recruitment may represent potential targets to develop Conclusions: A reliable BBB model was created which will new treatments for methamphetamine abuse in individuals be used for MO infection and transmigration studies follow- with HIV. ing PrEP±MVC. The results could provide a different paradigm and approach in preventing HIV-associated cognitive impairment. P78 CCR5-inhibitor combined with PrEP to potentially reduce HIV-infected monocyte trafficking across the blood brain P79 barrier APOE isoform specific effects on Tat-mediated HIV-1 LTR transactivation in astrocytes Joanna Kettlewell1,RobertOda2,ChristieNakamura3 (corresponding author: [email protected]) Nabab Khan, Jonathan D Geiger, Xuesong Chen (corresponding author: [email protected]) 1Department of Tropical Medicine, Medical Microbiology, and Pharmacology; Hawaii Center for AIDS; John A. Burns School of Medicine and Health Sciences, University of North School of Medicine-University of Hawaii at Manoa; Dakota 2Department of Molecular Biosciences & Bioengineering; University of Hawaii at Manoa; 3Department of Tropical Currently 36 million people worldwide are infected with HIV- Medicine, Medical Microbiology, and Pharmacology; John 1, and it is estimated that 50 % of those HIV-1 infected indi- A. Burns School of Medicine-University of Hawaii at Manoa viduals have HIV-1 associated neurocognitive disorders (HAND). Although unclear mechanistically, various forms Background: Morbidity from cognitive problems following of apolipoprotein E cholesterol carriers are known to affect HIV infection continues to be significant. Since HIV-1 enters HIV-1 replication and the prevalence of HAND: apoE4 allele J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S39 especially has been associated with higher steady-state viral cells expressing the LTR-driven luciferase (LTR-Luc), we de- loads and accelerated rates of cognitive decline. HIV-1 termined the extent to which modulating endolysosome pH transactivator of transcription (Tat) protein, a protein essential affects Tat-mediated HIV-1 LTR transactivation. We demon- for HIV-1 replication, can be secreted from infected or strated that endolysosome de-acidifying agents, transfected cells and can be taken up by bystander cells from Chloroquine and PI5P, enhance Tat-mediated HIV-1 LTR the extracellular environment via receptor-mediated endocy- transactivation; whereas endolysosome acidifying agents, tosis. Because HIV-1 Tat can bind to lipoprotein receptor ML-SA1 and PI(3,5) P2, blocks Tat-mediated HIV-1 LTR protein-1 (LRP-1) that is a major contributor to the uptake of transactivation. Our findings suggest that endolysosome apoE-cholesterol in brain, we determined the extent to which pH plays a key role in Tat-mediated HIV-1 LTR different isoforms of apoE affected HIV-1 Tat-mediated HIV- transactivation. (This work was supported by 1 LTR transactivation in U87MG astrocytoma cells express- R01MH100972, and R01MH105329.) ing LTR-driven luciferase. Compared to apoE2-cholesterol and apoE3-cholesterol, apoE4-cholesterol (co-incubation or post-incubation) is less effective in preventing Tat- P81 mediated HIV-1 LTR transactivation. Similar effects were Pre-insulin resistance predicts worsening cognitive observed when cells were treated with isoforms of apoE function in HIV-infected patients without the incorporation of cholesterol. Furthermore, we demonstrated that a small peptide that contains the apoE Saja Khuder1, SooAh Kim1,ScottLetendre2,Thomas receptor-binding region attenuated HIV-1 Tat-mediated Marcotte2,IgorGrant2, Ned Sacktor1, Justin McArthur1, LTR transactivation. These findings contribute to our un- Alex Dickens1, Norman Haughey3 derstanding of the effects of apoE on HIV-1 infection and (corresponding author: [email protected]) associated neurocognitive disorders and suggest that apoE mimetic peptides might be used as a therapeutic strategy 1The Johns Hopkins University School of Medicine; 2HIV against HIV-1 infection and associated neurocognitive Neurobehavorial Research Program and Department of disorders. (This work was supported by P30GM103329, Psychiatry; 3The Johns Hopkins University School of R01MH100972, and R01MH105329.) Medicine, The Johns Hopkins University School of Medicine, Department of Psychiatry

P80 HIV-infection and combination antiretroviral therapy (cART) Role of endolysosmal pH in Tat-mediated HIV-1 LTR are independently associated with increased risk of develop- transactivation in astrocytes ing lipodystrophy and metabolic abnormalities that include alterations in fat metabolism and insulin sensitivity. Nabab Khan, Jonathan D Geiger, Xuesong Chen Reduction in insulin sensitivity is associated with lipolysis (corresponding author: [email protected]) of adipocytes, increased release of fatty acid derivatives into circulation and subsequent storage in liver, skeletal muscle School of Medicine and Health Sciences, University of North and heart. This redistribution accelerates the development of Dakota insulin resistance. Although insulin resistance has been firmly associated with cognitive impairment in diabetes, the relation- Currently 36 million people worldwide are infected with HIV- ship of this metabolic syndrome with the development of cog- 1, and it is estimated that 50 % of those HIV-1 infected indi- nitive impairment in HIV is emerging. We have previously viduals have HIV-1 associated neurocognitive disorders shown that abnormalities in CNS lipid metabolism are asso- (HAND). Human immunodeficiency virus type 1 (HIV-1) ciated with the development and trajectory of cognitive im- transactivator of transcription (Tat) is essential for HIV-1 viral pairment in HIV infected subjects. Here, we sought to deter- replication. HIV-1 Tat can be secreted from infected or mine the associations of metabolic abnormalities with cART transfected cells and taken up by bystander cells from extra- and cognitive function in the setting of HIV infection. We cellular environment via receptor-mediated endocytosis. conducted untargeted lipidomic analysis, targeted clinical Upon internalization, Tat is then accumulated in and metabolic measures in longitudinal plasma samples from endolysosomes, from which it can be released into the cyto- HIV+ patients exhibiting temporal changes in cognitive status plasm and enters the nucleus to facilitate viral replication. It is (stably normal, stably impaired, declining and improving). still unknown the underlying mechanisms of Tat escaping Increased plasma levels of triacylglycerols (TAG), their ether endolysosomes and subsequent transactivation in the nucleus. derivatives monoalkyl-diacylglycerols (MADAG) and elevat- We postulate that endolysosome pH plays a key role in Tat- ed insulin were associated with stably impaired and declining mediated HIV-1 LTR transactivation. In U87MG astrocytoma cognitive status compared to stably normal. Subjects with S40 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 improving cognitive status exhibited decreased plasma levels (WT) mice (mortality at 12 days instead of 8 days post inoc- of TAG and MADAG compared to stably impaired subjects. ulation). Moreover, MALT1 KO mice exhibited reduced ex- High plasma c-peptide levels in subjects with stably im- pression of IL-1beta IFN-gamma and iNOS compared to WT paired and declining cognitive function suggested that an mice which might explain the delay in the appearance of clin- increase in pancreatic insulin production and secretion ical signs. Viral RNA loads were decreased, suggesting rather than a decrease in clearance was responsible for that inhibition of MALT1 somehow reduces virus replica- elevated plasma insulin. Low plasma adiponectin and tion and/or spread of the virus in the brain. Stronger T HDL in subjects with stably impaired and declining cog- lymphocyte and macrophage infiltration and microglia ac- nitive functions further implicated the involvement of a tivation were observed in WT mice compared to KO metabolic syndrome in cognitive decline. These results mice, which suggests that these cells contribute to patho- suggest that pre-insulin resistance in HIV is associated genicity rather than protection of the host. Daily mepazine with impaired or declining cognitive function. treatment also delayed the onset of disease, showing an Restoration of insulin sensitivity in the setting of HIV involvement of the MALT1 proteolytic activity. We are infection may improve cognitive function. currently further examining the impact of MALT1 deficiency in mice with conditional KO in specific cell types to better understand the role of MALT1 in rabies patho- P82 genesis. Our study shows that MALT1 inhibition has a MALT1 contributes to the pathogenicity of the virulent significant impact on the progression of rabies virus in- rabies virus CVS-11 in mice. fection and disease, which emphasizes the importance of inflammatory/immunological mechanisms on rabies dis- Elodie Kip1, Vanessa Suin2,JensStaal3, Rudi Beyaert3, ease development. Steven VanGucht2 (corresponding author: [email protected]) P83 1 National Reference Centre of Rabies, Viral Rift Valley fever virus is highly neurotropic and cytotoxic diseases,Communicable and Infectious Diseases, Scientific in a rat model of lethal encephalitis Institute of Public Health (WIV-ISP), Brussels and Unit of Molecular Signal Transduction in Inflammation, Michael Kujawa1, Aaron Walters1,TimOury2,Leonard Inflammation Research Center, VIB, Ghent and Gh; D’Aiuto3,AmyHartman1 2National Reference Centre of Rabies, Viral diseases, (corresponding author: [email protected]) Communicable and Infectious Diseases, Scientific Institute of Public Health (WIV-ISP), Brussels; 3Unit of Molecular 1Department of Infectious Diseases and Microbiology, Center Signal Transduction in Inflammation, Inflammation for Vaccine Research, University of Pittsburgh; 2Department Research Center, VIB, Ghent and Ghent University, of Pathology, University of Pittsburgh; 3University of Department of Biomedical Molecular Biology, Ghent, Pittsburgh Belgium Background: Rift Valley Fever (RVF) is a mosquito-borne Rabies virus is a neurovirulent RNAvirus, which causes about viral disease found primarily within Africa. In humans, lethal 59000 deaths in humans each year. The paracaspase MALT1 encephalitis is one of the most severe clinical outcomes. Due is crucial for immune and inflammatory cell activation by to the lack of a reproducible, immunocompetent mouse model different receptors, including antigen receptors, lectin recep- of RVF encephalitis, not much is known about how RVFV tors, and some GPCRs. MALT1 acts as a scaffold signalling causes neurological disease, despite the importance of this protein and a cysteine protease that cleaves substrates, pro- clinical outcome in human epidemics. Our lab recently devel- moting NF-kB signalling and mRNA stabilization. As a result, oped the Lewis rat model to study the neuropathogenic mech- MALT1 activation drives the expression of multiple immuno- anisms of RVFV. After inhalational exposure to RVFV, Lewis regulatory genes. Mepazine, an inhibitor of MALT1 proteo- rats develop a uniformly lethal encephalitic disease with neu- lytic activity, proved to have therapeutic effects in B cell lym- rological symptoms. The goal of this research study is to use phoma and experimental autoimmune encephalomyelitis in in vitro and in vivo methodology to understand virus-CNS mice. We examined the role of MALT1 in the development interactions. Methods: For in vivo studies: Lewis rats (infected of rabies disease using MALT1 knock-out (KO) mice or with ZH501 by aerosol exposure) were euthanized and viral mepazine treatment after infection with the rabies strain infection was assessed by q-RT-PCR and immunohistochem- CVS-11. The development of disease was significantly istry. For in vitro studies: viral infection and growth was slowed down in MALT1 KO mice compared to wild-type assessed in human NPCs, SH-SY5Y, HBMEC, Vero E6, and J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S41

BHK-21 cells. Results: Viral replication in the CNS begins in neuron-restricted measles virus infection in neonatal mice, the olfactory bulb (1–2 dpi) and proceeds posteriorly through IFN did not affect proliferation of nestin + NSPCs; however, the brain and spinal cord, while peripheral replication is very it reduced neurogenesis as measured by BrdU incorporation limited. Pathologic lesions in the brain include pervasive into newly-born neurons. These studies identify a role of IFN vasculitis and meningitis. Preliminary studies indicate that in modulating NSPC activity, which would identify potential neurons are the primary cells expressing antigen. In vitro, new targets that could salvage the developing CNS from dis- SH-SY5Y and HBMEC cells were both highly permissive ruptive effects of an anti-viral immune response. Funding for for viral growth compared to Vero E6 and BHK-21 cells. this work is provided by the NIH (1R15NS087606-01A1) and iPSC-derived primary human neurons were extremely per- Duquesne University’s Mylan School of Pharmacy. missive for viral infection, with near-100 % infection levels observed by 24 hpi followed by extensive cell death. Conclusions: Lewis rats are thus far the most appropriate rodent model for studying neurotropic RVFV. P85 In vitro and in vivo evidence shows the virus is highly Molecular and structural traits of IRS-1/LC3 nuclear neurotropic and cytotoxic, a previously unrecognized and structures and their role in glioblastoma drug resistance unappreciated finding. Our findings pave the way for future studies understanding the mechanism of Adam Lassak1, Dorota Wyczechowska1,AnnaWilk2, neuropathogenesis of RVFV. Mathew Dean1, Adriana Zapata1,LuisDelValle1, Francesca Peruzzi1, Krzysztof Reiss1 (corresponding author: [email protected]) P84 The antiviral cytokine interferon-gamma restricts neural 1Louisiana State University, Stanley S Scott Cancer Center; stem/progenitor cell proliferation through activation 2University of South Alabama, Mitchell Cancer Institute of STAT1 and modulation of retinoblastoma protein phosphorylation. Insulin receptor substrate 1 (IRS-1) is a common cytosolic adapter molecule, which transduces the signal from the insulin Apurva Kulkarni, Kristen N. Fantetti, Taylor J. Scully, Lauren and insulin-like growth factor I (IGF-I) receptors. We have A. O’Donnell previously reported that IRS-1 translocates to the nucleus ei- (corresponding author: [email protected]) ther in the presence of the human polyomavirus JC oncoprotein, large T-antigen, or in the presence of estrogen Mylan School of Pharmacy, Duquesne University receptors. Nuclear IRS-1 (nIRS-1) has been also detected in cells expressing the SV40 T-antigen, v-Src, and in breast can- Viral infections in the central nervous system (CNS) result in cer cells in association with estrogen receptor alpha, which all immune cell infiltration and release of pro-inflammatory cy- indicate that nIRS-1 may play a role in tumor cell biology. tokines. Interferon-gamma (IFN), a key anti-viral cytokine, is Here we report that nuclear IRS-1 can form unique ring-like required for non-cytolytic clearance of many viruses from the structures in a limited number of cells found in glioblastoma CNS. IFN-mediated cell signaling is responsible for altering biopsies and glioblastoma xenografts. In these structures IRS- cell survival and proliferation in different neural cells. Here, 1 localizes at the periphery and the center harbors a key au- we address the effects of IFN signaling on the proliferation tophagy protein, LC3. They appear more frequently after and differentiation of neural stem/progenitor cells (NSPCs). genotoxic stress, and can be reproduced by ectopic expression We hypothesized that IFN would inhibit NSPC proliferation of IRS-1 with nuclear localization signal. These IRS-1- and induce glial differentiation via the Janus activated kinase containing nuclear structures are highly dynamic, disassemble (Jak)/Signal Transducers and Activators of transcription during mitosis, rapidly exchange IRS-1 content with nucleo- (STAT) signaling pathways. We observed that IFN inhibited plasm, and interact with other nuclear suborganelles including NSPC proliferation and cell cycle progression in a STAT1- PML and Polycomb bodies. Remarkably, cells engineered to dependent manner characterized by inhibition of neurosphere form these ring-like structures acquired several new adapta- growth. Pulse-chase experiments with BrdU confirmed that tions including drugs resistance and growth advantage in sub- IFN slowed cell cycle progression as compared to untreated optimal conditions, which correlated with the recruitment of cells. IFN restricted cell cycle progression at the late G1/S Bmi1 to Polycomb bodies and repression of the canonical checkpoint, which was mediated by dephosphorylation of Polycomb Repressor Complex 1 gene target, Ink4a-Arf locus. the retinoblastoma protein (pRb) at serine 795 and decreased This is the first demonstration of a functional interplay be- cyclin E/cdk2 expression. In addition, IFN increased astrocyt- tween IRS-1 and LC3, which are known to have distinct roles ic differentiation and reduced neurogenesis in vitro. During a in cytoplasm; however, in the nucleus they form unique S42 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 structure, which according to our data could play a role in accelerate and exacerbate subsequent neuroinflammatory dis- controlling autophagy and acquired stress/drug resistance. ease through direct CNS infection and peripheral activation that may promote blood brain barrier permeability.

P86 Herpesvirus trigger accelerates a neuroinflammatory P87 demyelinating disease in a nonhuman primate model HIV-1 Tat causes regional differences in phagocytosis of multiple sclerosis by perivascular macrophages and microglia and causes disruption of the blood–brain barrier Emily Leibovitch, Breanna Caruso, Matthew Schindler, Nick Luciano, Seung-Kwon Ha, Nathanael Lee, Bridgette Billioux, Crystal Leibrand1,JasonParis2, Pamela Knapp3, Woong-Ki Joseph Guy, Xiaozhen Li, Cecil Yen, Pascal Sati, Afonso Kim4, Kurt Hauser2, MaryPeace McRae1 Silva, Daniel Reich, Steven Jacobson (corresponding author: [email protected]) (corresponding author: [email protected]) 1Department of Pharmacotherapy and Outcomes Science, NINDS School of Pharmacy, Virginia Commonwealth University; 2Department of Pharmacology and Toxicology, School of Viruses are implicated as triggers of disease onset/ Medicine, Virginia Commonwealth University; 3Department progression in multiple sclerosis (MS) and similar of Anatomy and Neurobiology, School of Medicine, Virginia neuroinflammatory disorders. Human herpesviruses (HHV) Commonwealth University; 4Department of Microbiology in particular are strongly associated, however the time be- and Molecular Cell Biology, Eastern Virginia Medical School tween viral acquisition and disease presentation complicates the study of such mechanisms. Using a small nonhuman The mechanisms by which HIV infection compromises primate, we demonstrate that prior intranasal (IN) inocula- the blood–brain barrier (BBB) are not completely under- tions with the HHV-6 viruses accelerate an experimental stood. This is important because HIV-infected macro- MS-like neuroinflammatory disease (experimental autoim- phages are thought to cross the BBB and infect resident mune encephalomyelitis (EAE)). In this study, marmosets microglia and astroglia within the CNS. We utilized a (n = 12) received intranasal HHV-6A, 6B or mock inocula- transgenic mouse model that conditionally expresses the tions, and then were induced with EAE 6 months later. Post HIV-1 regulatory protein, transactivator of transcription EAE induction, brain MRIs were performed every 2 weeks (Tat 1–86), in a GFAP-driven, doxycycline-dependent until predetermined clinical endpoints. Animals receiving in- manner, to examine the role of Tat in disrupting BBB tranasal virus alone were asymptomatic, and we observed integrity and triggering activation of perivascular macro- patterns of viral DNA detection that reflect the phages and resident microglia within several brain regions compartment-specific tropism observed in humans. in vivo. Inducible Tat(+) transgenic and control Tat(−) Moreover, in one 6B-inoculated marmoset that met endpoint mice received an intracerebroventricular (ICV) injection criteria before EAE induction, low levels of viral antigen of AlexaFluor 488-labeled dextrans 5 days (long-duration were demonstrated in the brain, suggesting that the olfactory exposure), followed by a second ICV injection of pathway is sufficient for viral entry into the CNS. Following AlexaFluor 594-labeled dextrans 4 h (short-duration EAE induction, virus-inoculated marmosets exhibited re- exposure) prior to sacrifice to assess potential changes in duced time to inflammatory perivascular brain lesions and macrophage/microglial turnover, and an intracardiac in- significantly reduced overall survival compared to uninfect- jection of 44-kDa horseradish peroxidase (HRP) 5 min ed, EAE-induced controls. By intracellular cytokine staining, prior to sacrifice. CNS HRP labeling was significantly levels of the most highly proinflammatory CD8 T cell subset greater in Tat(+) mice in comparison with Tat(−)controls, significantly correlated with post-EAE survival time in the consistent with impairment of BBB integrity. Within virus-inoculated marmosets, suggesting that a (viral?) caudate/putamen there was a significant increase in all antigen-driven expansion occurred in the periphery during labeled (short-, long-, and dual-labeled) perivascular mac- the EAE disease course. By histopathology, viral antigen in rophages and microglia among Tat(+) expressing mice the brain of virus-inoculated EAE marmosets was markedly compared to controls. No significant differences were ob- elevated and concentrated in lesions relative to the surround- served between the number of short- and long-labeled ing parenchyma. The observation of viral antigen localization macrophages within either intra-striatal or perivascular to brain lesions mirrors the findings linking HHV-6 with MS. spaces, indicating no differences in macrophage/ Collectively, we demonstrate that in a nonhuman primate microglial turnover. The number of long-labeled phago- model of MS, asymptomatic intranasal viral acquisition can cytic monocyte-derived cells was significantly increased J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S43 in the caudate/putamen of Tat(+) mice, but significantly viruses but not with R5 viruses. We hypothesize that decreased in Tat(+) mice in cortical regions, including the engagement of CXCR4 in the absence of CD4 could somatosensory cortex, agranular insular cortex, and occur by immature virus since the binding site for piriform cortex, compared to Tat(−) controls. No signifi- CXCR4 might be exposed within the gp120 of the vi- cant differences in phagocytic monocyte-derived cells rus. Before the virus is fully mature, the binding site were observed within nucleus accumbens, anterior cingu- should be available for interaction with CXCR4, thus late cortex, or primary motor cortex. These findings sug- mediating viral entry. These observations conclusively gest that expression of HIV-1 Tat disrupts the BBB and implicate astrocytes as a reservoir for HIV and demon- causes regional differences in the distribution of strate that the mechanism of infection requires immature perivascular macrophages and microglia in vivo. virus.

P88 HIV infection of astrocytes is detected in vivo P89 by RNAscope and occurs in vitro via a non-classical Human Endogenous Retrovirus-K envelope protein mechanism induces neurotoxicity by causing p53-dependent nucleolar stress response Guan-Han Li1, MyoungHwa Lee1, Eugene Major2, Avindra Nath1 Wenxue Li1, Myoung-Hwa Lee1,RichaTyagi1, Muzna (corresponding author: [email protected]) Bachani2, Joseph Steiner2, Avindra Nath1 (corresponding author: [email protected]) 1Section of Infections of the Nervous System, NINDS, NIH; 2Laboratory of Molecular Medicine and Neuroscience, 1Section of Infections of the Nervous System, National NINDS, NIH Institute of Neurological Disorders and Stroke, National Institutes of Health; 2Neurotherapeutics Unit, National Astrocytes are an ideal reservoir for HIV since once Institute of Neurological Disorders and Stroke, National infected the virus can stay dormant and thus reside in- Institutes of Health definitely without perturbing the cell. However, demonstration of HIV-infected astrocytes in vivo poses techni- About 8 % of the human genome is composed of cal challenges. Similarly, infecting the cells in vitro with retrovirus-like sequences called human endogenous retro- cell free virus has been very difficult. Now, we show virus (HERVs), yet their function is poorly understood. that both hurdles have been overcome. In this study, we Recent studies have shown that HERVs may play critical demonstrated unambiguous infection of astrocytes at roles in both pathological and physiological conditions. least in 2 of 6 brain tissues with HIV encephalitis by Our group found that HERV type K (HERV-K) was ac- RNAscope and GFAP co-immunostaining techniques. In tivated in the brain of patients with amyotrophic lateral the in-vitro experiments, we found that HIV infection in sclerosis (ALS). Overexpression of HERV-K envelope primary fetal astrocytes was blocked due to lysosomal protein (Env) caused neurotoxicity in a cell culture sys- degradation after the virus was endocytosed in a non- tem and motor neuron specific degeneration in a trans- CD4 receptor-mediated manner. In fact, we could con- genic mouse model. HERV-K Env caused nucleolar dys- sistently detect low levels of CD4 mRNA in astrocytes function in the transgenic mouse as determined by mi- and small amounts of CD4 protein by immunoprecipita- croarray analysis. Immunostaining showed translocation tion. Interestingly, CD4 mRNA but not protein was sig- of the nucleolar proteins into the cytoplasm. The Env nificantly up-regulated by pro-inflammatory cytokines. has a signal peptide (SP) that gets cleaved and Therefore, increased infection of HIV was not seen in translocated into the nucleolus. In vitro studies show that the cells pre-treated with pro-inflammatory cytokines. SP directly interacts with nucleolar proteins as deter- However, productive infection of astrocytes was consis- mined by immunoprecipitation studies. The binding of tently demonstrated in the transwell cultures loaded with SP to nucleolar proteins may disrupt their interaction HIV-infected lymphocytes. This infection was CD4- with MDM2, the negative regulator of p53 signal path- independent but CXCR4-dependent since it could be way. Indeed, transfection of SP upregulates p53 and its blocked by anti-CXCR4 antibody and AMD3100. We downstream target p21 protein, and causes neuronal screened a panel of laboratory strains and primary iso- death. In conclusion, HERV-K Env may induce neuro- lates of HIV including one isolated from the CSF and toxicity by causing p53-dependent nucleolar stress found that the infection occurred with X4 or R5X4 response. S44 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89

P90 MH108458 to W-KK and also by R01 AI097059 to Phosphorylation of SAMHD1 in Brain Macrophages MJK. Increases with Viral Loads in the Brains of SIV-Infected Rhesus Macaques P91 Allison Lindgren1, Adam Filipowicz1, Marcelo Kuroda2, The hCD59 transgenic rodent model Woong-Ki Kim1 of Intermedilysin-mediated cell ablation (corresponding author: [email protected]) Fengming Liu1,ShenDai1,AlisonKearns1,XiaoPeng1, 1Department of Microbiology and Molecular Cell Biology, Jennifer Gordon1, Sulie L Chang2,XuebinQin1 Eastern Virginia Medical School; 2Division of Immunology, (corresponding author: [email protected]) Tulane National Primate Research Center 1Department of Neuroscience, Center for Neurovirology, SAMHD1 is a host restriction factor that blocks the Lewis Katz School of Medicine at Temple University, replication of HIV-1 and SIV via its depletion of the Philadelphia, PA; 2Institute of NeuroImmune Pharmacology intracellular deoxynucleoside triphosphate (dNTP) pool. and Biological Sciences, Seton Hall University, South Recently, in vitro, it has been shown that phosphoryla- Orange, NJ tion of SAMHD1 (pSAMHD1) in cycling macrophages by cyclin-dependent kinase 2 acts to increase the dNTP Conditional and targeted cell ablation is powerful and pool, thereby increasing susceptibility to HIV-1 infec- widely used for studying specific cellular functions, as tion. We therefore investigated, using our rhesus ma- well as tissue repair and recovery. Current cell ablation caque SIV model, whether the level of SIV infection models have some inherent shortfalls that include of the brain corresponds to the level of pSAMHD1 ex- offäóñtarget effects, narrow pharmacological windows, pression in situ in brain macrophages. Using paraffin- and relatively slow onset of cell death. Thus their thera- embedded brain tissues of uninfected macaques (n =4) peutic applications are limited. Intermedilysin (ILY), a and SIV-infected macaques with (n = 5) or without en- cytolytic pore-forming toxin that is secreted by cephalitis (n = 5), we performed immunohistochemistry Streptococcus intermedius, lyses human cells exclusively to examine the expression of SAMHD1 or pSAMHD1. through the necrotic effect on the cells by binding to the Automated cell counting was performed using ImageJ. human complement regulator CD59 (hCD59). Taking ad- Viral loads from all of the animals and their correspond- vantage of these features, we previously reported a novel ing brain areas were also measured by qPCR and qRT- approach by generating hCD59 transgenic mice and rats. PCR. Phenotypic characterization of these SAMHD1- or Administering ILY these hCD59 transgenic strains could pSAMHD1-expressing cells was undertaken using im- achieve conditional, rapid and targeted cell ablation. munofluorescent microscopy for various markers, in- Recently, we established a Cre-inducible hCD59 transgen- cluding CD68, CD163, Ki-67, and SIV p28. All statis- ic mouse line (ihCD59) where hCD59 expression only tical analyses were carried out using GraphPad Prism 5. occurs after Cre-mediated recombination. Administration Both SAMHD1 and pSAMHD1 showed a significant of ILY to various lines of double transgenic (Cre + difference between uninfected controls and SIV-infected ihCD59+) mice resulted in the rapid and specific ablation animals with encephalitis (P < 0.01), but only of immune, liver, epithelial or neural cells without off- pSAMHD1 showed a significant difference between target effects. Importantly, ILY had a large pharmacolog- SIV-infected animals without encephalitis and animals ical window, which allowed us to perform dose-dependent with encephalitis (P < 0.01). Results from RNA and studies. Most recently, by crossing ihCD59 with endothe- DNA analysis show a strong correlation (r = 0.79) of lial specific Cre transgenic line (Tek-Cre), we have increasing pSMAHD1 with increasing viral loads, and established specific endothelial cell hCD59 expressing a mild correlation (r = 0.66) of increasing SMAHD1 mice (Tek-Cre+/ihCD59+). Injection of ILY at the non- with increasing viral loads. These cells were confirmed lethal doses to the Tek-Cre+/ihCD59+ had specifically to be perivascular macrophages (PVM) based on their severe damage on the brain blood barrier of which endo- cellular marker expression. Our results demonstrate that thelial cells are the key component. Together, ILY/hCD59 SIV infection increases the number of PVM expressing method is an important and versatile tool that can be used pSAMHD1 and Ki-67, suggesting that SAMHD1 phos- to study the effects of cell ablation in any organ system or phorylation during development of encephalitis renders cell type and therefore has universal application and value cycling PVM more susceptible to SIV/HIV infection. to investigators in multiple fields. This approach can be Supported by NIH grants R01 MH107333 and R21 also used to study the functions of individual cell J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S45 populations in multiple disease models including infec- differential protein expression and activation of key tious diseases and to investigate tissue injury and regen- endoplasmic reticulum response markers in astrocyto- eration including alcohol and drug abuse-associated ma cells inducing an anti-apoptotic response, while organ/cell damage & recovery. Gp120 Clade C caused activation of pro-apoptotic mechanisms on astrocytoma cells.

P92 Interclade HIV-1 Gp120 mediates Unfolded Protein P93 Response anti-apoptotic effects on glioma cells Development of a humanized astrocyte/human peripheral blood mononuclear cells NOD/scid-IL-2Rgc Sheila Lopez-Acevedo, Madeline Rodriguez-Valentin, null mouse model to assess the role of astrocytes Gabriel Valentin, Mariela Rivera-Serrano, Luis Cubano, in HIV infection Lilia Kucheryavykh, Nawal Boukli (corresponding author: [email protected]) Victoria Lutgen, Srinivasa Narasipura, Maureen Richards, Lena Al-Harthi Universidad Central del Caribe (corresponding author: [email protected])

Patients infected with HIV are more prone to develop Rush University cancers, including glioblastoma brain cancer (GBM). The median survival of GBM patients with HIV is HIV invades the brain shortly after infection and can significantly smaller compared to HIV-negative GBM become latent in cells such as astrocytes. Given the patients, receiving same treatment. This indicates that inability for invasive studies to examine the role of HIV infection influences GBM tumors’ responsiveness astrocytes in HIV in humans coupled with the inability to treatment. The purpose of this study is to reveal the of HIV to infect non-human astrocytes, we developed a mechanism of action of HIV-1 clades B and C shell novel humanized astrocyte/human peripheral blood proteins Gp120 and their effect on glioma resistance mononuclear cells (PBMCs) NOD/scid-IL-2Rgc null to apoptosis-inducing factors. We hypothesize that (NSG) mouse models using either neonatal pups or HIV-1 clades B and C glycoproteins Gp120, initiate adult mice. Neonatal (PND1) NSG pups were injected differential protein expression in human astrocytoma with normal fetal human astrocytes (NHAs) either un- cells modulating survival abilities of cancer cells. A infected or infected with HIV-GFP (NLENG1-IRES Tandem Mass tag (TMT) isobaric labeling quantitative 70), with or without VSVG pseudotyping. Rate of proteomic approach followed by oxidative stress HIV integration in microinjected astrocytes was at 1– H2DCFDA analysis, autophagy assay by Acridine 3%or20–30 % based on GFP expression from wild- Orange staining, western blot, and apoptosis assays type or VSVG pseudotyped virions, respectively. Pups based on Trypan Blue and Annexin 5/Propidium were weaned at 5 weeks of age and reconstituted with Iodide staining were used on HIV-1 clades B and C huPBMCs at 6 weeks. We found that at 10 weeks of treated U87 human astrocytoma cells. The study re- age, NHAs continue to survive, proliferate, and migrate vealed significant up-regulation of apoptotic, endoplas- throughout many areas of the forebrain and form pro- mic reticulum, oxidative stress, and chaperone- cesses. To circumvent the length of time required for mediated autophagic markers in U87 cells treated with the neonatal studies, we performed these studies in HIV-1 Gp120 for 24 h. Moreover, the study revealed adult (5–6 weeks old) NSG mice. Five days after bi- that HIV-1 Gp120 C and HIV-1 Gp120 B provided lateral microinjection of HIV-infected or uninfected different regulation on survival mechanisms in U87 NHAs into the striatum, mice were reconstituted and cells. HIV-1 Gp120 C induced nitrite release and pro- sacrificed 4 weeks later. NHAs survived and formed duction of reactive oxidative species, causing apopto- processes in adult NSG mice, however, there is less sis and tumor inhibition by an autophagy-induced cell proliferation and little migration at this time point. In death mechanism. HIV-1 Gp120 clade B protein in- both models, we have evidence for widespread HIV duced the expression of key endoplasmic reticulum dissemination from HIV infected astrocytes to blood molecular response and unfolded protein response and other tissues. Each model has its advantages, neo- markers such as: GRP78, Protein Disulfide nates have widespread engraftment whereas adult ex- Isomerase, Calreticulin, eIF2 alpha, and Bcl-2 leading periments have a shorter timeframe. Both models sup- to a protective and anti-apoptotic response in glioma port survival of human astrocytes up to 5 weeks after cells. Our data suggests that Gp120 B induces a injection. These novel models allow for further studies S46 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 to determine the role of astrocytes in brain/periphery P95 dynamics and HIV persistence/latency. Simian varicella virus reactivates in rhesus macaques after depletion of CD4 T cells

P94 Ravi Mahalingam1, Mary Wellish1, Meredith Hunter2, PROGRESSIVE DEFICITS IN SUSTAINED Amy Frieman1, Lindsay Hacker1,DonGilden1,Vicki ATTENTION WITH ADVANCING AGE IN THE HIV-1 Traina-Dorge2 TRANSGENIC (Tg) RAT (corresponding author: [email protected])

Charles F. Mactutus, Kristen A. McLaurin, Rosemarie M. 1Department of Neurology, University of Colorado Denver, Booze Aurora, CO; 2Division of Microbiology, Tulane University, (corresponding author: [email protected]) Tulane National Primate Research Center, Covington, LA

Program in Behavioral Neuroscience, Department of Simian varicella virus (SVV) infection of primates is the Psychology, University of South Carolina counterpart of human varicella zoster virus (VZV) infection. Intrabronchial inoculation of 5 rhesus macaques with Despite the great success of combination antiretroviral 4X 10 5 PFU of SVV resulted in varicella rash 10–14 days therapy (cART) in diminishing the most severe forms of post inoculation (p.i.). Eight months later, 4 monkeys neurocognitive impairment, antiretrovirals alone are not were treated with 50 mg/kg of anti-CD4 antibody. After sufficient to protect the brain as HAND remains as prev- 1 week, the percent of CD4-T cells in blood was reduced alent as before cART, afflicting up to 40–70 % of HIV-1 from 40–60 % to 5–30 % in treated monkeys and infected individuals. Attention and executive function are remained low for rest of the experiment. Seven days after the cognitive domains most affected in HAND. treatment, zoster rash developed in the absence of viremia Asymptomatic neurocognitive impairment conveys a 2- in the monkey with lowest (5 %) level of CD4-T cells. 6X risk for progression to symptomatic HAND. Zoster also developed in all other treated monkeys 28– Fundamental to research progress in this area is a well- 55 days post treatment (d.p.t.) and in the untreated mon- characterized model of neurocognitive deficits that per- key 10 months p.i. Examination of affected skin revealed mits a prospective approach to assess chronic, progres- SVV ORF 63 protein mostly in sweat glands. All mon- sive, impairments across the lifespan. Accordingly, we keys were euthanized 1 week after the time of their re- employed a longitudinal design, with both male and fe- spective zoster, and lymph nodes were analyzed by dual male HIV-1 Tg and Fischer-344 N control rats (back- color immunohistochemistry. SVVORF63 protein was ground strain), to test the hypothesis that with advancing found to be colocalized in macrophages. Our findings age, there will be an HIV-1 viral protein-induced progres- indicate that CD4-T cell immunity is important in SVV sive loss of neurocognitive function as indexed by accu- reactivation. racy of performance in a sustained attention task. Beginning at 60 days of age, sustained attention was assessed with a signal detection task (162 trials/session) P96 that were either reinforced (hit or correct rejection) or SIV Sequences in the Choroid Plexus Represent CNS non-reinforced (miss or false alarm) with sucrose pellets ViralReservoirsinRhesusMacaquesthatDevelopAIDS until a criterion was met (70 % accuracy for 5 consecutive with SIVE or 7 total days). The control rats learned and improved on the task when retested every 60 days, and with this dis- Jaclyn Mallard1, Brittany Rife2, Emily Papazian1, Arianna tributed practice, reached asymptotic criterion of ~6 days Noggle1,DavidJNolan2, Marco Salemi2, Kenneth C by 300 days of age. In contrast, the HIV-1 transgenic rats Williams1 displayed an initial learning impairment, an asymptotic (corresponding author: [email protected]) criterion performance of ~7.5 days at 300 days of age, and deterioration of performance beginning at 360 days 1Department of Biology, Boston College, Chestnut Hill, MA; of age. The neurocognitive impairment was most promi- 2Emerging Pathogens Institute, University of Florida, nent in HIV-1 Tg female rats. The demonstration of pro- Gainesville, FL gressive neurocognitive deficits in the HIV-1 Tg rat offers a translational model for HAND as well as the testing of HIV-associated neurocognitive disorders (HAND) and SIV- novel therapeutic approaches. Funded by NIH grants associated encephalitis (SIVE) are characterized by DA013137, HD043680, & MH106392 monocyte/macrophage (Mo/MΦ) accumulation and virus in J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S47 the CNS. Mo/MΦ traffic is critical for CNS viral reservoirs. T specific treatment. Insulin resistance is also a major complica- cells and Mo/MΦ enter the CNS from cerebrospinal fluid tion of contemporary HIV/AIDS care. Studies have shown (CSF) via the choroid plexus (CP) or via the blood brain that insulin treatment suppressed HIV-1 replication in leuko- barrier. CSF is the only route to sample CNS virus. CNS cytes. Herein, we investigated the actions of insulin using Mo/MΦ s are the predominantly infected CNS cells in ex vivo and in vivo models of HAND. Primary human mi- HAND and SIVE. We hypothesized CP and CSF viral se- croglia (HFM) were infected with HIV-1 and treated quences represent CNS, Mo/MΦ and T cell-derived se- with/without insulin, followed by mRNA analyses. Primary quences, but CP and not CSF virus seed CNS viral reservoirs. human neurons were exposed to viral protein R (Vpr) and We assessed CNS, CP and peripheral (CSF, plasma, T cells treated with/without insulin followed by toxicity assays. In and Mo/MΦ) tissues and cells from 19 SIVmac251-infected an in vivo model of HAND, cats infected with a neurovirulent and 2 uninfected rhesus macaques (11 CD8-depleted, 10 non- feline immunodeficiency virus (FIV) strain, were treated daily depleted). SIV+ animals included: 4 without AIDS, 6 AIDS for 6 weeks with intranasal (IN) insulin or PBS. Increased with SIVE and 9 AIDS without SIVE (SIVnoE). Double-label neuroinflammatory gene expression was observed in brains in situ hybridization and immunohistochemistry was done to from patients with HIV/AIDS. The insulin receptor was de- count SIV-infected T cells and Mo/MΦ in CP. SIVE animals tected on both neurons and glia but its expression was unaf- had higher numbers of Mo/MΦ, not T cells in CP compared to fected by HIV-1 infection. Insulin treatment of HIV-infected SIVnoE animals. Mo/MΦ andTcellsinCPcontainedSIV- HFM suppressed supernatant HIV-1 p24 levels, reduced RNA+ subsets. Phylogenetic analysis of SIV gp120 CXCL10 and IL-6 transcript levels while PPAR-gamma ex- cDNAwasusedtocompareCSF,CPandCNSviruswith pression was induced. Insulin treatment of primary human peripheral virus. CSF viral sequences were dispersed be- neurons prevented HIV-1 Vpräóñmediated cell process re- tween CNS and peripheral sequences. CP and CNS viral traction and death. In FIV-infected (FIV[+]) cats, daily IN sequences clustered together and were highly compart- insulin treatment (20.0 IU/200 μl for 6 weeks) reduced mentalized in SIVE animals, but not in SIVnoE animals. CXCL10, IL-6 and FIV RNA detection in brain although Detection of SIV-RNA+ T cells and Mo/MΦ underscores PPAR-gamma in glia was increased, compared to PBS- the CP as a source of CSF virus. Dispersed phylogeny of treated FIV[+] control animals. These molecular changes CSF viral sequences among peripheral and CNS se- were accompanied by diminished glial activation in cere- quences indicates that CSF is not a viral reservoir. Mo/ bral cortex and white matter of insulin-treated FIV[+] an- MΦ accumulation and compartmentalized CP and CNS imals, with associated preservation of cortical neurons. virus suggests infected Mo/MΦ in these tissues are the Moreover, IN insulin treatment improved neurobehavioral source of CNS viral reservoirs. Increased compartmental- performance including both memory and motor functions ization of CP and CNS virus with SIVE highlights the in FIV[+] animals. Thus, insulin exerted ex vivo and importance of viral reservoirs in SIVE pathogenesis. in vivo antiviral, anti-inflammatory and neuroprotective effects in models of HAND, representing a new therapeutic option for patients with neurodegenerative disorders P97 including HAND. Insulin signaling suppresses virus replication and neuroinflammation in HIV/AIDS. P98 Manmeet Mamik1, Eugene Asahchop1,WingChan1,Yu A CNS IRIS-like phenotype in SIV-infected pigtailed Zhu41 William Branton1, Brienne Mckenzie2,EricCohen3, macaques on cART Christopher Power4 (corresponding author: [email protected]) Lisa Mangus, Sarah Beck, Suzanne Queen, Elizabeth Engle, Joseph Mankowski 1Department of Medicine, University of Alberta; 2Department (corresponding author: [email protected]) of Medical Microbiology & Immunology, University of Alberta; 3Department of Microbiology, Infectiology and Johns Hopkins University Immunology, Universite de Montreal; 4Departments of Medicine and Medical Microbiology & Immunology, Histologic examination of the central nervous system (CNS) University of Alberta of simian immunodeficiency virus (SIV)-infected pigtailed macaques treated with combination antiretroviral therapy HIV-1 infection of the brain results in neuroinflammation and (cART) revealed that animals with persistent low viral load contributes to the development of HIV-associated in plasma and/or CSF (n = 9 macaques) frequently developed neurocognitive disorder (HAND), for which there is no a unique moderate to severe lymphocyte-rich encephalitis that S48 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 was morphologically distinct from SIV-encephalitis (SIVE). modest reductions in excitatory proteins and structures. In CNS lesions in cART-treated macaques consisted of multifo- contrast, Tat markedly decreased synaptotagmin 2 (Syt2) cal perivascular infiltrates of lymphocytes and plasma cells levels, while increasing gephyrin protein levels reflecting a with fewer macrophages and no multinucleated giant cells. disruption in GABAergic transmission within CA1. Further Further, while SIV RNA is abundant in the CNS of untreated investigation of the GABAergic cells within CA1 revealed animals with SIVE, brain viral RNA levels in cART-treated that parvalbumin (PV) positive cells of the pyramidal layer animals with lymphocytic lesions were typically low or unde- (putatively bistratified cells), SST positive cells of the stratum tectable by digital droplet PCR (ddPCR) and in situ hybridi- oriens (putatively O-LM cells), and nNOS positive, NPY neg- zation. Immunophenotyping demonstrated that the infiltrates ative cells of the pyramidal layer and stratum radiatum (IS3s were comprised of a mixture of CD20+ B cells, CD3+ T cells, and neurogliaform cells respectively) were preferentially vul- and CD68+ macrophages, and that approximately 30 % of nerable to Tat. These interneurons form a microcircuit within cells were positive for Ki67, indicating active proliferation. CA1 that has a demonstrated role in spatial memory forma- Western blot analysis of CSF from these animals revealed tion. Electrophysiological analysis of CA1 pyramidal cells intrathecal antibodies targeting multiple SIV proteins. in slice preparations from Tat-expressing or control trans- Immunostaining for Epstein Barr nuclear antigen genic mice treated with morphine or a placebo showed (EBNA) to detect associated gamma-herpesviral infection that Tat increases the resting membrane potential, and was negative; no evidence of other opportunistic infec- increases the amount of current needed to depolarize cells tions (OI) in the CNS or other organ system was found. to their firing threshold. Surprisingly, this was unchanged Taken together, these findings may represent an enhanced in pyramidal cells from Tat and morphine exposed mice. adaptive immune response to residual low level SIV rep- However, numbers of action potentials were reduced if lication or viral protein production in the brain. While the morphine was removed from the recording bath. These morphologic features resemble those reported in HIV pa- results suggest a compensatory upregulation of GABAA tients with CNS immune reconstitution inflammatory syn- receptors (GABAR) in pyramidal cells, which offsets the drome (CNS-IRIS) in the absence of OI, we have not effects of morphine to inhibit mu opiate receptor-express- identified a correlation of the syndrome with CD4 nadir. ing-PV+ basket cells and consistent with the observed In addition, the preponderance of B lymphocytes in the increase in gephyrin. Additional electrophysiological macaque lesions is unique. Further research into the path- studies will utilize GABAR antagonists to determine ogenesis of these lesions may aid our understanding of the whether Tat and morphine reduce spontaneous inhibitory consequences of residual CNS HIV replication in the set- post synaptic currents (iPSCs) as predicted by diminished ting of an intact immune system. interneuronal presynaptic inputs.

P99 P100 Interaction between HIV-1 Tat and morphine causes Sex Differences in HIV Effects on Mechanisms disruption of GABAergic systems within CA1 of Executive Dysfunction Among Drug Users

William D Marks1, Aaron J Barbour2,JasonJParis3, Christina Eileen Martin1, Michael Keutmann2,RaulGonzalez3, Pauline JSchier3, Melissa D Denton3, Sylvia Fitting4,ARory Maki2,LeahRubin2 McQuiston2, Pamela E Knapp2, Kurt F Hauser3 (corresponding author: [email protected]) (corresponding author: [email protected]) 1Department of Psychiatry, Rush University Medical Center; 1Department of Pharmacology and Toxicology; 2Department 2Department of Psychology, University of Illinois-Chicago; of Anatomy and Neurobiology, Virginia Commonwealth 3Department of Psychology, Florida International University University; 3Department of Pharmacology and Toxicology, Virginia Commonwealth University; 4Department of Rationale. Performance on tests of verb (action) fluency (AF) Psychology and Neuroscienc, University of North Carolina is a robust predictor of functional independence and CSF ab- Chapel Hill normalities among persons living with HIV/AIDS. AF performance is strongly correlated with neurocognitive measures of The spatial memory deficits associated with HIV can be traced executive function and is critically dependent on integrity of back to hippocampal dysfunction caused in part by the actions prefrontal-striatal circuitry, but has not been compared sys- of HIV-1 Tat. Previous investigations found that Tat exposure tematically between HIV+ men and women or among sub- significantly decreased LTP in CA1 in HIV transgenic mice; stance dependent individuals (SDIs). We compared AF per- however, the loss in function was accompanied by only formance of HIV+ and HIV- male and female SDIs and J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S49 investigated the relationship between AF and performance on be greater for those with a drug history. HIV+ individ- measures of two different types of executive function with uals with a drug history showed hyper BG activity when known sensitivity to HIV. Methods. We tested 329 HIV- and task demands required them to ignore the reward infor- 148 HIV+ substance dependent individuals (SDIs), primarily mation, indicating reward salience was high. HIV+ indi- users of cocaine and alcohol, enrolled in a larger study of sex viduals without a drug history showed no such BG ac- and HIV serostatus effects on neurocognitive function among tivity. Thus, HIV+ individuals with a drug history may SDIs. The sample was approximately 70 % women and 85 % face the greatest challenge in ignoring reward informa- African American. Subjects were free of AIDS-defining and tion and, consequently, controlling risky behaviors in other neurologic illness or injury and HIV+ subjects were pursuit of that reward. ambulatory and stable on cART. All subjects performed a timed measure of verb fluency requiring speeded retrieval of names of human activities. Subjects also completed P102 two executive function measures of verbal learning strat- Alpha-synuclein restricts West Nile virus in neurons egy and decision making under risk. Subjects were veri- during exocytosis and contributes to neuronal subtype fied abstinent from drugs and alcohol at testing and were susceptibility to infection well matched on demographic, substance use, and comorbid variables. Results. HIV+ men retrieved significantly Aaron Massey1, Katherine Shives2, J David Beckham3 more words than HIV- men and HIV+ women, p =.002. (corresponding author: [email protected]) Decision making was significantly correlated with AF for HIV+ men only, p < .001, but verbal learning strategy cor- 1Department of Medicine, Division of Infectious Diseases, related significantly with AF performance for HIV+ wom- University of Colorado; 2Department of Immunology and en, p = .003. Conclusions. Different types of executive Microbiology, University of Colorado; 3Division of function contribute to verb fluency performance among Infectious Diseases HIV+ men and women, suggesting possible sex differences in brain circuitry affected by neuroAIDS. Introduction: West Nile Virus infection can cause sub- Supported by the National Institute on Drug Abuse. cortical encephalitis with injury to specific regions of the brain including the basal ganglia, substantia nigra, and cerebellum. Given the injury of dopaminergic P101 pathways, patients can present with Parkinsonian Identifying the Neurocognitive Determinants of HIV-Risk symptoms. Alpha-synuclein (Asyn) is known to cause Behaviors Parkinson’s disease but the function of native Asyn expression in neurons is not known. Recent evidence Cherie Marvel1, Brian Anderson2, Jordan Mandel1,Ned suggests that Asyn interacts with Rab proteins and N- Sacktor1 ethylmaleimide-sensitive factor attachment protein re- (corresponding author: [email protected]) ceptor (SNARE) proteins in membranes to alter vesicle transport in neurons. Thus, we determined the role of 1Johns Hopkins University; 2Texas A&M University Asyn in WNV pathogenesis in neurons. Methods & Results: In a primary neurons and a mouse model of HIV-risk behavior is often motivated by the possibility of WNV encephalitis, we have shown that Asyn restricts immediate reward (e.g., unprotected sex). Moreover, re- viral growth and prevents neuronal injury and disease ward stimuli co-opt one’s attention. Our previous study in the brain. To determine the mechanism of Asyn- showed that attention to reward was stronger in HIV+ induced restriction of WNV, primary cortical and individuals with vs. without a drug history. In this pre- striatal neurons from E18 mice were inoculated with liminary follow-up study, we have tested 10 HIV+ indi- mock or WNV (MOI 1) and analyzed using immuno- viduals(5withand5withoutadrughistory),using fluorescence analysis. We found that Asyn localizes fMRI combined with a task that measures the ability to with WNV envelope and Rab1-positive membranes. learn a stimulus-reward association and then, subsequent- WNV-inoculated primary cortical neurons exhibit sig- ly, to ignore that reward when presented in a novel, nificantly higher levels of Asyn co-localization with irrelevant context. The basal ganglia (BG), a key target Rab1 compared to primary striatal neurons. Thus, we for damage by HIV infection, play a central role in reexamined primary cortical neurons for sensitivity to ward processing and addiction. We hypothesized that if WNV infection and injury. Using wild-type and Asyn reward salience was high and difficult to ignore, BG knockout neurons, we found that Asyn expression activity would increase in response. This effect would protected primary cortical neurons from WNV growth S50 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 and injury compared to primary striatal neurons. alterations in molecular expression at both the mRNA Conclusions: Our findings suggest that Asyn- and protein levels. dependent resistance to WNV infection is associated with Rab1-dependent vesicle trafficking and contributes to neuron subtype susceptibility to infection. P104 Lipocalin-2 contributes via a CCR5-dependent mechanism to neuronal degeneration in an in vivo model P103 of HIV-associated brain injury In vitro modeling of blood–brain barrier deregulation by HIV-1 Tat and opioids RickY Maung1,DanielOjeda1, Ana Sanchez1,MarcusKaul2 (corresponding author: [email protected]) Monique Maubert1, Katherine Kercher1,MarianneStrazza1, Vanessa Pirrone1,WeiLin2, Rui Feng2, Brian Wigdahl1, 1Infectious & Inflammatory Disease Center, Sanford Michael Nonnemacher1 Burnham Prebys Medical Discovery Institute, La Jolla, (corresponding author: [email protected]) CA 92037; 2Infectious & Inflammatory Disease Center, Sanford Burnham Prebys Medical Discovery Institute, 1Department of Microbiology and Immunology, Institute for La Jolla, CA 92037, Department of Psychiatry, Molecular Medicine and Infectious Disease, Drexel University of California, San Diego, 9500 Gilman Drive, University College of Medicine; 2Department of San Diego, CA 92093 Biostatistics and Epidemiology, Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania Infection with HIV-1 often triggers neurocognitive im- School of Medicine pairment and degenerative brain injury. Viral proteins, such as the envelope protein gp120, trigger neuroinflam- Many of the pathological observations made in human mation and production of neurotoxins by microglia and immunodeficiency virus type 1 (HIV-1)-associated macrophages. We recently observed in a transgenic neurocognitive disorders (HAND) have been attributed model of HIV-associated brain injury induced by a to compromise of blood–brain barrier (BBB) integrity, CXCR4-utilizing viral envelope gp120 (gp120tg) that and selected viral proteins have been implicated in de- the genetic knockout of CCR5 ameliorated microglial regulation of the BBB, including the transactivator of activation and abrogated neuronal damage and behavior- transcription (Tat). In addition, illicit drug use is a al impairment. A microarray analysis of CNS RNA ex- known comorbidity in exacerbation of disease in HIV- pression showed that brains of CCR5 wild-type (WT) 1-infected individuals. Importantly, opioid abuse within and CCR5KO gp120tg mice express markers of an in- this population confounds disease progression in multi- nate immune response and neuroinflammation. The ple ways, including increased viral replication and pe- acute phase protein lipocalin-2 (LCN2) was one of the ripheral viral load, and enhanced incidence and severity most up-regulated factors. In vitro, LCN2 was itself of neurocognitive impairment including dementia, as neurotoxic in a CCR5-dependent fashion in compared to non-users. Studies have suggested that ex- cerebrocortical cell cultures comprising neurons, astro- posure to both HIV-1 Tat protein and mu-opioids dis- cytes and microglia. As expected, inhibition of CCR5 rupts BBB homeostasis and permeability in primary alone failed to abrogate neurotoxicity of the CXCR4- cells, including an increased pro-inflammatory state, as utilizing gp120, but paradoxically it rescued neurons well as augmented cellular transmigration, and enhanced from gp120 toxicity in combination with LCN2 while barrier leakiness. In this study, a human brain microvas- concomitantly reducing activation of microglial cells, cular endothelial cell line, hCMEC/D3, was utilized to thus recapitulating the finding in CCR5-deficient establish an in vitro model of the BBB to investigate gp120tg brains. Moreover, in vivo, the genetic knockout the effects of Tat or morphine exposure on BBB com- of LCN2 in gp120tg mice protected neurons from promise. Changes in mRNA transcripts of tight junction gp120-induced injury without reducing astrocytosis. proteins (TJP) were observed throughout the course of Altogether, our study provided evidence for a combined exposure. Differences in TJP expression and localization and interdependent contribution of the acute phase pro- were also observed at the protein level following cellu- tein LCN2 and CCR5 to microglial activation and HIV lar fractionation and western immunoblot analysis. gp120-induced brain injury. Supported by grants from These studies demonstrate that exposure to Tat or mor- the NIH, MH087332, MH104131, MH105330 and phine compromises BBB integrity by inducing DA026306 (to M.K.) J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S51

P105 underlying ventricular arrhythmias and sudden cardiac Reduced Resting-State Connectivity death. between the Ventromedial Prefrontal Cortex and Insula Corresponds to Longer QTc Interval in HIV+ and HIV- Individuals P106 Roger McIntosh1, Dominic Chow2,CoreyLum3, fMRI Correlates of Cardioautonomic Melissa Hidalgo1 , Cecilia Shikuma2 ,Kalpana and Endothelial Dysfunction in Postmenopause Kallianpur2 Women with HIV (corresponding author: [email protected]) Roger McIntosh1, Melissa Hidalgo1, Tali Naibryf2,Neha 1Department of Health Psychology, University of Rajan1, Judith Lobo1 Miami; 2Hawaii Center for AIDS, Department of (corresponding author: [email protected]) Medicine, John A. Burns School of Medicine; 3Division of Cardiology, Department of Medicine, 1Department of Psychology, Unversity of Miami; University of Hawaii John A. Burns School of 2Department of Psychology, University of Chicago Medicine Endothelial dysfunction is associated with a predomi- Chronic human immunodeficiency virus (HIV) infec- nance of sympathetic nervous system activity, particu- tion is associated with increased risk of cerebrovascu- larly after menopause. The central nervous system, lar and cardiovascular disease. Prolongation of the QT which is susceptible to HIV infection, has direct influ- interval is a precursor to fatal cardiac arrhythmias ob- ence on parasympathetic and sympathetic balance. served in HIV. Although resting-state functional con- Postmenopause is a period concomitant with endothe- nectivity of neural networks (RSFC) is altered in per- lial, cardioautonomic and functional brain connectivity sons with HIV, the RSFC of brain structures that reg- impairment, however, it is unclear how these markers ulate cardio-autonomic function has not been assessed. are related in HIV. Seven postmenopausal women on This study examined the relationship between heart stable antiretroviral therapy and no history of coronary rate-corrected QT interval (QTc) and RSFC of the left artery disease performed a deep breathing test, from and right ventromedial prefrontal cortex (VMPFC). which E:I ratio was calculated from largest RR inter- Eighteen HIV+ patients on stable antiretroviral therapy val, during expiration, and smallest RR interval, during [median age = 52 years; median CD4 count = 457 cells/ inspiration. Blood was drawn to assess the proportion μl; 14 with plasma HIV RNA <50 copies/ml; median of classical, intermediate, and non-classical peripheral QTc = 412 msec] and no history of coronary artery dis- blood mononuclear cells along with colony-forming ca- ease were compared to 26 HIV-negative participants pacity of endothelial progenitor cells (EPCs), i.e., pro- with similar demographic and cardio-metabolic vari- portion of the number of tunneling nanotubules to EPC ables. Contrasts of seed-based RSFC of the left and colonies after 7 and 14 days. A 7 min resting state right VMPFC revealed lower anterior-posterior connec- brain scan was also recorded on a subsequent visit. tivity in patients than in comparison subjects. Whole- There was a significant (p < .001) positive correlation brain regression analyses of RSFC of the VMPFC between E:I ratio and EPC colony forming capacity seeds showed that longer QTc interval was associated at 7 days; a negative (p < .001) correlation between with reduced RSFC between (a) left VMPFC and both duration of HIV infection and colony forming capacity the left insula and right dorsal cingulate cortex at 14 days; and a trending (p = .089) negative correla- (p < 0.001), and (b) right VMPFC and both left insula tion between the proportion of intermediate (CD14++ and left dorsal cingulate (p < 0.001). After controlling CD16+) mononuclear cells and EPC colony forming for age, longer QTc interval corresponded to decreased capacity at 7 days. The E:I ratio was then regressed RSFC between the left VMPFC and (a) right cingulate on a map of brain areas showing functional connectiv- (R = −0.33, p < 0.05) and (b) left insula (R = −0.40, ity with the left anterior insula - a structure commonly p < 0.01). Reduced right VMPFC connectivity with the activated during parasympathetic response. Greater left insula was also associated with longer QTc connectivity between the left anterior insula and two (R = −0.37, p < 0.05), and for HIV+ individuals, was structures implicated in the generation of sympathetic linked to decreased CD4 count (R =0.45, p =0.06). response, i.e., right posterior insula and cingulate were RSFC appears sensitive to clinical markers of cardio- found in patients with higher E:I ratios. This study autonomic function and may shed light on mechanisms provides preliminary evidence for a neurogenic model S52 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 of cardioautonomic and endothelial dysfunction in HAND. Supported by NIH grants R01 DA018633, postmenopause women with chronic HIV infection. K02 DA027374, K99 DA039791, and T32 DA007027.

P108 P107 INTACT SENSORY AND MOTOR SYSTEM Alterations in hippocampal CA3 synaptodendritic FUNCTION THROUGH ADVANCING AGE IN THE structure in the HIV-1 Tat transgenic mouse HIV-1 TRANSGENIC RAT

Virginia McLane, William Marks, Pamela Knapp, Kurt Kristen McLaurin, Rosemarie Booze, Charles Mactutus Hauser (corresponding author: [email protected]) (corresponding author: [email protected]) Program in Behavioral Neuroscience, Department of Virginia Commonwealth University Psychology, University of South Carolina

Nearly one-third of human immunodeficiency virus Understanding the progression of HIV-1 associated (HIV)-1 patients develop HIV-associated neurocognitive disorders (HAND) is a critical need as neurocognitive disorders (HAND). HIV-1 rapidly in- the prevalence of HIV-1 in older individuals fects the central nervous system (CNS), where neuro- (>55 years) is markedly increasing due to the advent toxic HIV-1 proteins such as transactivator of transcrip- of combination antiretroviral therapy (cART). tion (Tat) are expressed by infected glia. Tat exerts Longitudinal experimental designs, used to assess age- direct and indirect toxic effects on sensitive neurons related disease progressioninHIV-1,however,arede- such as those in the hippocampus, a region critical pendent upon intact sensory and motor system function for learning and memory. Previously, we observed re- through advancing age. Three experimental paradigms duced apical dendritic spines and disrupted synaptic were employed to assess the integrity of the sensory integrity in pyramidal CA1 neurons in an inducible andmotorsystemsintheagingHIV-1transgenic(Tg) HIV-1 tat transgenic mouse model. This dendritic pa- rat. The HIV-1 Tg rat, which resembles HIV-1 seropos- thology correlated to deficits in Barnes Maze perfor- itive individuals on cART, expresses 7 of the 9 HIV-1 mance and alterations in CA1 interneuron microcircuit- genes, is non-infectious, and has been promoted for ry. In the current work, we investigated the effect of investigating the effect of long-term HIV-1 viral pro- Tat expression on pyramidal neuron dendrite structure tein exposure on neurocognitive deficits, as in HAND. and total volume in the CA3 region of the hippocam- HIV-1 Tg and control animals were assessed at ad- pus. Male mice (60–80 days old) expressing GFAP- vancing ages (i.e., ≥ 6 months of age) in all experi- driven reverse tetracycline transcriptase activator mental paradigms. First, the integrity of the auditory, (rtTA) + and Tat transgenes received 14 days of doxy- visual, and tactile sensory systems was assessed using cycline chow (6 mg/g). GFAP-rtTA+, Tat- mice served cross-modal prepulse inhibition (PPI). HIV-1 Tg and as controls. Tat expression correlated to a 27 % reduc- control animals exhibited robust inhibition of the audi- tion in dendritic spine density in the Golgi-impregnated tory startle response to all sensory modalities, indicat- neurons of Tat + mice as compared to Tat- controls ing the ability to detect stimuli. Second, gustatory sys- (9.13 ± 0.53 vs. 12.50 ± 0.91 spines/10 μm, p = 0.012). tem function was assessed using a sucrose taste pref- Using stereological methods, we observed no signifi- erence test, controlling for bottle position and test day cant change in the volume of four major hippocampal using a Latin-square experimental design. As sucrose subregions: CA1 (Tat- 10.39 ± 0.47 mm^3 vs. Tat + concentration increased, a linear increase in mean con- 10.16 ± 0.77 mm^3), CA2 (Tat- 1.24 ± 0.12 mm^3 vs. sumption was observed regardless of genotype. Third, Tat + 1.43 ± 0.06 mm^3), CA3 (Tat- 7.35 ± 0.33 mm^3 motor system function was assessed using locomotor vs. Tat + 7.57 ± 0.47 mm^3), and the dentate gyrus activity. Through advancing age, HIV-1 Tg animals (Tat- 6.72 ± 0.41 mm^3 vs. Tat + 6.57 ± 0.33 mm^3). displayed significantly greater ambulatory activity rela- Overall, this work suggests that Tat induction causes tive to control animals, suggesting no motor system synaptodendritic injury and subtle alterations in hippo- impairment. Thus, the present studies provide strong campal network connectivity, rather than gross neuro- evidence for intact sensory and motor system function nal losses, in area CA3. The findings also suggest Tat through advancing age in the HIV-1 Tg rat indicating mediates key aspects of the pathology cause by HIV-1 the utility of the HIV-1 Tg rat for longitudinal studies that underlie the cognitive deficits associated with investigating the progression of neurocognitive J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S53 impairment in HAND. Funded by NIH grants morphological alterations in vitro. Overall, these analyses DA013137, HD043680, and MH106392 seek to determine a correlation between GDS and the prevalence of Tat variants in asymptomatic or symptomatic neurocognitively impaired patients, which may prove use- P109 ful characterizing HIV-1 neuropathogenesis. HIV-1 Tat variation correlates with neurocognitive impairment in the Drexel Medicine CARES cohort P110 Anthony Mele1,GregoryAntell2, William Dampier1, Vanessa Higher hypocretin (orexin) levels correlates with better Pirrone1, Benjamas Aiamkitsumrit1, Jean Williams1,Shendra motor skills in HIV+ women Passic1, Katie Kercher1, Wen Zhong1, Zsofia Szep3, Jeffrey Jacobson4, Brian Wigdahl1, Uri Hershberg2, Michael Raissa Menendez-Delmestre1, Claribel Gonzalez1,Rosa Nonnemacher1 Rodrí_guez-Benitez2, Miriam Matos1,RichardNoel3, (corresponding author: [email protected]) Valerie Wojna1 (corresponding author: [email protected]) 1Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease, Drexel 1University of Puerto Rico, Medical Sciences Campus; University College of Medicine; 2School of Biomedical 2University of Puerto Rico, Rio Piedras Campus; 3Ponce Engineering, Science and Health Systems, Drexel Health Sciences University University; 3Division of Infectious Disease and HIV Medicine, Department of Medicine, Drexel University The hypocretin (orexin) system is associated with multiple College of Medicine; 4Department of Medicine, Section of brain functions including alertness and central motor con- Infectious Disease, Lewis Katz School of Medicine, Temple trol due to the many projections of the hypocretin-secreting University neurons. These neurons are located specifically in the lateral hypothalamus, an area known to be dysfunctional in In recent years, HIV-1 mortality has decreased; however, HIV-positive individuals suffering from HIV-associated the prevalence of neurocognitive impairment has in- neurocognitive disorders (HAND). HAND is characterized creased. Previous studies have examined the HIV-1 protein by cognitive, behavioral and motor dysfunctions. In this Tat and demonstrated that expression continues even in study we investigated the correlation of hypocretin-1 patients currently on antiretroviral therapy (ART) with (hcrt-1) levels with cognitive performance in HIV- low-to-undetectable viral titers. Tat has also been shown positive women. In a retrospective study, we measured to be neurotoxic and a causative agent of central nervous serum and CSF hcrt-1 levels of HIV-seropositive women system (CNS) inflammation. The current studies seek to without a history of drug abuse evaluated for identify and characterize the predominant variations within neurocognitive performance. Cognitive performance was HIV-1 Tat correlated with the neurocognitive impairment. determined using the HAND criteria where eight cognitive HIV-1 Tat sequences were obtained from the Drexel domains were evaluated. Serum and CSF hcrt-1 levels Medicine CNS AIDS Research and Eradication Study were determined using the fluorescent immunoassay kit (CARES) Cohort. The sequences were amplified from pe- (Phoenix Pharmaceuticals), with an intra- and inter-assay ripheral blood mononuclear cells, translated, and aligned to validity of 10 and 15 % respectively. Non-Parametric sta- the HIV-1 subtype B consensus reference genome to com- tisticswereusedtodeterminecorrelationwithapvalue pare amino acid substitutions. Multiple positional hotspots less than 0.05 to determine significance. A positive corre- of high variation in Tat were identified within the cysteine- lation was found between Hcrt-1 levels and age in the HIV- rich domain, glutamate-rich domain, and second exon. seropositive group with normal cognition (Serum: Statistical analyses were applied to amino acid positions p = 0.020; CSF: p = 0.026). Serum and CSF hcrt-1 levels and variants associated with the neurocognitive impair- were positively correlated (p = 0.022). After adjusting for ment status of each patient. To assess the functional impli- age, a significantly positive correlation was observed be- cations of these results, ten patients, five asymptomatic and tween CSF hcrt-1 levels and better performance in the mo- five symptomatic for neurocognitive impairment, were se- tor skills domain (p = 0.0016), mainly due to the effect of lected based on their Global Deficit Score (GDS). Tat exon the grooved pegboard test when performed with the non- fractions were quantified based on the frequency in each dominant hand (CSF: p = 0.008). This was also true for the patient. The predominant Tat exon 1 and 2 sequences were CSF/serum hcrt-1 ratio (motor skills domain: p =0.047; reconstructed into expression vectors. These will be uti- grooved pegboard non-dominant: p = 0.024). No other cor- lized to identify and characterize functional and relations were observed between hcrt-1 levels and other S54 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 cognitive domains or individual neuropsychological tests. accuracy was 90 % for HAND and 82 % for MCI. This work These results suggest that the hcrt system may have a role has clinical relevance as the HIV-infected population ages and in the motor skill performance of HIV-positive women. has increased vulnerability to neurodegenerative diseases. Further investigations need to be conducted to determine Recognizing these distinct profiles might be useful in a clini- the role of the hcrt system in cognitive performance of cal setting in identifying HIV-infected patients whose pattern HIV-positive individuals. of impairment is not typical of HAND and who may require further evaluation or monitoring.

P111 Discriminant analysis of neuropsychological testing P112 differentiates HIV-associated Neurocognitive Disorder Neuropsychological Phenotypes Among Men with and (HAND) from Mild Cognitive Impairment (MCI) due Without HIV Disease in the Multicenter AIDS Cohort to Alzheimer’s disease in HIV over age 60 Study

Benedetta Milanini1, Isabel Allen2, Shireen Javandel1,Joanna Samantha A. Molsberry1, Yu Cheng2,LawrenceKingsley3, Hellmuth1,RobertPaul3, Victor Valcour1 Lisa Jacobson4, Andrew Levine5, Eileen Martin6, Cynthia (corresponding author: [email protected]) Munro7, Ann Ragin8,NedSacktor9,JamesT.Becker10,For the Neuropsychology Working Group of the MACS 1Memory and Aging Center, Department of Neurology, (corresponding author: [email protected]) University of California San Francisco, San Francisco; 2Department of Epidemiology & Biostatistics, University of 1Population Health Sciences Program, Harvard University California San Francisco, San Francisco; 3Missouri Institute Graduate School of Arts and Sciences; 2Department of of Mental Health, University of Missouri, St. Louis Statistics, University of Pittsburgh; 3Departments of Infectious Diseases and Microbiology and Epidemiology, Differentiating HIV-associated neurocognitive disorder University of Pittsburgh Graduate School of Public Health; (HAND) from Alzheimer’s disease (AD) in older HIV- 4Department of Epidemiology, Johns Hopkins Bloomberg infected individuals has become a major clinical challenge. School of Public Health; 5Department of Neurology, Here we hypothesized that the clinical expressions of University of California Los Angeles; 6Department of HAND and Mild Cognitive Impairment (MCI) due to AD Psychiatry, Rush University School of Medicine; are characterized by distinct neuropsychological profiles. All 7Department of Psychiatry, Johns Hopkins University participants underwent a comprehensive neuropsychological School of Medicine; 8Department of Radiology, battery. We performed a discriminant function analysis includ- Northwestern University Feinberg School of Medicine; ing a set of cognitive measures and demographic factors (i.e., 9Department of Neurology, Johns Hopkins University age, gender, education and premorbid functioning) to identify School of Medicine; 10Departments of Psychiatry, the best model to differentiate HAND from MCI. Logistic Neurology, and Psychology, University of Pittsburgh regression analyses were performed to identify tests associated with group membership; only these tests were included Background: Since the beginning of the HIV epidemic, it when collinearity was noted. The sample included 95 individ- has been clear that HIV infection affects brain function. uals with HAND (79 % cognitively symptomatic according to Prior to the development of combination anti-retroviral Frascati criteria) with an average age of 65 (5), average edu- therapies, HIV infected individuals performed poorly on cation of 15 (2.2) and CD4 T-cell count of 545 (245.4), and 72 tests of cognition and often became severely cognitively non-HIV individuals with MCI with an average age of 69 impaired. Since the development of these therapies, severe (10.5) and education of 18 (3.3). Most of the HIV-infected impairment is rare but milder impairment remains com- individuals were on antiretroviral therapy (97 %) and were mon. The objective of this study was to identify virologically suppressed (83 %). Results revealed a cognitive participant-based latent structure of cognitive function phenotype of HAND characterized by significantly higher using cluster analysis to identify subgroups within the discrimination index (difference between hits and false posi- Multicenter AIDS Cohort Study and to build a model to tives on the California Verbal Learning Test), better perfor- predict cluster membership. Methods: We conducted k- mance on the Digit Backward, as well as worse performance means cluster analysis using MACS participantsäó» neu- on the Mini Mental State Exam, Trails A, Modified Trails, and ropsychological domain scores from the time of their first Digit Symbol (p’s < 0.05). Our model correctly classified cognitive classification. Exploratory analyses were used to 86 % of the cases using this combination of measures identify factors associated with cluster membership and (Wilks’ Lambda = 0.54, p < 0.001). The classification stepwise regression was performed to select variables for J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S55 a multinomial logistic regression model to predict cluster neuropsychological composite score (NPZ-7). Secondary membership. Results: The k = 4 solution was selected, gen- endpoints included specific cognitive measures and domains, erating clusters characterized as follows: cluster 1 per- as well as functional outcomes (daily functioning, emotional formed poorly for every domain, cluster 2 performed and quality of life variables). Safety analysis covered frequen- above average on learning and memory and below average cy of adverse events and changes in laboratory test results. for all other domains; cluster 3 performed above average Results: Seventy-six subjects were screened and 29 finally for executive function, speed, and working memory, and recruited. All groups showed increased cognitive out- below average on all other domains; and cluster 4 per- comes, although they failed to reach significant change. formed highly for every domain except learning. After The rivastigmine group showed the greatest trend (mean conducting exploratory analyses and stepwise regression, NPZ-7 [SD], baseline vs wk 48): rivastigmine: −0.47 the variables retained for the final model were age, test (0.22) vs −0.11 (0.29), p = 0.06; lithium: −0.50 (0.40) vs language, CESD score, race, and ever having syphilis. −0.26 (0.21), p = 0.22; control: −0.52 (0.34) vs −0.32 When tested on a separate subset of the data, the model (0.52), p = 0.44. Regarding the primary efficacy endpoint, predicted cluster membership poorly. Conclusions: There there were no differences among groups (mean NPZ-7 is an identifiable pattern of neuropsychological domain change [SD]): 0.35 (0.14), 0.25 (0.40), 0.20 (0.44); scores among MACS members but we were unable to de- p = 0.78. No significant changes were also observed with velop a model that accurately predicts cluster membership. regard to secondary outcomes. Severe adverse events Neither HIV serostatus nor HIV-related biomarkers were were not recorded and blood tests revealed no relevant related to cluster membership, which is consistent with changes in blood count, biochemistry or immunology. other recent findings that patterns of neuropsychological Conclusions: In this small randomized trial, transdermal test performance do not map directly onto HIV serostatus. rivastigmine was safe and well-tolerated in HIV-infected patients on stable antiretroviral therapy after a 48-week follow-up, although no significant benefits were observed P113 in cognitive status or functional parameters. A larger ran- Transdermal Rivastigmine as Adjuvant Therapy domized trial should contrast these findings. for HIV-Associated Neurocognitive Disorders

Jose A. Munoz-Moreno1, Anna Prats1, Nuria Perez-Alvarez1, P114 Jose Molto1, Maite Garolera2, Crisanto Diez-Quevedo3, Accuracy of the NEU Screen to Detect Cognitive Cristina Miranda1, Carmina R. Fumaz1, Maria J. Ferrer1, Impairment in Older People with HIV Bonaventura Clotet4 (corresponding author: [email protected]) Jose A. Munoz-Moreno1, Estela Lopez-Masramon1, Anna Prats1,NuriaPerez-Alvarez1, Maite Garolera2,MariaJ. 1Lluita Contra la SIDA Foundation - Germans Trias i Pujol Ferrer1,BonaventuraClotet3 University Hospital; 2Consorci Sanitari Terrassa Hospital; (corresponding author: [email protected]) 3Germans Trias i Pujol University Hospital; 4Lluita Contra la SIDA Foundation - Germans Trias i Pujol University 1Lluita Contra la SIDA Foundation - Germans Trias i Pujol Hospital - IrsiCaixa Foundation - Universitat de Vic University Hospital; 2Consorci Sanitari Terrassa Hospital; 3Lluita Contra la SIDA Foundation - Germans Trias i Pujol Background: Adjuvant therapies could serve as supportive University Hospital - IrsiCaixa Foundation - Universitat de treatments for improvement of HIV-associated neurocognitive Vic disruption. To date, no studies have been reported investigating the effects of transdermal rivastigmine in people with HIV Background: The NEU Screen is a proposed tool for practical infection and cognitive impairment. We developed a random- detection of HIV-associated cognitive impairment in the clin- ized controlled pilot trial to study the safety and efficacy of ical setting. This method includes 3 paper-based neuropsycho- transdermal rivastigmine as potential adjuvant therapy for logical measures and has an expected administration time of HIV-associated neurocognitive disorders. Methods: We en- ≤10 min. We investigated its precision in a sample of aging rolled HIV-infected patients with cognitive impairment on HIV population. Methods: A total of 368 HIV-infected people stable antiretroviral therapy in a 48-week follow-up, random- with available neurocognitive data were studied. All subjects ized, controlled, pilot trial. Participants received transdermal had undergone a comprehensive neuropsychological battery rivastigmine (9.5 mg daily), lithium (400 mg twice daily, ti- (2–3 h, 7 domains) and had accessible demographic and clin- trated progressively), or did not start a new treatment (control ical information. Presence of cognitive impairment was group). The primary efficacy endpoint was change in a established as gold standard, and the accuracy of the NEU S56 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89

Screen was compared according to several age cutoffs (spe- Among ART-experienced HIV-1-infected adults, cerebrospi- cifically 30, 40, 50 and 60 years). Sensitivity, specificity, and nal fluid (CSF) viral escape has been associated with neuro- positive and negative predictive values (PPV, NPV) were cal- inflammation, drug resistance-associated mutations (DRMs), culated. Results: Subjects were mostly men (80 %), with a and neurological symptoms. In a retrospective case-series median (interquartile range) age of 43 (37; 48) years old, from Boston hospitals and the National NeuroAIDS Tissue undetectable plasma viral load (70 %), median CD4 cell Consortium, we identified 41 HIV-1-infected patients on count of 511 (375; 720) cells/μL,andmediannadirCD4 ARTwith discordant CSF and plasma HIV-1 RNA levels from cell count of 223 (101; 360) cells/μL. Frequency of cog- 2005 to 2016. Discordance was defined as detectable CSF nitive impairment was 51 %, with a comorbidity rate of HIV-1 RNA when plasma levels were <50 copies/ml, or 39 %. When age cutoffs were compared, both sensitivity HIV-1 RNA >0.5-log higher in CSF than plasma. The estimat- and specificity appeared to be greater as the age was ed prevalence of CSF viral escape was 4.7 %; median baseline higher, starting at 75.43 % and 73.75 %, respectively. age was 50, and duration of HIV-1 infection was 17 years The highest accuracy was found at the 60 years cutoff (IQR 13–22). Median CD4 count and nadir were 329 cells/ (≥60 vs <60 years): sensitivity: 90.91 % vs 74.01 %; mm^3 (IQR 217–388), and 21 cells/mm^3 (IQR 2–60), re- specificity: 92.31 % vs 71.26 %; PPV: 90.91 % vs spectively; 76 % were on boosted-protease inhibitors. 73.18 %; NPV: 92.31 % vs 72.12 %. This was confirmed Neurological symptoms were present in 29 cases; 3 cases by a significantly greater correct classification at the same had a concurrent diagnosis of PML or lymphoma. The cohort cutoff (91.67 % vs 72.67 %; p = 0.022). Conclusions: The was classified into subgroups based on plasma HIV RNA NEU Screen offers a remarkable good sensitivity and levels in the preceding 24 months: high-level viremia (HLV: specificity to detect cognitive impairment in people with >1000 copies/ml), low-level viremia (LLV: 51–1000 HIV, particularly those aged 60 years or older. We pro- copies/ml), and plasma suppression with CSF blip or escape pose the use of this brief and feasible method to help in (CSF HIV-1 RNA ≤200 or >200 copies/ml, respectively). the screening of HIV-associated neurocognitive disorders, Subjects classified as HLV reported more substance considering specially its benefits in the elderly HIV abuse, while those classified as LLV or plasma suppres- population. sion were more frequently neurosymptomatic (81 % vs. 53 %). The majority of DRMs in Boston cases and published reports were detected in both plasma and CSF; P115 M184V/I mutations were more frequent in CSF than plas- Cerebrospinal Fluid Viral Escape in ART-experienced ma. Characteristics frequently observed in HIV-1-infected HIV-1-Infected Adults individuals with CSF viral escape include HIV-1 infection >15 years, low CD4 nadir, boosted-protease inhibitor use, Shibani Mukerji1,2,VikasMisra2,DavidLorenz3,Anna and LLV in the preceding 6 months. M184V/I genotype is Cervantes-Arslanian4, Jennifer Lyons5, Spyridon Chalkias6, another factor that may predispose to CSF escape. Alysse Wurcel7, Deirdre Burke7, Nagagopal Venna2,Susan Classification of CSF escape subtypes based on longitu- Morgello8, Igor Koralnik9, Dana Gabuzda1 dinal plasma HIV RNA levels provides a conceptual (corresponding author: [email protected]) framework that will enable better understanding of viral andhostfactorsleadingtoCSFviralescape. 1Department of Cancer Immunology and Virology, Dana- Farber Cancer Institute, Boston, MA; 2Department of Neurology, Massachusetts General Hospital, Boston, MA; P116 3Department of Cancer Immunology and Virology, Dana- Getting in to the Brain: Potential of Nanotechnology Farber Cancer Institute, Boston, MA; 4Departments of to Manage Neuro-AIDS and Drug Addictions Neurology and Neurosurgery, Boston Medical Center, Boston, MA; 5Department of Neurology, Brigham and Madhavan Nair Womens Hospital, Boston, MA; 6 Division of (corresponding author: [email protected]) NeuroImmunology, Beth Israel Deaconess Medical Center, Boston, MA; 7Department of Geographic Medicine and Department of Immunology- Herbert Wertheim College of Infectious Diseases, Tufts Medical Center, Boston, MA; Medicine- Florida International University 8Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY; 9 Division of 2014 report suggests that more than 36.9 million people are NeuroImmunology, Beth Israel Deaconess Medical Center, living with HIV/AIDS in the world today that includes more Boston, MA; Department of Neurological Sciences, Rush than 1.2 million people from US. Current studies also show University Medical Center, Chicago, IL that more than 247 million people are affected with substance J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S57 abuse in the world that includes more than 24 million reconstitution, animals were sacrificed. HIV-1 was measured Americans. Reports also show that more than 3–4 million in blood, spleen and lymph nodes using Taqman realtime PCR people are co-affected with HIV and illicit drug use. and GFP detection by microscopy or flow cytometry. We Although highly active anti-retroviral therapy (HAART) has found HIV in all of the tissues tested in about 80 % of the resulted in remarkable decline in the morbidity and mortality infected animals. Viral outgrowth assays of the huPBMCs in AIDS patients, inadequate delivery of HIV drugs across the isolated from spleen demonstrated replication compe- blood–brain barrier (BBB) to the brain results in HIV persistence of the virus. Injection of free virus into the brain tence. Drugs of abuse such as opiates act synergistically with did not lead to HIV detection in or outside of the brain. HIV-1 to potentiate the HIV-related neurotoxicity that leads to These data demonstrate that HIV-1 infected astrocytes development of Neuro-AIDS. In recent years, use of nano- are capable of harboring replication competent virus technology has shown exciting prospect for development in vivo that can egress from the brain to seed the periph- of novel drug delivery systems. We herein report the de- ery. Given that astrocytes make up ~60 % of the brain velopment of a Magneto-Electric Nanocarrier (MEN) to cells and rate of infection is around 1–2%,astrocytes deliver and release on demand of HIV drugs and opiate can be a significant reservoir for HIV that is not com- antagonist which are otherwise impenetrable to brain and partmentalized in the brain but can disseminate to other inhibit HIV and reverse opiate mediated adverse neurolog- tissues. ical effects. The proposed nanocarrier is anticipated to simultaneously reduce Neuro-AIDS and opiate addiction in HIV-1 infected opiate addicts. Further, this invented/ P118 patented new technology will have universal applicability Brain delivery of the glutamine antagonist for targeting and controlled release of drugs against a 6-Diazo-5-oxo-L-norleucine (DON) via prodrug variety of other CNS diseases such as Parkinson’s, approach: a potential treatment for HIV-associated Alzheimer’s, brain tumors etc. neurocognitive disorders (HAND)

Michael Nedelcovych1,Boe-HyunKim2, Rana Rais1, Andrej P117 Jancarik3,LukasTenora3,JesseAlt4, Jennifer Kelschenbach5, HIV-1 infected astrocytes support HIV dissemination Pavel Majer3,DavidVolsky5, Barbara Slusher6 into blood and other tissues (corresponding author: [email protected])

Srinivas Narasipura, Victoria Lutgen, Maureen Richards, 1Department of Neurology, Johns Hopkins Drug Discovery, Lena Al-Harthi Johns Hopkins School of Medicine; 2Department of (corresponding author: [email protected]) Medicine, Icahn School of Medicine at Mount Sinai; 3Institute of Organic Chemistry and Biochemistry, Academy Rush University of Sciences of the Czech Republic v.v.i.; 4Johns Hopkins Drug Discovery, Johns Hopkins School of Medicine; 5Department Ample evidence demonstrates that HIV infects the brain dur- of Medicine, Icahn School of Medicine at Mount Sinai; ing acute stage, setting the stage for inflammatory responses, 6Departments of Neurology, Psychiatry, Neuroscience, and leading to HIV-Associated Neurocognitive Disorders. The Medicine, Johns Hopkins Drug Discovery, Johns Hopkins brain is a reservoir for HIV, yet whether this reservoir is com- School of Medicine partmentalized or HIV can egress from it to re-seed the periphery is not clear. We evaluated here the role of HIV-1 in- Recent studies revealed that cART-treated patients with HIV- fected astrocytes in disseminating virus to other tissues out- associated neurocognitive disorders (HAND) have increased side of the brain. We used a chimeric humanized astrocyte/ levels of cerebrospinal fluid (CSF) glutamate compared to human peripheral blood mononuclear cells (hu-astro/PBMCs) those without neurocognitive impairment, suggesting that al- NSG mice to address this question. Primary normal human terations in glutamate homeostasis may contribute to HAND astrocytes (NHA) were infected in vitro with full infectious pathogenesis. The glutamine antagonist 6-Diazo-5-oxo-L- HIV with GFP under the control of IRES. Rate of infection norleucine (DON) attenuates glutamate release from activated was 20–30 % when pseudotyped with VSVG or 1–3%when microglia/macrophages exposed to HIV, and reverses cogni- non-pseudotyped. Neonate and adult NSG mice were injected tive deficits in preclinical models of CNS viral infection, but with HIV-1 infected NHAs or free virion. Astrocyte engraft- has not yet been tested in a model of HAND. Furthermore, the ment and survival was assessed using flurescent microscopy. clinical utility of DON is limited by GI toxicity. We thus tested Six weeks (neonatal) or 5 days (adult) post engraftment, mice DON in a murine model of HAND and subsequently rational- were reconstituted with huPBMCs and 4 weeks post ized a targeted DON prodrug design to enhance its brain/ S58 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 plasma ratio, limiting systemic exposure and GI toxicity. effects meta-analysis was used to estimate individual study DON was evaluated in the EcoHIV-infected murine model standardized mean differences along with between and within of HAND. Cognitive performance in radial arm water maze study sources of variation. Next, cART effects were studied (RAWM) was measured in mice administered DON (1 mg/kg, with DTI in a separate sample of HIV infected participants and i.p., q.o.d) beginning before (prevention) or after (treatment) controls studied before, and 3 and 6 months after beginning EcoHIV inoculation. CSF glutamate was measured in the treatment. Results: Meta-analysis revealed standardized mean treatment paradigm. To enhance DON’s therapeutic index differences of −0.56 for FA (p <0.01)and0.21(p = 0.06) for for HAND, we then utilized a dual promoeity prodrug strategy MD. Nevertheless, estimates of between study heteroge- to increase lipophilicity and brain delivery. DON prevented neity showed that 62–76 % of the observed variance was and reversed cognitive impairment as measured by RAWM between studies. In a separate longitudinal sample that performance. DON also normalized EcoHIV-induced in- showed some evidence of mild neuropsychological im- creases in CSF glutamate. However, DON-treated mice suf- pairment, but no evidence of AIDS dementia, pre- fered systemic side effects such as lethargy and weight treatment FA was higher and MD lower in HIV infected loss. In an attempt to synthesize DON prodrugs with en- participants than controls (p < .001), and measures hanced brain/plasma ratio and improved tolerability, alkyl remained stable over 6 months. Conclusions: Published esters were added to DON’s carboxylate functionality studies of white-matter microstructure following HIV in- with additional masking of DON’s amine group by fection reveal substantial between study variations that N-(acyloxyalkoxycarbonyl) derivatives. Dual promoiety could result from image acquisition, image processing or DON prodrugs were metabolically stable in human, mon- cohort selection differences. Low temporal variation in FA key, and pig plasma and provided >10 fold enhancement and MD in our longitudinal cohort demonstrates repro- in CSF/plasma ratio after in vivo administration in mon- ducibility of imaging biomarkers when uniform acquisi- key and pig. These studies provide the first evidence that tion and processing methods are used. Use of standardized glutamine antagonism may be useful in the treatment of acquisition and processing methods and longitudinal HAND. DON prodrugs may provide an innovative and study designs might better isolate sources of biological safe clinical path for this therapeutic strategy. variationinmeasureddiffusionparameters.

P119 P120 Brain Microstructural Changes in HIV Infection: A Blood Brain Barrier Integrity Compromised by PBMC Meta-analysis and longitudinal study from HIV Positive Individuals

Erin O’Connor1, Assia Jaillard 2, Felix Renard2,Thomas Robert Oda1, Joanna Kettlewell1, Christie Nakamura1, Zeffiro3 Melissa Agsalda-Garcia1,CeciliaShikuma2, Nancy Hanks2, (corresponding author: [email protected]) Bruce Shiramizu1 (corresponding author: [email protected]) 1Lewis Katz School of Medicine at Temple University; 2Centre Hospitalier Universitaire Grenoble; 3Temple 1Department of Molecular Biosciences & Bioengineering, University College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa; 2Hawaii Center for AIDS; Purpose: Because of strong histopathological evidence John A. Burns School of Medicine; University of Hawaii at documenting HIV effects on cerebral white matter, numerous Manoa studies have used diffusion tensor imaging (DTI) to measure how HIV affects white-matter microarchitecture. While many Objectives: For HIV infected individuals who are on stable have reported reduced fractional anisotropy (FA) and in- antiretroviral therapy (ART) with maximally suppressed HIV creased mean diffusivity (MD) in HIV infection, the quantita- RNA levels, HIV associated cognitive impairment (HACI) tive inconsistencies observed across studies motivated us to: continues to persist. Data from our group suggest that addition (1) do a cross-sectional literature meta-analysis of DTI mea- of entry inhibitor drug Maraviroc (MVC) can improve HACI sures in HIV infection and then (2) examine white-matter mi- in patients on stable ART. We assessed the effect of peripheral crostructural changes in a small longitudinal study of HIV blood mononuclear cells (PBMC) on blood brain barrier infected participants studied before and after beginning com- (BBB) integrity before introduction of MVC. Methods: The bination anti-retroviral therapy (cART). Methods: The meta- BBB was established with astrocytes and endothelial cells on analysis included 15 cross-sectional studies reporting FA and an insert. Inserts were cultured in growth medium for 7 days 11 studies reporting MD in the corpus callosum. Random prior to trans-migration studies. PBMC isolated from J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S59 consented patients were placed on the BBB and allowed to in vivo, in acute and chronic rodent models of HIV-1-gp120- transmigrate for 24 h before the flow through was collected. associated brain inflammation. Primary cultures of rat astro- Confluence of the barriers was analyzed using trans endothe- cytes were exposed to gp120, and inflammatory and oxidative lial electronic resistance (TEER) and permeability was stress markers (TNFalpha, IL-1beta, iNOS) were measured assessed using a 0.45 % concentration of Evanäó»s Blue dye using qPCR. For the acute in vivo model, rats were adminis- in bovine serum albumin (EBA). Results: PBMC from 4 tered an icv injection of gp120 and an intraperitoneal (ip) HIV-positive (HIV+), cognitively impaired individuals injection of PPARgamma agonist (rosiglitazone) or co- before Maraviroc intensification were collected and administration with PPARgamma antagonist (GW9662). placed on the BBB. TEER values decreased from pre- qPCR and immunoblot analysis were applied to measure in- migration (123.1 ± 2.305 Ω/cm2, n = 14) to post-migration flammatory markers, glutamate transporter-1 (GLT-1) and (90.72 ± 4.57 Ω/cm2, n = 14) and EBA permeability values PPARgamma. For the in vivo chronic model, rats were increased from pre-migration (3.538 ± 0.8056 %, n =13)to administered icv injection of adeno-associated viral post-migration (15.93 ± 2.833 %, n =14), p-values (AAV) vector expressing GFP (positive control) or <0.0001 and 0.0004 respectively. Compared to post trans- gp120, 3 weeks post icv, immunohistochemical and migration data from HIV-negative subjects (n =4, qPCR analysis were used to measure transgenes and in- TEER = 112.5 ± 6 Ω/cm2, EBA = 3.925 ± 1.153 %), post flammatory markers. In primary cultures of rat astrocytes transmigration data from HIV+ PBMC showed decreased and the in vivo acute model, gp120 exposure resulted in confluence (p = 0.0313) and increased permeability an elevation of inflammatory markers, and a decrease in (p = 0.0425). Conclusions: Preliminary results indicated a GLT-1 which were attenuated with rosiglitazone treat- significant compromise in BBB integrity upon exposure ment. In the in vivo chronic model, AAV-GFP adminis- to HIV+ PBMC. The impact of MVC on PBMC and BBB tered rodents, showed efficient transduction throughout integrity will be compared to entry results after partici- the hippocampus while AAV-gp120 administration result- pants have reached the study time point. Results of the ed in elevated inflammatory markers. Our data suggest study could provide valuable insight into the impact of that targeting PPARgamma may provide a novel therapeu- MVC on BBB integrity. tic option for preventing/treating HIV-associated brain inflammation.

P121 Role of peroxisome proliferator activated receptors P122 (PPARs) in the regulation of HIV-1 gp120 associated brain Allopregnanolone attenuates combined neurotoxicity inflammation: implications in HIV-1 neuropathogenesis associated with morphine and HIV-1 Tat in human cells and its treatment Jason J. Paris, Joyce M. Balinang, ShiPing Zou, Pamela E. Amila Omeragic, Tozammel Hoque, David Hampson, Reina Knapp, Kurt F. Hauser Bendayan (corresponding author: [email protected]) (corresponding author: [email protected]) Virginia Commonwealth University Leslie Dan Faculty of Pharmacy, University of Toronto Human immunodeficiency virus (HIV) infection is associated Despite the use of combination antiretroviral therapy for the with neurocognitive impairment that can be exacerbated by treatment of HIV-1 infection, cognitive impairments remain opioid drug use. One virotoxin that may contribute to these prevalent due to persistent viral replication and associated effects is the HIV-1 regulatory protein, trans-activator of tran- brain inflammation. Primary cellular targets of HIV-1 in the scription (Tat). Opioids, such as morphine, are observed to brain are microglia and astrocytes which in response to infec- exert additive or synergistic effects on HIV-1 Tat, promoting tion release inflammatory markers, viral proteins [i.e., glyco- neuroinflammation and neurotoxicity. We have observed the protein 120 (gp120)] and exhibit impaired glutamate uptake. progesterone metabolite and neurosteroid, 5alpha-pregnan- Peroxisome Proliferator-Activated Receptors (PPARs) are 3alpha-ol-20-one (i.e., allopregnanolone), to ameliorate Tat- members of the nuclear receptor superfamily of ligand- mediated dysregulation of intracellular calcium homeostasis activated transcription factors. Compelling evidence suggests in microglia and neurons, as well as microgliosis and neuronal that PPARs exert anti-inflammatory properties in neurological cell death, in murine cells in vitro. However, it was not known disorders. The goal of this study was to examine the role of whether these effects would extend to human cells, nor was it PPARgamma in the context of HIV-1 gp120 induced inflam- known whether the protective effects of allopregnanolone mation in vitro, in primary cultures of rat astrocytes and would be sustained when Tat-toxicity was catalyzed by an S60 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 opioid challenge. Differentiated human neuroblastoma cells decrease in PEBP1 expression, this decrease was further aug- (SH-SY5Y) or neurons derived from fetal neural progenitors mented by Tat and meth synergy. Interestingly, pretreatment were incubated with media that did, or did not, contain with ibudilast prevented this down regulation of PEBP1 by allopregnanolone (0.1–100 nM) in the absence or presence both Tat and meth. Given the localization of PEBP1 on of morphine (500 nM) and/or HIV-1 Tat1-86 (100 nM) or dendritic spines that are actin rich and regulate synaptic HIV-1BaL (25–500 pg/ml p24). Tat significantly increased function, we tested a more causal effect of PEBP1 on intracellular calcium, depolarized mitochondrial inner mem- actin dynamics at the synapse. While Tat and meth alone brane potential, increased reactive oxygen species, and pro- decreased the expression of F-actin in cultures treated moted loss of neurons. While morphine was not toxic on its with siRNA against PEBP1, this effect was significantly own, the combination of morphine and Tat displayed ad- augmented during Tat and meth synergy. This marked ditive effects for the observed neurotoxic profile associat- dramatic reduction in F-actin levels was ameliorated by ed with HIV-1 Tat. Allopregnanolone conferred a pretreatment with ibudilast thus suggesting a novel role of concentration-dependent protection against Tat- or PEBP1 in modulating actin dynamics associated with a morphine/Tat-mediated toxicity. The addition of morphine neuroinflammatory state due to HIV and meth synergy. shifted the concentration curve for allopregnanolone pro- Ongoing studies are aimed at further dissecting down- tection to the left, revealing 10 nM to be the more effica- stream mechanisms of PEBP1 down regulation on actin cious than the previous 100 nM concentration (which was dynamics during HIV/meth synergy at the synapse. efficacious in the absence of morphine). These data support the notion that neurosteroid-based therapeutics may confer prophylactic advantages for opioid-catalyzed HIV- P124 neurotoxicity. HERV-W envelope expression in microglia from Multiple Sclerosis patients: a lifelong role in neuroinflammation, demyelination and axonal lesion. P123 Targeting inflammation at the synapse in neuroAIDS : Herve Perron1, David Kremer2,RanjanDutta3, Jack Role of Synaptic PEBP1 VanHorssen 4, Sadie Deckard3, Sandra Amor4,BruceTrapp3, Patrick Kury5 Gurudutt Pendyala1,StevenLisco1, Rick Bevins2, Shilpa (corresponding author: [email protected]) Buch1 (corresponding author: [email protected]) 1Geneuro; 2Dusseldorf University Hospital; 3Cleveland Clinic; 4VU-University of Amsterdam; 5University of 1University of Nebraska Medical Center; 2University of Dusseldorf Nebraska at Lincoln Multiple Sclerosis (MS) associates genetic predisposition and A characteristic hallmark associated with HIV and metham- environmental factors. Human genome studies have investi- phetamine (meth) synergy induced brain dysfunction is neu- gated exons encoding somatic proteins and their regulatory roinflammation. Our focus is on understanding the role of sequences, which only generated variable panels of suscepti- inflammation especially at the synapse including application bility genes. Attempts to identify a causative agent of MS of novel and efficacious treatment strategies to treat progres- from the environment have consistently failed. Since the sion of neuroAIDS. One emerging anti-inflammatory drug is 1990’s, a new horizon arose from studies of MS cells from ibudilast, a phosphodiesterase inhibitor that modulates the ac- which a human endogenous retrovirus (MSRV) and its related tivity of glial cells by suppressing the production of proinflam- multicopy family (HERV-W) were identified. Endogenous matory cytokines. We recently demonstrated for the first time retroviruses and their relatives occupy approximately 8 % of a novel role for ibudilast’s anti-inflammatory effect at the syn- the human genome and are part of the broader category of apse and its ability to attenuate meth seeking during absti- “mobile genetic elements”, which together account for one nence. Furthermore our proteomics screen on purified synap- half of the human genome. Cumulated results from numerous tosomes from animals that self-administered meth identified studies over past decades brought converging and consistent the synaptic signaling protein phosphatidylethanolamine- observations and provide a global picture suggesting that en- binding protein 1 (PEBP1) whose decreased expression by vironmental infectious agents may trigger the expression of meth was reversed by ibudilast. Extending this initial obser- certain HERV-Welements in MS, thereby engaging pathogen- vation in the context of HIV and meth synergy, we employed ic pathways leading to final pathognomonic features. These an in vitro mixed cerebrocortical cultures comprising of neu- scientific advances are now providing a new perspective of rons and glia. While both Tat and meth led to a significant “ gene-environment interplay” underlying the J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S61 etiopathogenesis of MS. Within this global understanding, it with both mean genetic diversity as well as the absolute dif- now appears that microglia is the lifelong and key pathogenic ference in genetic diversity between R5- and X4-genome spe- player of neuroinflammation, recruiting T-cells in RRMS cific trends. As expected, the V3 domain of the gp120 protein phase and causing chronic innate immune activation, as well was enriched with statistically divergent positions. as of myelin and axonal lesions. We shall present how this Statistically divergent positions were also identified in microglial activity is strongly associated with the expres- Tat amino acid sequences within the transactivation and sion of the HERV-W envelope protein (MSRV-Env) (i) in TAR-binding domains, and in nucleotide positions microglia within areas of active demyelination, from ear- throughout the LTR. We further analyzed LTR sequences liest lesions to late progressive plaques, and (ii) in mi- for putative transcription factor binding sites using the croglia directly involved in axonal lesions. This endoge- JASPAR transcription factor binding profile database and nous retroviral protein is likely to play a role in the con- found several putative differences in transcription factor text of pro-inflammatory activation of microglia contrib- binding sites between R5 and X4 HIV-1 genomes, specif- uting to myelin damage, oligodendrocyte cell death and ically identifying the C/EBP sites I and II, and Sp site III, ultimately axonal degeneration in progressive MS. Along among others, to differ with respect to sequence configu- with previous and still ongoing studies, these observations ration for R5 and X4 LTRs. These observations support the suggest that MSRV-ENV can be an appropriate therapeu- hypothesis that co-receptor utilization coincides with spe- tic target in MS. cific genetic signatures in HIV-1 Tat and the LTR, likely due to differing transcriptional regulatory mechanisms and selective pressures applied within specific cellular targets P125 during the course of productive HIV-1 infection. Utilization of HIV-1 envelope V3 to identify X4- and R5-specific Tat and LTR sequence signatures P126 Vanessa Pirrone1, Gregory Antell2,WilliamDampier1, Expression profile of cathepsin B, cystatins B / C, Benjamas Aiamkitsumrit1, Michael Nonnemacher1, Jeffrey and related proteins in CD14+ monocyte subpopulations Jacobson3, Wen Zhong1, Katherine Kercher1, Shendra and HAND severity Passic1, Jean Williams1, Gregory Schwartz2, Uri Hershberg2, Fred Krebs1,BrianWigdahl1 Marines Plaud/Valentin1,YamilGerena1, Valerie Wojna1, (corresponding author: [email protected]) Richard Skolasky2,LoydaMMelendez1 (corresponding author: [email protected]) 1Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease, Drexel 1University of Puerto Rico Medical Sciences; 2Johns Hopkins University College of Medicine; 2School of Biomedical University Engineering, Science, and Health Systems, Drexel University; 3Department of Medicine, Section of Infectious HIV-1 infection affects approximately 34 million people Disease, Lewis Katz School of Medicine, Temple University worldwide and is associated with neurocognitive disorders (HAND). Particularly its milder forms, HAND, remains prev- HIV-1 entry is a receptor-mediated process directed by the alent even in patients receiving combined antiretroviral thera- interaction of the viral envelope with the host cell CD4 mol- py. There is a need for early diagnosis and establishment of ecule and one of two co-receptors, CCR5 or CXCR4. The therapeutic agents that can halt HAND. Previous studies using amino acid sequence of the third variable (V3) loop of the our Hispanic women cohort indicated that intracellular cathep- HIV-1 envelope is highly predictive of co-receptor utilization sin B and cystatin B levels are elevated in CD14+ monocytes preference during entry, and machine learning predictive al- of HIV+ women with HIV associated dementia (HAD). It is gorithms have been developed to characterize viral sequences unknown if these proteins increase in monocytes since the as CCR5-utilizing (R5) or CXCR4-utilizing (X4). It was hy- asymptomatic stage to help in predicting the development of pothesized that while the V3 loop is predominantly responsi- HAD. We hypothesize that the expression of cathepsin B, ble for determining co-receptor binding, additional compo- cystatin B, increased in early HAND. Cystatin B, cathepsin nents of the HIV-1 genome may contribute to overall replica- B interacting protein partners (APOC2, APP, SAPC) and re- tive properties following viral entry and display sequence sig- ceptor (IGF2R) were analyzed in CD14+ resting (CD16-) and natures associated with co-receptor utilization. The accessory activated (CD16low and high+) monocytes from HIV+ protein Tat and the HlV-1 long terminal repeat (LTR) were Hispanic women. Peripheral blood monocytes from 40 pa- analyzed with respect to genetic diversity and compared by tients (14 N, 13 ANI, and 13 HAD) controlled for age, viral Jensen-Shannon divergence which resulted in a correlation load, and therapy were immunolabeled for CD14 and CD16 S62 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 surface markers. For detection of intracellular protein levels, temperature at pH7.4 for several days led to apparition of long cells were permeabilized using the BD Cytofix/Cytoperm kit, fibrillar and annular structures, with reduced alpha-helical incubated with antibodies for cystatins B and C, cathepsin B, structure. We showed that Tat and its aggregates directly APOC2, APP, SAPC, and IGF2R followed by fluorescence interacted with amyloid beta peptide. With Tat, uniform am- labeled secondary antibodies. The mean fluorescence inten- yloid fibrils turned into double twisted fibrils followed by sity of each protein in CD14/CD16 subsets was deter- populations with thick unstructured filaments and aggre- mined by flow cytometry. We found elevated levels of gated large patches. The fibers became more rigid and cystatins B in CD14highCD16low (p =0.010) and CD14 mechanically resistant. Tat attached externally to fibrils, high CD16 high subpopulations (p = 0.017), and APOC2 causing their lateral aggregation into thick multifibrilar in CD14high/CD16 low cells (p = 0.005) correlating with structures, that presented enhanced adhesion. As result, HAND severity. No differences were found with cystatin the neurotoxic properties of Tat and amyloid beta aggre- C,cathepsinsB,APP,SAPC,andIGF2R.Theseresults gates were strongly synergistic when complexed together. indicate that cystatin B increases in CD14+ monocytes These data suggest that Tat is a multimeric protein with regardless the activation state, while cathepsin B protein strong adhesive properties but sensitive to storage condi- partner, APOC2 increased in CD16low monocytes. tions. When complexed with amyloid beta, the increased Evaluation of these two biomarkers for predictive value rigidity, mechanical resistance and adhesion of the com- will be performed using the outcome from subsequent plexes led to neuronal damage, likely through pore for- visits. mation in membranes.

P127 P128 HIV-Tat protein: molecular structure, aggregation The PD- 1:PD-L1 pathway promotes development of CNS and interaction with amyloid beta TRM cells following acute viral encephalitis

Alina Popescu Hategan1, Mario Bianchet2,Elena Sujata Prasad, Shuxian Hu, Wen S Sheng, Priyanka Chauhan, Karnaukhova3, Joseph Steiner1, Emilios Dimitriadis4, James R Lokensgard Avindra Nath1 (corresponding author: [email protected]) (corresponding author: [email protected]) Depatment of Medicine, University of Minnesota Medical 1NINDS, Section of the Infectious of the Nervous System; School 2Johns Hopkins School of Medicine, Department of Neurogogy; 3FDA, CBER; 4NIBIB, Scanning Probe Initial T-cell-mediated immune responses to infection with Microscopy Unit cytomegalovirus (CMV) trigger generation of a large number of short-lived effector cells (SLEC), as well as a pool of mem- Numerous studies show that Tat protein plays a critical role in ory precursor effector cells (MPEC). These MPEC subse- HIV neuropathogenesis however, little is known about its quently develop into tissue resident memory (TRM) cells, structure, propensity for aggregation and for interaction with which could be critical in controlling viral reactivation, partic- other proteins. Highly purified recombinant Tat protein was ular in the tissues like the brain. Previously we reported that studied by atomic force microscopy. Only 8 % of the protein during acute infection microglial cells and astrocytes modu- was in monomeric state. The remaining Tat aggregates ranged late antiviral T-cell effector responses through the PD1: PD- from dimers, trimers, tetramers to large oligomers (50-mers L1 pathway, thereby controlling neuroinflammation. Here, we and larger) and all structures presented globular shape. evaluated the role of the PD-1: PD-L1 pathway in the devel- Circular dichroism and force spectroscopy showed that 20– opment of TRM within the murine CMV (MCMV)-infected 29 % of the sampleäó»s structure was alpha-helical, which brain. Flow cytometric analysis of brain-infiltrating cells was likely represents the aggregated state. Force spectroscopy performed at 7, 14, and 30 d post-infection (dpi) to assess the showed that 44 aminoacids long domains could be mechani- transition from SLEC to MPEC, as well as generation of cally unfolded. Adhesion forces of 88 pN showed the in- TRMs. In wild-type (WT) animals, we observed a switch in creased adhesion capacity of Tat and its aggregates, as com- the phenotype of brain-infiltrating CD8+ T-cells from pared to adhesion forces between cells. The reducing agent KLRG1 + CD127- (SLEC) to KLRG1-CD127+ (MPEC) dur- dithiothreitol dissociated the aggregates into smaller aggre- ing transition from acute through chronic phases of infection. gates, but structures smaller than the monomer were also The majority of CNS-infiltrating CD8+ T-cells expressed found, indicating that the molecule is prone to rupture. High CD127, a marker of memory cells at 14 and 30 dpi. In con- salt reduced the size of aggregates, and storage at room trast, fewer CD8+ T-cells expressed CD127 within the brains J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S63 of infected, PD-L1-KO animals. Notably, in WT mice a large was significantly higher in participants with mild cognitive population of CD8+ T-cells was phenotyped as CD103+, a motor disorder (mean = 4.43 ng/mL, p = 0.008) and HIV- marker of TRM and striking differences were observed in associated dementia (mean = 5.73 ng/mL, p = 0.0005) com- the numbers of these TRM cells when compared to PD-L1- pared to those with normal cognition (mean = 1.2 ng/mL). KO animals. Further immunohistochemical studies revealed CSF Gal-9 levels inversely correlated with NP global that the majority of brain-resident CD103+ TRM cells were composite z-scores (r = −0.63,p = 0.015), and subdomain localized to the brain parenchyma. Interestingly, in explant z-scores in executive function (r = −0.70,p =0.005),work- reactivation studies, reactivated virus was more efficiently re- ing memory and concentration (r = −0.65,p =0.011), mo- covered from the brains of latently-infected PD-L1-KO ani- tor skills (r = −0.65,p = 0.010), and psychomotor speed mals, which contained less TRMs, when compared to WT (r = −0.66, p = 0.009). Moreover, Gal-9 in both CSF and animals. In conclusion, our results indicate that the PD1: plasma directly correlated with HIV DNA copy number in PD-L1 pathway is critical in the generation of brain TRMs, peripheral blood mononuclear cells (r = 0.79,p = 0.001; which control spread of reactivated, latent virus. r =0.55,p = 0.036 respectively), and CSF levels of the macrophage activation marker sCD163 (r = 0.59,p = 0.024; r = 0.78,p = 0.001 respectively). P129 Collectively, these data suggest that Gal-9 may be in- Cerebrospinal Fluid Galectin-9 is a Novel Biomarker volved in the neuropathogenesis of NCI and serve as a for Neurocognitive Impairment Among Elderly HIV-1 novel indicator of HIV-associated NCI. The effects of vi- Infected Adults ral suppression on the relationship between circulating Gal-9 and NCI would be of further interest. Thomas Premeaux1,MichelleD’Antoni1, Bruce Shiramizu1, Melissa Agsalda-Garcia1, Cecilia Shikuma2,MohamedAbdel Mohsen3, Satish Pillai4,LishomwaNdhlovu1 P130 (corresponding author: [email protected]) In vivo visulation of HIV transmission and viremia rebound in humanized mice 1Department of Tropical Medicine, Medical Microbiology, and Pharmacology, Hawaii Center for AIDS, John A. Burns Xiying Qu, Won-Bin Young School of Medicine, University of Hawaii; 2Hawaii Center for (corresponding author: [email protected]) AIDS, John A. Burns School of Medicine; 3Blood Systems Research Institute, UCSF School of Medicine; 4Blood Department of Radiology, University of Pittsburgh School of Systems Research Institute, UCSF Department of Medicine Laboratory Medicine, UCSF-GIVI Center for AIDS Research Knowing where HIV-infected cells are located allows for The prevalence of HIV-associated neurocognitive impairment monitoring of the infection spread from the infection site to (NCI) remains high in the HIV-infected population despite other sites in the body. The whole-body examination or viral suppression with combination antiretroviral therapy monitoring dynamics of HIV infection in real time over (cART). Circulating cell-associated HIV DNA and markers the course of infection provides an opportunity for inves- of macrophage activation have been suggested as potential tigating where the viral infection starts, where the sanctu- indicators of NCI, however more sensitive and reliable bio- aries are during suppressive ART and, when and where markers are needed. We previously identified Galectin-9 (Gal- viremia rebound will occur following ART withdraw. We 9), a soluble lectin with immunomodulatory properties, as a previously demonstrated that bioluminescence imaging component of the first wave of the plasma cytokine storm (BLI) based on an enhanced firefly luciferase is sensitive during HIV acquisition. Gal-9 is known to be released by for visualizing as few as 5 single cells in a niche (Song astrocytes and elevated in the cerebrospinal fluid (CSF) of et al., 2015). In this study, we engineered replication- patients with progressive multiple sclerosis. We assessed the competent HIV-BaL (CCR5/macrophage tropism) carrying relationship between Gal-9 and HIV-associated NCI in 15 this luciferase reporter for infecting humanized Bone elderly HIV-1 infected individuals with detectable CSF viral marrow-Liver-Thymus (BLT) mice via vaginal mucosa or loads (VL) enrolled in the Hawaii Aging with HIV Cohort intraperitoneal injection. Compared to the intraperitoneal (86.7 % male; age range: 50–58 years; mean: log10 plasma/ route, penetration of mucosal barrier by HIV vaginal trans- CSF VL = 4.33/2.99 copies/mL, CD4 count/nadir = 392/285 mission requires at least 17 fold more virus. Infection in cells/μl; on cART: 6/15). Plasma and CSF Gal-9 levels were the female reproductive tract (FRT) could be visualized in measured by ELISA and analyzed with available neuropsy- the vaginal vulva/canal 48 h post inoculation (p.i.). Robust chological (NP) test assessments and clinical data. CSF Gal-9 infection from FRT to initiate a systemic infection was S64 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 visualized as early as 21 days, p.i., presumably an eclipse neurocognitive functioning was determined with a battery of phase during local HIV expansion. Compared to intraperi- standardized tests. Global neurocognitive functioning calcu- toneal inoculation, the spatial-temporal dynamics of HIV lated by averaging all scores. Monocytes were isolated from mucosal transmission is significantly different, which is whole blood via Rosette separation. Global gene expression unlikely to be observed via conventional measurement of determined with the Illumina HT-12 v4 Expression BeadChip. viral load in the blood. Treating the inoculated hu-BLT Neurocognitive functioning was re-evaluated 2 years later. mice with combination ART, consisting of Truvada plus Genes showing the greatest associations with change in global Raltegravir, resulted in declined HIV dissemination in only neurocognitive functioning after adjustment for age and race 2 weeks. HIV rebound was observed in the neck lymph were subjected to DAVID gene set enrichment analysis. node 2 weeks after ART withdrawal. We also demonstrated Results were adjusted using FDR for multiple comparisons. that HIV can escape from the clinically approved Pre- Results: The strongest DAVID category positively associ- Exposure Prophylaxis (PrEP) Truvada, if not adhering to ated with improved neurocognitive function was the Wnt daily PrEP schedule, resulting in a systemic HIV infection signaling pathway (enrichment score = 2.24, FDR = 0.026), 3weeksafterthePrEP. as well as several other Wnt-related pathways. Categories enriched in genes negatively associated with improving neurocognitive functioning were largely related to tran- P131 scription and mRNA processing (enrichment score = 2.83, Peripheral blood monocyte transcriptome at baseline FDR = 0.0005), as well as NF-kappaB (enrichment implicates Wnt-signaling pathway in later neurocognitive score = 1.83, FDR = 0.0004). Conclusion: While cross- change sectional transcriptome studies of HAND have implicated oxidative stress and inflammation processes, the most no- Austin Quach1, Stefan Horvath2,DimitriosVatakis2,Otoniel table finding from this longitudinal analysis is that Wnt- Martinez-Maza3, Paul Shapshak4, Mallory Witt5,Elyse signaling pathway activity at baseline is prognostic for Singer6,AndrewLevine7 neurocognitive change 2 years later. These findings lend (corresponding author: [email protected]) support to in vitro and animal studies demonstrating that the Wnt-pathway is a repressor of HIV transcription in 1Departments of Biostatistics and Human Genetics, David multiple cell types in the periphery and in the CNS. Geffern School of Medicine at the University of California Los Angeles; 2Department of Medicine, Division of Hematology-Oncology UCLA AIDS Institute, David Geffen P132 School of Medicine at the University of California Los SIV Can Emerge from the CNS Despite Sustained Angeles; 3Department of Obstetrics & Gynecology and Peripheral Suppression in ART-treated Macaques Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine at the Suzanne Queen, Sarah Beck, Lisa Magnus, Lucio Gama, University of California Los Angeles; 4Department of Janice Clements, Joseph Mankowski Medicine, Division of Infectious Disease & International (corresponding author: [email protected]) Medicine, University of South Florida, Morsani College of Medicine; 5Los Angeles Biomedical Research Institute at Department of Molecular and Comparative Pathobiology, Harbor-UCLA Medical Center; 6Department of Neurology, Johns Hopkins University National Neurological AIDS Bank, David Geffen School of Medicine at the University of California Los Angeles; Despite successful suppression of HIV with antiretroviral 7Department of Neurology, David Geffen School of therapy (ART), HIV associated neurocognitive disorders Medicine at the University of California Los Angeles (HAND) remain prevalent. Additionally, the brain remains an unappreciated reservoir of replication-competent virus. Background: Cross-sectional transcriptome studies have im- The SIV macaque model of HIV neurologic disease is widely plicated a variety of genes and biological pathways associated recognized as an excellent model of HAND because it reca- with HIV-associated neurocognitive impairment and demen- pitulates the various stages of HAND development. Using this tia. To further develop such methods towards prognostic bio- model, we treated 4 SIV-infected pigtailed macaques with markers, we sought to determine if gene expression character- CNS penetrant ART consisting of once daily dolutegravir istics of peripheral blood monocytes at a baseline visit are (2.5 mg/kg), PMPA (20 mg/kg), and FTC (40 mg/kg) begin- predictive of neurocognitive changes 2 years later. Methods: ning at day 12 post-inoculation. Virus suppression (<50 Eighty-two HIV+ participants in the Multicenter AIDS copies/mL) in both CSF and plasma occurred by 44 days after Cohort Study in Los Angeles completed the study. Baseline initiating treatment. Despite maintenance of undetectable J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S65 virus for 2 months, one pigtail (PT4) rebounded only in the 7.60 × 10^–7 per year for cART-treated HIV/AIDS patients CSF then had persistent detectable SIV RNA in CSF until without comorbid neurological disorders. The transmission euthanasia, approximately 3 months later. Examination of vi- rate for SIV infection among untreated macaques was 6.40– ral loads in tissues showed replicating virus in the brain of 9.45 times greater than cART treated HIV-1 patients. An im- PT4, but no detectable virus in peripheral tissues (spleen, lung provement in cART efficacy (11–21 %) would suppress HIV- and mesenteric LN). PT4 also had replication-competent SIV 1 infection in patients without neurological disorders. Among in cultured primary microglia. The remaining 3 pigtailed patients with advanced disease, a substantial improvement macaques in the study had no detectable SIV RNA in in cART efficacy (50 %) would eradicate HIV-1 provirus either the CNS or periphery. Four additional macaques from the brain within 9–11 years in patients without neu- were SIV-inoculated and treated under the same ART pro- rological disorders, whereas 20–26 years of efficacious tocol but with addition of twice daily oral maraviroc cART would be required for HIV/AIDS patients with co- (200 mg), a CCR5 antagonist. In addition to suppressing morbid neurological disorders. Conclusions: HIV-1 and virus in both CSF and plasma 14 days earlier, these 4 SIV provirus burdens in the brain increase over time. A macaques receiving maraviroc had no detectable SIV moderately efficacious antiretroviral therapy regimen RNA in CSF or plasma throughout the experiment; no could eradicate HIV-1 infection in the brain that was de- SIV RNA was detected in CNS or periphery at terminal pendent on brain macrophage lifespan and the presence of time-points. This study demonstrates that 1) SIV can neurological comorbidity. emerge from the CNS despite suppression in the periphery and 2) addition of the CCR5 antagonist maraviroc poten- tiates suppression of SIV. P134 Sex Differences in the Expression and Activity of HAND Neurotoxic Factor, Cathepsin B. P133 Modeling brain lentiviral infections during antiretroviral Lester Rosario1, Krystal Colon1, Loyda Melendez2 therapy in AIDS (corresponding author: [email protected])

Weston Roda1, Michael Li1, Michael Akinwumi1, Eugene 1University of Puerto Rico, Medical Sciences Campus; Asahchop2, Benjamin Gelman3, Kenneth Witwer4, 2University of Puerto Rico, Medical Sciences Campus and Christopher Power2 RCMI Translational Proteomics Center (corresponding author: [email protected]) HIV-associated neurocognitive disorders (HAND) are preva- 1Department of Mathematical and Statistical Sciences, lent despite combined antiretroviral therapy. Women account University of Alberta; 2Division of Neurology, Department for more than a half of the 35 million people living with HIV of Medicine, University of Alberta; 3Texas NeuroAIDS worldwide, and also represent a risk factor for HAND. Research Center and Department of Pathology, University of Cathepsins and cystatins are lysosomal proteins secreted by Texas Medical Branch; 4Department of Molecular and macrophages and microglia that play important roles in Comparative Pathobiology, Johns Hopkins University neuroregulatory responses. Our laboratory, has demonstrated School of Medicine increased secretion and neurotoxicity of cathepsin B from in- vitro HIV-infected women monocyte-derived macrophages, Objective: Understanding HIV-1 replication and latency in including increased expression in postmortem brain tissue different reservoirs is an ongoing challenge in the care of with HIV encephalitis and HAND. However, cathepsins and patients with HIV/AIDS. A mathematical model was created cystatins expression and their neuroregulatory responses have that predicted HIV-1 and SIV infection dynamics within the not being studied in men. We hypothesize that HIV-positive brain during effective combination antiretroviral therapy men express similar levels of cathepsin B and its inhibitors as (cART). Design: By developing a two compartment mathe- HIV-positive women. Monocyte-derived macrophages were matical approach, a predictive model was generated from isolated from peripheral blood of 7 HIV seronegative women existing empiric data. Methods: Based on previous reports and 5 HIV seronegative men. Cells were infected with HIV-1- quantifying total viral DNA levels in brain from HIV-1 and ADA at 0.1 MOI and, at day 11 post-infection, supernatants SIV infections, estimates of proviral DNA burden were made, were collected, and ELISA pro-cathepsin B and cystatin C which were fit to a mathematical model predicting viral ac- assays were performed, including a cathepsin B activity assay. crual in brain macrophages from primary infection. Results: Our results show that HIV-1-positive men significantly The annual rate at which susceptible brain macrophages be- expressed lower levels of pro-cathepsin B and cystatin C com- come HIV-1 infected was estimated to be in the range 6.72– pared to HIV-1-positive women. However, cathepsin B S66 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 activity was higher in HIV-1-positive men than HIV-1- to controls. Proper electrode placement was later confirmed positive women, and significantly increased in HIV-1- via Nissl stain. Together, these experiments establish the role positive men compared to non-infected men. Moreover, of the dopamine system in contributing to, if not mediating, HIV-positive men had significant lower levels of cystatin C potential abuse liability of MPD in the treatment of ADHD in than HIV-positive women. These results suggest that HIV- HIV-1+ youths. Funded by: DA013137, MH106563, positive men express less pro-cathepsin B than HIV- MH106392, HD043680 positive women, with increased activity after HIV infection. In conclusion, sex-linked differences were observed in the secretion of pro-cathepsin B, and HIV-1 infection P136 is mediating cathepsin B activation in men outside of the Low dose hydrocortisone has acute enhancing effects cell. Future studies on the relationships of sex hormones on verbal learning in HIV-infected men with cathepsin B secretion and activation, along with neurotoxicity assays will lay the groundwork for the de- Leah Rubin1, Luan Phan1,SheilaKeating2, Kathleen Weber3, velopment of treatments for men and women against Pauline Maki1 HAND. (corresponding author: [email protected])

1Department of Psychiatry, University of Illinois at Chicago, P135 Chicago, IL; 2Department of Laboratory Medicine, University Increased oral self-administration of methylphenidate of California San Francisco, San Francisco, CA; Blood and decreased dopamine release in the nucleus accumbens Systems Research Institute, San Francisco, CA; 3The Core core region of HIV-1 transgenic rats Center, Cook County Health and Hospitals System and Hektoen Institute of Medicine, Chicago, IL Robert F. Roscoe Jr.1, Srimal Samaranayake2,Parastoo Hashemi2,StevenB.Harrod1, Hailong Li1,CharlesF. Background: Glucocorticoids are released in response to Mactutus1, Rosemarie M. Booze3 stress and alter cognitive performance and brain function (corresponding author: [email protected]) through both rapid, nongenomic mechanisms and slow, genomic mechanisms. Administration of glucocorticoids 1Dept. of Psychology, University of South Carolina; 2Dept. of in the form of low dose hydrocortisone generally enhances Chemistry, University of South Carolina; 3Dept. of cognition in individuals with trauma, but impairs cognition Psychology, Bicentennial Endowed Chair for Behavioral in healthy individuals. Here we target the time-dependent Neuroscience, University of South Carolina effects of glucocorticoids on learning and memory in HIV- infected men, a group that generally reports higher than HIV+ youths have an increased risk for mental health disor- average levels of stress and trauma. Methods: In a dou- ders, including ADHD. As a result, HIV+ children have twice ble-blind, placebo-controlled, cross-over study, we exam- the odds of receiving psychostimulant medications, such as ined the time-dependent effects of a single low dose of methylphenidate (MPH, Ritalin®). Unfortunately, little is hydrocortisone (10 mg; LDH) on learning and memory in known regarding the etiology of this psychopathology and 45 HIV-infected men between the ages of 18 and 45. potential for psychostimulant abuse in HIV-1+ adolescents. Participants were randomized to receive either LDH or First, we examined oral self-administration of methylpheni- placebo, and 1 month later were given the opposite treat- date (MPH) in female OVX F344 (n =20)andfemaleOVX ment. At each intervention session, cognition was assessed HIV-1 Tg rats (n = 19). Animals were given access for 14 days, both30min(assessingnongenomiceffects)and4h on a FR1 schedule, to MPH/sucrose (max dose 4 mg/kg/day). (assessing genomic effects) after pill administration (LDH A significant increase in MPH self-administration was found vs. placebo). Verbal learning and memory, attention/con- during the first week for HIV-1 Tg animals, relative to F344 centration, executive functioning, and visuospatial abilities controls (p < 0.05), indicating escalation in MPH dosing dur- (only at 30 min) were assessed. Self-reported stress and ing drug initiation in HIV-1 Tg animals. Second, we evaluated anxiety and cortisol and cytokines in saliva were measured dopamine release from the nucleus accumbens core (NAcc) repeatedly throughout each session. Results: Compared to region using an electroanalytical technique, fast-scan cyclic placebo, LDH increased salivary cortisol levels by 201 %. voltammetry. Animals (F344 n = 8 males/6 females; HIV-1 Cortisol levels returned to baseline 4 h post administration. Tg n = 7 males/7 females) were fully anesthetized and the At the 30-min assessment, LDH enhanced verbal learning stimulating electrode was positioned in the medial forebrain compared to placebo; the greater the increase in cortisol, bundle. HIV-1 Tg rats of both sexes exhibited a significant the greater the enhancement in verbal learning. LDH did decrease in dopamine release in the NAcc, (p <0.05),relative not affect subjective stress, anxiety, or any other cognitive J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S67 outcomes at the 30-min or 4-h time point. Conclusion: The in MOG35-55-induced progressive, as well as PLP138- rapid effects of LDH on verbal learning in HIV-infected 151-induced relapsing-remitting experimental autoim- men suggest a nongenomic mechanism by which glucocor- mune encephalomyelitis (EAE) mice. The decline in both ticoids can enhance cognition. The non-enduring nature of progression and relapse of EAE occurred as a result of this enhancement may limit its clinical utility, but provides reduced demyelination and myeloid cell infiltration into novel insights into mechanisms underlying the effects of the CNS tissue. DC numbers were restored in the spleen acute glucocorticoids on learning. of C57BL/6 and peripheral blood of SJL/J mice along with a decreased TH17 phenotype within CD4+ T-cells. The effects of CLEC12A blocking were further validated P137 using CLEC12A knockout (KO) animals wherein EAE Antibody blockade of CLEC12A delays EAE onset disease induction was delayed and reduced disease sever- and attenuates disease severity by impairing myeloid cell ity was observed. These studies reveal the utility of a CNS infiltration and restoring positive immunity DC-specific mechanism in designing new therapeutics for MS. Divya Sagar1,NarendraP.Singh2, Rashida Ginwala1, Xiaofang Huang3, Ramila Philip3, Mitzi Nagarkatti4, Prakash Nagarkatti5,KonstantinNeumann6, Jurgen Ruland6, P138 Allison Andrews7,ServioRemirez7,ZafarK.Khan1, Pooja Cocaine promotes both the initiation and the elongation Jain1 phases of HIV-1 transcription by activating NF-kappaB, (corresponding author: [email protected]) MSK1 and P-TEFb, besides inducing selective epigenetic modifications at HIV-1 LTR 1Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA; Geetaram Sahu1, Kalamo Farley1,NaziraEl-Hage2, Benjamas 2Department of Pathology, Microbiology and Immunology, Aiamkitsumrit1, Ryan Fassnacht1, Fatah Kashanchi3, School of Medicine, University of South Carolina, Jonathan Karn4,GarySimon1,KurtHauser2,MuditTyagi1 Columbia, SC; 3Immunotope Inc., Pennsylvania (corresponding author: [email protected]) Biotechnology Center, Doylestown, PA; 4Department of Pathology, Microbiology and Immunology, School of 1Division of Infectious Diseases, Department of Medicine, Medicine, University of South Carolina and William George Washington University; 2 Department of Jennings Bryan Dorn VA Medical Center, Columbia, SC; Pharmacology and Toxicology, Virginia Commonwelth 5Department of Pathology, Microbiology and Immunology, University; 3NCBID, George Mason University; School of Medicine, University of South Carolina, 4Department of Molecular Biology and Microbiology, Case Columbia, SC; 6Institut fur Klinische Chemie und Western Reserve University Pathobiochemie, Klinikum rechts der Isar, Technische Universitat Munchen, Munich; 7Department of Pathology Illicit drug users are a high risk population for infection with and Laboratory Medicine, Lewis Katz School of Medicine, the Human Immunodeficiency Virus (HIV). A strong correla- Temple University, Philadelphia, PA tion exists between prohibited drugs use and an increase rate of HIV transmission. Cocaine is one of the most widely The mechanism of dendritic cells (DCs) recruitment abused drugs in the United States, which both impairs the across the blood brain barrier (BBB) during neuroinflam- normal functioning of brain cells and also activate HIV ex- mation has been the least explored amongst all leuko- pression in central nervous system (CNS). Cocaine accelerates cytes. For cells of myeloid origin, while integrins function HIV replication by altering specific cell-signaling and epige- at the level of adhesion, the importance of lectins remains netic pathways. We have elucidated the underlying molecular unknown. Here, we identified functions of one C-type mechanisms through which cocaine exerts its effect in mye- lectin receptor, CLEC12A, in facilitating DC binding loid cells, a major target of HIV-1 in CNS. We demonstrate and transmigration across the BBB in response to CCL2 that cocaine treatment promotes HIV gene expression by ac- chemotaxis. This process involved Src homology region 2 tivating both nuclear factor-kappa B (NF-kappaB) and domain-containing phosphatase (SHP)1/2-mediated sig- mitogen- and stress-activated kinase 1 (MSK1). MSK1 sub- naling in coordinating actin polymerization in DCs that sequently catalyzes the phosphorylation of histone H3 at ser- express the WASP Interacting Protein (WIP). To test func- ine 10, and p65 subunit of NF-kappaB at 276th serine residue. tion of CLEC12A in an animal model of multiple sclero- We demonstrate that a short-term (acute) cocaine treatment sis (MS), we administered blocking antibody to promotes HIV-1 transcription by activating both nuclear CLEC12A that significantly ameliorated disease scores factor-kappa B (NF-kappaB) and MSK1. However, during S68 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 longer-term or chronic cocaine treatment MSK1 is the main vs. gp120 SAL: e.g. GFAP, HTR6, SYNPHILIN, CDK5; facilitator of HIV1 transcription. These activation events en- 3) WT SAL vs. gp120 METH: e.g. GFAP, BDNF, DRD5, hance the interaction of NF-kappaB with histone acetyl- CASP3, HTR1D/7; 4) WT METH vs. gp120 SAL: e.g. transferases (HATs) and promote the recruitment of the GFAP, HTR6, CDK5; 5) WT METH vs. gp120 METH: positive transcription elongation factor b (P-TEFb) to the e.g. GFAP, B2M, HTR1A/1D/1 F/2C/4/7, VMAT1; 6) HIV-1 LTR, supporting the development of an gp120 SAL vs. gp120 METH: e.g. GFAP, DRD5, open/relaxed chromatin configuration, and facilitating HTR1A/1D/2C/4/7. In summary, histopathology and im- both the initiation and elongation phases of HIV-1 tran- paired spatial learning and memory due to METH expo- scription. Results are also confirmed in primary monocyte sure and HIV-1 gp120 expression are associated with sig- derived macrophages (MDM). Overall, our study provides nificant alterations in the four major neurotransmission detailed insights into cocaine-driven HIV-1 transcription systems of the brain. and replication.

P140 P139 Role of autophagy and extracellular vesicles HIV-1/gp120 protein and methamphetamine induce in neurotoxicity associated with HIV-1 Nef alterations in dopaminergic and serotonergic neurotransmission systems Sami Saribas, Stephanie Cicalese, Taha Ahooye, Kamel Khalili, Shohreh Amini, Ilker Sariyer Ana Sanchez1, Ricky Maung1, Marcus Kaul1,TMARC (corresponding author: [email protected]) Group1 (corresponding author: [email protected]) Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine at Temple University, 1Sanford Burnham Prebys Medical Discovery Institute, Philadelphia, PA Infectious and Inflammatory Disease Center; 2UCSD, Translational Methamphetamine AIDS Res. Ctr. HIV-associated neurological disorders (HAND) affect the majority of AIDS patients and are a significant problem among Individuals infected with human immunodeficiency virus HIV-1-infected individuals who live longer due to combined type-1 (HIV-1) frequently use methamphetamine (METH). anti-retroviral therapies (cART). The virus utilizes viral pro- The incidence of both HIV-1 gp120 viral protein and METH teins and subsequent cytokine inductions to unleash its toxic- in the central nervous system (CNS) is suspected to exacerbate ity on neurons. Among these viral proteins, Nef is a small HIV-associated neurocognitive disorders (HAND). In addi- HIV-1 protein expressed abundantly in astrocytes of HIV-1- tion METH is a psychostimulant drug that compromises sev- infected brains and has been suggested to have a role in the eral neurotransmitter systems, including the dopaminergic and pathogenesis of HAND. In order to explore its effect in the serotonergic network. However, the combined effects of HIV- CNS, Nef was expressed in primary human fetal astrocytes 1 and METH on the brain are incompletely understood at the (PHFA) using an adenovirus. Our results revealed that Nef is molecular level. We recently treated 3–4 months old HIV-1/ released in extracellular vesicles (EVs) derived from PHFA gp120 transgenic (gp120tg) and wild type (wt) mice with an cells expressing the protein. Interestingly, Nef release in EVs escalating METH binge regimen for 25 days. At 10– was enriched significantly when the cells were treated with 12 months of age, HIV-1/gp120tg and METH-exposed ani- perifosine, MG-132, LY294002, and wortmannin suggesting mals showed significant impairment in spatial learning and a novel role of autophagic signaling in Nef release from astro- memoryand neuropathology. METH-exposed and cytes. Next, Nef-carrying exosomes were purified from astro- HIVgp120tg animals also displayed changes components of cyte cultures and neurotoxic effects on neurons were ana- the glutamatergic and GABAergic neurotransmission sys- lyzed. We observed that Nef containing extracellular vesicles tems. METH-treated HIVgp120tg mice were the most severe- were readily taken up by neurons as evidenced by immuno- ly affected. In order to further investigate underlying mecha- cytochemistry and immunoblotting. Furthermore, treatment of nisms in the brain, we used in the present study RT-qPCR neurons with Nef -carrying EVs induced oxidative stress as arrays to assess expression of genes related to the dopaminer- evidenced by a decrease in glutathione levels. To further in- gic and serotonergic neurotransmission systems. Six compar- vestigate its neurotoxic effects, we expressed Nef in primary isons between the four experimental groups revealed signifi- neurons by adenoviral transduction. Intracellular expression cant gene regulation due to METH exposure and chronic HIV- of Nef caused axonal and neurite degeneration of neurons. 1/gp120 expression: 1) WT Saline (SAL) versus (vs.) WT Furthermore, expression of Nef decreased the levels of METH: e.g. BDNF, AKT1, VMAT2, SERT1; 2) WT SAL phospho-tau while enhancing total tau in primary neurons. J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S69

In addition, treatment of primary neurons with Nef- localization of ORF2 into nucleus also occurs implicating carrying exosomes suppressed functional neuronal firing regulatory roles in the several aspects of the viral life activityassessedbymulti-electrodearraystudies(MEA). cycle including, replication, transcription or in virion Collectively, these data suggested that HIV-1 Nef can be a biogenesis. We are currently further investigating their formidable contributor to neurotoxicity along with other roles in viral life cycle. factors which leads to HAND in HIV-1 infected AIDS patients. P142 Agnoprotein of JC Virus, BK Virus and Simian virus 40 is P141 Released from Cells: Importance of the Dimerization Discovery and Characterization of the Novel ORFs Domain for Release Resulting from Trans-Splicing of the Human Polyomavirus JC Late Transcripts A. Sami Saribas, Jason Saredy, Martyn White, Mahmut Safak (corresponding author: [email protected]) A. Sami Saribas, Jason Saredy, Martyn White, Mahmut Safak (corresponding author: [email protected]) Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine at Temple University, Department of Neuroscience, Center for Neurovirology, Philadelphia, USA Lewis Katz School of Medicine at Temple University, Philadelphia, USA JC virus (JCV) is the etiological agent of the fatal disease, progressive multifocal leukoencephalopathy RNA splicing is a highly regulated nuclear processing (PML), in the central nervous system (CNS), seen in a event where various combinations of exons from pre- subset of patients with underlying immunosuppression, mRNA molecules are jointed together to generate multi- including lymphoma and AIDS. JCV specifically infects ple protein isoforms by cis-and trans-splicing. The JC oligodendrocytes and astrocytes and causes an extensive virus genome is known to transcribe two primary tran- myelin loss in CNS leading to the neurological manifes- scripts from its early and late coding regions and pro- tations of PML. PML is a rare disease, but the inci- duces several predicted alternatively spliced products by dence of PML dramatically increased in the era of the cis-splicing, capable of encoding regulatory and structur- AIDS epidemic (5–7 %). PML is also steadily increas- al proteins. Our recent NMR-based mutational analysis ing among a group of patients with autoimmune disor- of the agnoprotein dimerization domain transcripts by ders, including multiple sclerosis (MS) and Crohn’sdis- RT-PCR has led us discover two new open reading ease, who are treated with immunomodulatory antibody- frames (ORF1 and ORF2) by trans-splicing. These unex- based therapies. Agnoprotein is an important regulatory pected ORFs results from (i) a trans-splicing of the 5’- protein of several polyomaviruses including JCV, BKV short coding region of VP1 between the coding regions and SV40 and these viruses are unable to replicate ef- of both agnoprotein and VP2 after replacing the intron 1 ficiently in the absence of this protein. Agnoprotein and (ii) a further frame-shift occurring within the 5’-short forms highly stable dimers/oligomers through its Leu/ coding sequences of VP2. ORF1 and ORF2 have the Ile/Phe-rich alpha-helix domain, which plays a critical capacity to generate 58 and 72 aa long proteins respec- role in stability of the protein. In this report, we have tively. Initial characterization of these ORFs by RT-PCR investigated whether agnoprotein of BKV and SV40 ex- utilizing total RNA isolated from both the infected pri- hibits similar characteristics with respect to its release mary tissue culture cells and the PML brain tissue sam- from cells as was previously reported for JCV. Our data ples has confirmed that both ORFs are transcribed not showed that agnoprotein of BKV and SV40 is also re- only in vitro but also in vivo. Mutagenesis analysis of leased from cells. We further investigated the regions the ORF1 in the viral background suggests that it may and amino acid residues responsible for this release by play a role in transport of viral capsids into nucleus. employing mapping and site-directed mutagenesis stud- Subsequent analysis of the possible protein coding ca- ies. These studies revealed that amino acid residues pacity of the ORF1 by immunoblotting studies using Phe31 and Asp38 located within the dimerization do- newly raised polyclonal antibodies against its predicted main of JCV agnoprotein play important roles in re- amino acid coding sequence has revealed that it does lease. In addition, treatment of cultured cells with a indeed encode a small protein. Immunocytochemistry recombinant agnoprotein demonstrated a strong interac- studies revealed that both ORFs mostly localize to the tion of agnoprotein with the cell surface, suggesting a cytoplasmic compartment of the cells, but a noted novel role for this protein during initiation of the next S70 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 round of the viral infection cycle. This study has been P144 made possible by grants awarded to M. Safak from NIH Serum Inflammatory Markers and Risk of Dementia (1R01NS090949-01A1) and Temple University Drug and Neuropathy among HIV+ adults in Rakai, Uganda Discovery Initiative (161398). Deanna Saylor1, Anupama Kumar1, Gertrude Nakigozi2, Noeline Nakasujja3, Kevin Robertson4, Carlos Pardo- Villamizar1,RonaldGray5,MariaWawer6 P143 (corresponding author: [email protected]) Pur-Alpha Induces JCV Gene Expression and Viral Replication by Suppressing SRSF1 in Glial Cells. 1Department of Neurology, Johns Hopkins University School of Medicine; 2Rakai Health Sciences Program; 3Department Ilker Sariyer, Rahsan Sariyer, Jessica Otte, Jennifer Gordon of Psychiatry, University of Makarere; 4Department of (corresponding author: [email protected]) Neurology, University of North Carolina - Chapel Hill; 5Department of Epidemiology, Johns Hopkins Bloomberg Department of Neuroscience, Center for Neurovirology, School of Public Health; 6Department of Epidemiology, Lewis Katz School of Medicine at Temple University, Johns Hopkins University Bloomberg School of Public Health Philadelphia, USA Background: Systemic inflammation has been linked to risk of PML is a rare and fatal demyelinating disease of the CNS HIV-associated neurocognitive disorder (HAND) western caused by the human polyomavirus, JC virus (JCV), which HIV+ cohorts. Its relationship to HIV-associated peripheral occurs in AIDS patients and those on immunosuppressive neuropathy has not been investigated. Objective: We investi- monoclonal antibody therapies (mAbs). We sought to iden- gated the association between serum interleukin-6 (IL-6) and tify mechanisms that could stimulate reactivation of JCV in D-dimer with HAND and neuropathy among HIV+ adults in a cell culture model system and targeted pathways which rural Uganda. Methods: 400 HIV+ antiretroviral therapy naíve could affect early gene transcription and JCV T-antigen pro- adults were included. All participants completed a duction, which are key steps of the viral life cycle for sociodemographic survey, neurological examination, blocking reactivation of JCV. Two important regulatory neurocognitive assessment, and venous blood draw. partners we have previously identified for T-antigen include Neuropathy was defined as the presence of >1 subjective neu- Pur-alpha and SRSF1 (SF2/ASF). SRSF1, an alternative ropathy symptom and >1 objective sign of neuropathy on ex- splicing factor, is a potential regulator of JCV whose over- amination. Presence of dementia was determined using locally expression in glial cells strongly suppresses viral gene ex- derived normative data and Frascati criteria. Serum D-dimer pression and replication. Pur-alpha has been most extensive- levels were determined using ELISA, and serum IL-6 levels ly characterized as a sequence-specific DNA- and RNA- were determined using singleplex assays. Means of log- binding protein which directs both viral gene transcription transformed IL-6 and D-dimer were compared using t-tests. and mRNA translation, and is a potent inducer of the JCV Quartiles of IL-6 and D-dimer levels were compared using early promoter through binding to T-antigen. Pur-alpha and Wilcoxon rank sum tests. Univariate and multivariate logistic SRSF1 both act directly as transcriptional regulators of the regression analyses were used to compare odds of dementia by JCV promoter and here we have observed that Pur-alpha is serum markers. Results: Participants were 53 % male, mean capable of ameliorating SRSF1-mediated suppression of age 35 + 8 years, and mean education 5 + 3 years. Half of par- JCV gene expression and viral replication. Interestingly, ticipants had CD4 counts <200 and half had CD4 counts 350– Pur-alpha exerted its effect by suppressing SRSF1 at both 500. Neuropathy was present in 19 % (n = 76), and 16 % the protein and mRNA levels in glial cells suggesting this (n = 66) had dementia. Participants with CD4 <200 had effect can occur independent of T-antigen. Pur-alpha and higher levels of IL-6 (1.77 vs. 1.01; p < 0.001) and a trend SRSF1 were both localized to oligodendrocyte inclusion toward higher D-dimer levels (13.16 vs. 12.15; p =0.06). bodies by immunohistochemistry in brain sections from pa- IL-6 was higher among participants with dementia (1.83 tients with HIV-1 associated PML. Interestingly, inclusion vs. 1.32; p = 0.03) but not those with neuropathy. D-dimer bodies were typically positive for either Pur-alpha or did not vary by dementia or neuropathy status. Odds of SRSF1, though some cells appeared to be positive for both dementia increased by 39 % for every quartile increase in proteins. Taken together, these results indicate the presence IL-6 (OR 1.39; 95 % CI [1.04, 1.85]; p = 0.02) after con- of an antagonistic interaction between these two proteins in trolling for CD4 count and D-dimer. Conclusions: regulating of JCV gene expression and viral replication and Systemic inflammation as measured by IL-6 is associated suggests that they play an important role during viral reac- with an increased risk of dementia but not neuropathy in tivation leading to development of PML. HIV+ adults in rural Uganda. J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S71

P145 the mechanisms of epileptogenesis in PML remain unclear. Activation of SLC1A2 promoter using CRISPR/Cas9 gene We aimed to determine the incidence and risk factors for sei- editing technology zures in PML. In a prospective observational study of PML patients at BIDMC, we reviewed demographics, symptoms, Masoud Shekarabi, Prasun Datta etiology, CSF studies, JCV PCR, brain biopsy results, and (corresponding author: [email protected]) CD4 + T-cell counts. We measured JCV-specific T cell response in their blood by intracellular cytokine staining Department of Neuroscience, Center for Neurovirology, (ICS). Patients underwent neurologic exam, MRI of the brain Lewis Katz School of Medicine at Temple University, (including arterial spin labeling and MR spectroscopy) and Philadelphia, USA 256-channel EEG at enrollment and at 3, 6, and 12 months. EEG was acquired on a Geodesics EEG system 400 with 256 Solute carrier family 1, member 2 (SLC1A2) encodes the channels Hydrocel Geodesic sensor net. EEG findings were glutamate transporter 2 (EAAT2) protein primarily co-registered to the 3D rendition of the brain on MRI. Of 29 expressed in astrocytes that reuptakes excess glutamate PML patients, 48 % of patients had only one enrollment visit, from the synaptic cleft to prevent excitotoxicity. EAAT2 and 52 % were followed for 3–12 months. These included plays an essential role in cognitive functions and decreased 10 % of patients with clinical seizures prior to enrollment expression of EAAT2 protein is observed in NeuroAIDS. and 38 % during the study. EEG showed focal or multi- In the current study, we investigated whether engineered focal slowing in 21 patients (72 %), both focal and diffuse transcriptional activation systems based on CRISPR/Cas9 slowing in 7 (24 %), and no abnormalities in 1 (3 %). can be harnessed to activate HIV-1 Tat mediated dysregu- Focal slowing was concordant with location of PML de- lation of EAAT2 expression in astrocytes. We have devel- myelinating lesions on MRI in 24 patients (83 %). oped a stable astrocytic cell line that expresses the Epileptiform discharges were present on at least one deactivated Cas9 (dCas9) protein which includes dCas9 EEG in 13 patients (45 %), most commonly in temporal cassette in frame with the catalytic domain of p300 pro- (10/13, 77 %) and frontal (3/13, 23 %) regions. tein. We designed guide RNAs to target the promoter and Epileptiform discharges were concordant with PML lesion induce the expression of EAAT2 protein. Using multiple location in 12/13 patients (92 %) and were not associated techniques, we show that the SLC1A2 promoter is induced with a history of IRIS. (p = .70). Focal EEG slowing and by the gRNAs in presence dCas9-p300 in the human gli- epileptiform activity was concordant with demyelinating oma cell line. Induction of SLC1A2 promoter led to in- lesions of PML on MRI in almost all patients. crease in EAAT2 mRNA and protein expression. In addi- Demyelinating lesions appear to be associated with corti- tion, we show that co-transfection of gRNAs with dCas9- cal irritability and epileptogenesis. Correlation with perfu- p300 can mitigate HIV-1 Tat induced downregulation of sion MRI, MR spectroscopy and results of the cellular EAAT2 protein expression in primary human fetal brain immune response to JCV will be presented. astrocytes. Collectively, these results demonstrate that CRISPR/Cas9 system can be used for potential induction of EAAT2 expression not only in NeuroAIDS but also in P147 other neurodegenerative diseases such as ALS and Protective Role of Smoothened Agonist in HIV-associated Alzheimer’sdisease. Neuro-cognitive Disorder.

Meera V Singh1, Vir B Singh1, Santhi Gorantla2,LarisaY P146 Poluektova1, Sanjay B Maggirwar1 Epileptogenesis in Patients with Progressive Multifocal (corresponding author: [email protected]) Leukoencephalopathy 1Department of Microbiology and Immunology, University of Fabian Sierra Morales 1, Susan Herman2,Mary-Ann Rochester Medical Center; 2Department of Pharmacology and Dobrota2, Dhanashri P. Miskin2, Igor J. Koralnik 1 Experimental Neuroscience, University of Nebraska Medical (corresponding author: [email protected]) Center

1Rush University Medical Center; 2Beth Israel Deaconess Sonic Hedgehog (Shh) signaling is critical during Medical Center neurogenesis, however; recently, it has also been identified to play an important role in the maintenance of blood brain Although up to 44 % of progressive multifocal barrier (BBB) homeostasis in adult CNS. Previous work from leukoencephalopathy (PML) patients will develop seizures, our lab suggests that Human Immunodeficiency Virus (HIV) S72 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 infection causes impaired BBB. Excessive migration of mount against infectious agents undergo age-dependent inflammatory/infected leukocytes across the compromised changes. Microglial cells are the main immune-competent BBB in to the CNS can exacerbate HIV-associated neuro-cog- cells of the CNS forming the first line of defense against nitive disorder (HAND), a chronic neuro-inflammatory con- invading pathogens in case of injury or disease of the brain. dition, which afflicts nearly 50 % of HIV-infected individuals Human brain tissue autopsy samples from the frontal and on antiretroviral therapy (ART). In this report, humanized temporal lobes of 25 elderly subjects and 25 controls were mice were used as a model for HIV infection, to demonstrate used in this study. The inclusion criteria were the involvement of impaired Shh signaling in HAND. Mice pathomorphological signs of an unspecified encephalopa- were chronically infected for 10 weeks and were treated with thy on tissue examination. Whole slide scanning, conven- Smoothened Agonist (SAG) for the last week. Results showed tional immunohistochemistry using anti-HHV-6 and anti- reduced expression of Shh and Gli-1 in the brains of infected CD68 antibodies and confocal microscopy were applied. mice along with astrogliosis, microglial activation, and re- Immunostaining intensity was assessed with an additional duced dendritic arbor. However upon SAG administration, quantitative estimation of immunopositive cells. Brain tis- mice showed increased expression of tight-junction pro- sue samples were assayed for HHV-6 using nested and teins (Claudin5, Occludin) and reduced astrogliosis, indic- real-time PCR. SPSS 23.0 program was used for statistical ative of better BBB integrity and decreased inflammation. analysis. The HHV-6 DNA was detected in the frontal and Further the ability of SAG to prevent leukocyte infiltra- temporal lobes respectively of the following number of tion into the brain during acute infection was assessed. cases - encephalopathy group - 36 % (9/25), 44 % (11/ Mice were pre treated with SAG followed by infection 25); and control group - 16 % (4/25), 38.8 % (7/25). In with HIV for 2–3 days. Interestingly, SAG significantly both regions studied, there were significantly higher reduced the number of leukocytes extravasating into the (p = <0.001) number of HHV-6 positive gray matter astro- brain indicating that these mice may eventually have re- cytes, oligodendrocytes, microglial cells and neurons in duced viral burden. Our results emphasize a neuroprotec- the encephalopathy group when compared to controls. tive role for Shh signaling, while revealing SAG as a By contrast, there were significantly (p = <0.001) more potential therapeutic agent. These results are also consis- HHV-6 positive oligodendrocytes and microglial cells tent with the outcome of clinical trial (RV254/SEARCH found in the white matter when compared to the gray 010), which showed HIV entry into the CNS as early as matter. An increased number of activated microglial cells 8 days of infection. Reduced viremia during this phase, were detected in the white matter of the frontal and tem- possibly via SAG treatment, might subsequently result in poral lobes of the encephalopathy group. More neuronal a better prognosis for HAND. and oligodendroglial HHV-6 positivity was detected in the gray and the white matter, respectively, demonstrating heterogeneity of damage to the cortex and subcortical P148 white matter. Assessing the correlation between the grey and white matter damage and HHV-6 involvement in the frontal and temporal lobes of the brain of the elderly P149 In vivo visualization and quantification Sandra Skuja1,AneteZieda1, Kristine Ravina2,Svetlana of Spatial-Temporal kinetics of Chimeric HIV infection Chapenko3, Silvija Roga4, Ojars Teteris5, Valerija Groma1, and the efficacy of Truvada as PrEP Modra Murovska3 (corresponding author: [email protected]) Jiasheng Song1, Alexander G. White1, Yonggang Zhang2,Fei Yu 1, Wenhui Hu2, Won-Bin Young1 1Institute of Anatomy and Anthropology, Riga Stradins (corresponding author: [email protected]) University; 2Department of Neurosurgery, Stanford University School of Medicine; 3A. Kirchenstein Institute of 1Department of Radiology, University of Pittsburgh School of Microbiology and Virology, Riga Stradins University; Medicine; 2Department of Neuroscience, Temple University 4Department of Pathology, Riga 1st Hospital; 5Latvian State School of Medicine, Philadelphia, PA, USA Centre for Forensic Medical Examination Visualizing primary sites of HIV-1 infection and viral reser- Aging is an important risk factor for developing neurode- voirs longitudinally would be extremely useful for the devel- generative disorders. High percentage of world’spopulation opment of antiretroviral drugs and vaccines. Currently, surro- is seropositive for human herpesvirus 6 (HHV-6), most com- gate animal models are the major platform for testing newly monly the elderly. The immune responses the host can developed antiretroviral therapy (ART) and vaccines against J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S73

HIV infection. To establish a lower-cost high-throughput drug primates. SP increases HIV infection in macrophages through screening platform in conjunction with studying how viral interaction with NK1R. The SP effect on immune system is reservoirs are formed and maintained, we have employed a both pro- and anti-inflammatory and includes upregulation chimeric HIV-1 virus which contains a replacement of the of a number of cytokines and cell receptors. We determined HIV gp120 protein with gp80 from the ecotropic murine leu- whether there is interplay between monocyte exposure to kemia virus. This allows for infection of conventional mice SP and their recruitment into CNS. We demonstrated that without the need of expensive humanized mice. Insertion of exposure of both human monocytes/macrophages and the enhanced firefly luciferase gene in this chimeric HIV en- PBMC to SP leads to increased production of chemokines ables the infected cells to be visualized via in vivo biolumi- includingMCP-1whichisexpressedonlybycellsofmy- nescence imaging. We systemically imaged the spatial- eloid lineage. This effect was inhibited by the NK1R an- temporal biodistribution of the infected cells to reveal tagonist, aprepitant. Exposure to conditioned media from the viral infection kinetics during the first 24 h and SP treated PBMC resulted in increased monocyte migra- followed the infection for 107 days. The biodistribution tion through semipermeable membrane and in vitro human of infected cells, mainly neutrophils, was visualized and BBB model. Cell migration was blocked by anti-MCP-1 quantified longitudinally using bioluminescence imaging antibodies. Conclusions: The exposure of monocyte/ to illustrate the migration of infected cells in vivo. Ex macrophages to a greater levels of SP during HIV infection vivo imaging revealed the infected cells reside in liver, may lead to increased MCP-1 production in the CNS and gastrointestinal (GI) tract, spleen, peripheral lymph nodes, promote further monocyte migration into CNS thus con- and nasal mucosa. The viral RNA in the spleen was line- tributing to HIV infection of the brain. The transmigrated arly correlated to the photon flux output measured from monocytes may be a major source of the CNS reservoir in the light emission from the spleen during whole animal HIV infected individuals and factor contributing to CNS imaging. We also showed that this imaging/chimeric HIV inflammation. Supported by NIH PO1 MH-105303, UO1 platform can be used directly to evaluate the efficacy of MH-090325, R21 AI-108296, P30 MH-097488 to SDD Truvada as Pre-Exposure Prophylaxis against HIV infection in living animals. P151 A role for BACE1 in HIV-associated neurotoxicity P150 Substance P Mediated Chemokine Production Promotes Anna Stern1, Patrick Gannon1, Benjamin Gelman2, Dennis Migration of Human Monocytes Kolson1, Kelly Jordan-Sciutto1 (corresponding author: [email protected]) Sergei Spitsin1, Steven D. Douglas2 (corresponding author: [email protected]) 1The University of Pennsylvania; 2University of Texas Medical Branch 1Division of Allergy and Immunology, The Children’s Hospital of Philadelphia Research Institute, Philadelphia, HIV-associated neurocognitive disorder (HAND) persists in PA; 2Division of Allergy and Immunology, The Children’s 30–50 % of HIV+ patients despite effective viral suppres- Hospital of Philadelphia Research Institute, Department of sion by combined antiretroviral therapy. Though a distinct Pediatrics, Perelman School of Medicine, University of disorder, HAND has symptoms and neuropathological fea- Pennsylvania, Philadelphia, PA tures in common with Alzheimer’s Disease (AD). A hallmark of AD pathology is accumulation of amyloid-beta Substance P (SP) is a neuropeptide which plays a role in (Abeta), which is generated by cleavage of amyloid precur- neurotransmission in the central and peripheral nervous sys- sor protein (APP) by the beta-secretase BACE1. BACE1 is tem and has immunomodulatory properties. SP is a member of increased in AD brain, and BACE1 inhibitors reverse neu- the tachykinin family of neuropeptides and acts through inter- ronal loss and cognitive decline in animal models of AD action with neurokinin-1 receptor (NK1R) which is expressed with human trials underway. Although some studies have by a variety of cells including lymphocytes and monocyte/ found evidence of altered APP processing in HIV+ patients, macrophages. SP has physiological and pathophysiological it is unknown whether increased BACE1 or accumulation of roles in both CNS and peripheral tissues and is involved in oligomeric Abeta are a feature of HAND. As HIV does not cross talk between nervous and immune systems in various infect neurons, neurotoxicity is attributed to factors released conditions including HIV and SIV infection. Increased SP from HIV-infected macrophages including glutamate, and levels were demonstrated in plasma of HIV positive individ- NMDA receptors mediate HIV-associated neurotoxicity uals as well as in the CNS of SIV infected non-human in vitro. Herein, we hypothesize that HIV-associated S74 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 neurotoxicity is mediated by NMDA-dependent elevation of P153 BACE1 and subsequent altered processing of APP. In sup- EVALUATION OF COMBINATION LONG-ACTING port of this hypothesis, we have observed elevated levels NANOFORMULATED ANTIRETROVIRAL of BACE1 and Abeta oligomers in CNS of HIV+ patients. THERAPY IN EARLY HIV-1 INFECTED huPBL As a model of HIV-induced neurotoxicity, we treated pri- NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ MICE mary rat neurons with supernatants from HIV infected monocyte derived macrophages (HIV/MDMs). We ob- Hang Su, Prasanta Dash, Mariluz Arainga, Benson Edagwa, served elevation of BACE1 protein levels in both JoEllyn McMillan, Larisa Poluektova, Santhi Gorantla, HIVMDM- and NMDA-treated neurons, and HIVMDM- Howard Gendelman induced BACE1 increases were blocked by NMDA recep- (corresponding author: [email protected]) tor inhibition. Furthermore, blocking BACE1 activity with a BACE1 inhibitor abrogated HIV/MDM-induced Department of Pharmacology and Experimental and NMDA-induced neurotoxicity. These findings suggest Neuroscience, University of Nebraska Medical Center that increased BACE1 and resultant Abeta production may contribute to HAND neuropathology, and inhibition Early clinical intervention with combination antiretroviral of BACE1 may have therapeutic potential for HAND therapy (ART) in human immunodeficiency virus type one patients. (HIV-1) infected people was demonstrated to have sustained benefits by lowering viral set points, protecting CD4+ T lymphocytes and limiting viral reservoir pools that include central nervous system (CNS), which holds P152 limited ART excess, persistent viral replication and cellular Research Training in Neurovirology: Supporting activation, leading to continued neuropsychological impair- Pathways to Success ment, causing HIV-associated dementia (HAD). Notably and as shown in our prior studies cell-targeted David Stoff nanoformulated ART (nanoART) improves pharmacokinet- (corresponding author: [email protected]) ic and pharmacodynamics profiles and reduces local and systemic drug toxicities in experimental models of HIV/ National Institute of Mental Health, Division of AIDS AIDS. In addition, with optimized size, shape and protein Research and lipid coatings, nanoART can circumvent the blood– brain barrier (BBB) to facilitate CNS-directed drug deliv- This session represents our annual efforts at the current ery. These drugs are being considered in a step-wise drug ISNV meeting to support research training in regimen for viral eradication. In the present study, we neurovirology. The session is designed to provide new administrated long-acting nanoformulated combinations of and early stage research investigators with the tools nec- folic acid-decorated cabotegravir, lamivudine and abacavir essary to continue along the path of competitive re- to human peripheral blood lymphocyte (PBL) reconstituted search support and transition to independence. As in NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice 24 or previous meetings, active T32 and other predoctoral 72 h after HIV-1 infection. Replicate mice received free and/or postdoctoral trainees will briefly present data drugs at equivalent doses. Animals were sacrificed 2 weeks and research directions for their current mentored re- after HIV-1ADA infection with blood and tissues harvest- search projects and will be offered feedback for im- ed for viral measurements. NanoART-treated animals dem- provements. Preceding and subsequent to the trainee onstrated 75 % recovery of CD4+ T lymphocytes calcu- presentations, the symposium content will include: (1) lated from a total CD3+ cell pool. Immunohistochemical Introductory presentation on neurovirology research tra- tests showed HIV-1p24+ cells (~80/1000HLA-DR+ cells) jectories, with special attention to barriers faced by only in untreated infected animals. NanoART suppressed emerging young investigators to success in HIV-1gag RNA expression to <10copies as assessed by neurovirology at various levels (individual, institutional, real-time RT-PCR in brain, spleen, live, lung and kidney organizational, systemic), and (2) Panel discussion in- in 90 % animals while total viral DNA remained detect- cluding predoctoral, post-doctoral and faculty investiga- able. Limited tissue proviral DNA was seen in nanoART tors on strategies for successfully navigating obstacles treated animals with significant reduction, up to or exceed- and developing potential solutions on the journey to a ing 85 %, in plasma viral RNA levels was observed in successful research career. One of the intentions of this 24 h nanoART-treated group (<20 copies/ml). Early treat- roundtable discussion will be to facilitate mentoring re- ment of combination nanoART in infected PBL mice can lationships at multiple levels. effectively suppress HIV-1 growth in both central and J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S75 peripheral viral sanctuaries while preventing CD4+ T lym- This observation correlates with the increased viral load phocyte loss. and persistence associated with the 108G change in specific HIV-1-infected individuals. These results demonstrate that mutations are occurring in individuals on anti- P154 retroviral therapy that are likely clinically and functional- Clinical and functional characterization of viral single ly important. nucleotide polymorphisms (SNPs) within the HIV-1 LTR associate with increased virus persistence in the Drexel Medicine CARES Cohort P155 IFNbeta PROTECTS NEURONS IN A Neil Sullivan1, Michael Nonnemacher1, Vanessa Pirrone1,Rui CCL4-DEPENDENT FASHION AGAINST HIV-1 Feng2, Brian Moldover3, William Dampier1,ShendraPassic1, GP120-INDUCED INJURY Jean Williams1, Benjamas Aiamkitsumrit1,WenZhong1, Brandon Blakey1,SoniaShah1, Zsofia Szep4, Jeffrey Victoria Thaney1,AlanO’Neill1, Melanie Hoefer1,Ricky Jacobson5, Brian Wigdahl1 Maung1,MarcusKaul1 (corresponding author: [email protected]) (corresponding author: [email protected])

1Department of Microbiology and Immunology, Institute for 1Sanford Burnham Prebys Medical Discovery Institute Molecular Medicine and Infectious Disease, Drexel University College of Medicine; 2Department of Human immunodeficiency virus-1 (HIV-1) invades the Biostatistics and Epidemiology, Center for Clinical central nervous system (CNS) soon after peripheral infec- Epidemiology and Biostatistics, University of Pennsylvania tion and can cause HIV-associated neurocognitive disor- School of Medicine; 3B-Tech Consulting, Ltd; 4Division of ders (HAND). Type I interferons are critical mediators of Infectious Diseases and HIV Medicine, Department of anti-viral immune responses and interferon-beta (IFNbeta) Medicine, Drexel University College of Medicine; has been implicated in the control of HIV and SIV infec- 5Department of Medicine, Section of Infectious Disease, tion in the brain. However, whether IFNbeta has neuropro- Lewis Katz School of Medicine, Temple University tective activity in the context of HIV-mediated neuronal injury is unknown. In the present study, we used The HIV-1 LTR is continuously under selective pressure HIVgp120 transgenic mice (HIVgp120tg) as a model for and LTR SNPs can alter viral transcription in a cell-type HIV-induced brain injury that may underlie the develop- dependent manner. To elucidate the clinical and functional ment of HAND. Analysis by reverse transcription- impact of HIV-1 SNPs, the Drexel Medicine CNS AIDS quantitative polymerase chain reaction (RT-qPCR) detected Research and Eradication Study (CARES) Cohort con- an IFN response in gp120 transgenic brains with IFNbeta ducted a prospective, longitudinal study on >500 HIV-1- mRNA significantly increased at 1.5 months, but not at 3 infected patients. Numerous SNPs were strongly correlat- and 6 months. Therefore, we examined whether recombi- ed with clinical disease parameters, such as CD4+ T-cell nant IFNbeta treatment can prevent neuronal injury and/or count and viral load. Of interest, LTR position 108, a glial activation in vivo and in mixed neuronal-glial COUP/AP1 binding site, increased in frequency in pa- cerebrocortical cell cultures. In vivo, we administered tients with high viral loads and low CD4+ T-cell counts. IFNbeta intranasally in order to maximize IFNbeta deliv- Electrophoretic mobility shift assays (EMSAs) functional- ery to the brain and to minimize side effects in peripheral ly demonstrated differential transcription factor (TF)/DNA tissues. We found that a 4-week IFNbeta treatment trig- binding profiles with Jurkat T-cell and monocytic U-937 gered an IFN response that included increased CCL4 ex- nuclear extract (NE) when the nucleotide at position 108 pression. Neurohistological analysis revealed that IFNbeta is changed. The binding site with an A at position 108 treatment prevented loss of neuronal dendrites and pre- (108A) formed 3 complexes while a G at this position synaptic terminals in the frontal cortex and hippocampus (108G) formed 4 complexes with Jurkat NE. Three com- in gp120tg brains. Similarly, treatment of mixed neuronal- plexes were formed with both constructs when U-937 NE glial cerebrocortical cells with IFNbeta provided complete were used in the EMSAs. JASPER and supershift EMSAs neuronal protection against recombinant gp120 toxicity. support the presence of GATA-2, ETS-1, AP-1, and Neuroprotection was dependent on IFNalpha receptor 1 COUP binding to the sequences surrounding 108. (IFNAR1) and CCL4, as IFNAR1 deficiency and neutral- Transient expression analyses with an LAI LTR contain- izing antibodies against CCL4, respectively, abolished the ing a 108G showed increased transcription as compared to neuroprotective effects of recombinant IFNbeta. Taken to- 108A in monocytic U-937 cells but not in Jurkat T cells. gether, these results identify IFNbeta as a neuroprotective S76 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 factor that can ameliorate HIV gp120-induced brain injury induced activation of microglia involved sequential acti- via induction of CCL4 and be delivered in vivo using vation of mitophagy with defective clearance. In summa- intranasal application. Supported by NIH R01 ry, Tat activates microglia by both increasing mitochon- MH087332, MH104131 and MH105330 drial damage and defective mitophagy. Interventions aimed at blocking mitochondrial dynamics could thus provide promising therapeutic targets for abrogating Tat- P156 mediated neuroinflammation. (Supported by DA040397; HIV-1 Tat protein activates microglia through defective DA035203) mitophagy

Annadurai Thangaraj, Palsamy Periyasamy, Shilpa Buch P157 (corresponding author: [email protected]) Reduced cellular energy as a potential marker of HIV brain latency related neuropathogenesis: Department of Pharmacology and Experimental a pilot study Neuroscience, University of Nebraska Medical Center, Omaha, NE, ZIP: 68198 Michael Tobia1, Simon Jones2,MichaelLovelace2, Caroline Rae1, Lucette Cysique1, Bruce Brew1 HIV-1 invades CNS early in the course of infection (corresponding author: [email protected]) and primes a cascade of neuroinflammatory responses by activating immune cells. Since HIV-1 transactivator 1UNSWAustralia; Neuroscience Research Australia; 2UNSW protein, Tat has been well-documented to activate glial Australia; Applied Neurosciences Program, Peter Duncan cells, the objective of this study was to explore wheth- Neuroscience Research Unit, St. Vincent’sCentrefor er Tat-mediated activation of microglia also involved Applied Medical Research mitophagy. Our results demonstrated that exposure of microglia to Tat resulted in cellular activation by alter- Background: In virally suppressed HIV-infected (HIV+) ing the mitochondrial membrane potential and by in- the cause of brain injury is not known. One hypothesis creasing the expression of mitophagy markers, Pink1 is that the brain acts as a reservoir for latent HIV in- andParkininbothBV-2cellsandmouseprimary fection, and that the burden of HIV brain latency microglial cells. Increased Pink1 accumulation on the causes a cascade of neuropathological events. In the defective mitochondria has been shown to recruit cyto- current study we explore whether major brain metabo- solic Parkin to the site of damaged mitochondria for lites may serve as markers of brain HIV latency, de- their clearance via mitophagy. We also showed that fined by CSF BCL11b (a microglia cell transcription exposure of microglia to Tat resulted in increased ex- factor that inhibits HIV transcription). Methods: The pression of DRP1, thereby escalating the fission of study included 26 HIV+ men (mean age 57 years old; damaged mitochondria. Tat exposure also upregulated stable on cART ≥6 months, nadir CD4+ lymphocyte the expression of autophagosome markers, Beclin1 and count ≤350 cells/mm3, HIV infection ≥5years;96% LC3-II, indicating thereby that increased levels of undetectable <50 cp/mL; 100 % <100cp/mL). All un- Pink1, Parkin, and DRP-1 proteins likely tether the dertook lumbar puncture; a 1H Magnetic Resonance damaged mitochondria to the LC3-positive phagophore Spectroscopy (MRS) scan. CSF samples were analyzed for mitophagosome formation. These findings were val- for BCL11b, neopterin, NFL, and tat. All were had idated by imaging studies which demonstrated in- undetectable HIVRNA at <50 cp/mL. 1H MRS includ- creased co-localization of damaged mitochondria with ed measurements of N-acetyl Aspartate (NAA), choline, the LC3 puncta. Intriguingly, Tat exposure also in- creatine, Myo-inositol, glutamine/glutamate in the fron- creased the expression of p62, signifying thereby a possi- tal white matter, posterior cingulate cortex, and caudate ble blockade of mitophagy flux, that in turn, results in nucleus area. MRS spectra were measured with refer- accumulation of mitophagosomes. This phenomenon was ence to the unsuppressed water signal and quantified further validated by mRFP-EGFP-LC3 transfection using JMRUI V.03. Results: In a series of adjusted wherein the results confirmed Tat-mediated defective (for neopterin, NFL and tat) regression models; we mitophagy. Interestingly, Tat-mediated activation of mi- found that a higher CSF BCL11b was consistently as- croglia correlated with increased expression of TNF-al- sociated with lower frontal white matter creatine (ad- pha, IL-1beta, and IL-6. Using both pharmacological justed for neopterin, Std beta = −.30; p = .15; adjusted and gene-silencing approaches to block either autopha- for NFL, Std beta = −.51; p = .03; adjusted for tat, Std gy/mitophagy, our findings demonstrated that Tat- beta = −.47; p = .02). Conclusions: Frontal white matter J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S77 reduced cellular energy may indicate ongoing HIV P159 brain latency related neuropathogenesis. These pilot da- The epidermal growth factor counteracts the effects ta need to be further tested in a larger sample. of HIVtat on the expression of endogenous retroviruses of the W family in human astrocytes

Elena Uleri, Gabriele Ibba, Claudia Piu, Maurizio Caocci, P158 Caterina Serra, Antonina Dolei Neutralization of interferon-alpha by B18R (corresponding author: [email protected]) in a mouse model of HIV associated neurocognitive disorders Department of Biomedical Sciences, University of Sassari

William Tyor1, Rajeth Koneru2, Heather Bimonte-Nelson3, The pathogenic mechanisms of HIV-related neurodegen- Woldeab Haile4, Vincent Ciavatta5, Jennifer Ward6,Leonard eration are not fully elucidated, and include both host and Maroun6 viral mechanisms. The epidermal growth factor (EGF) has (corresponding author: [email protected]) been proposed to control the phenotypic features of adult astrocytes. Activation of EGFR, the EGF receptor, was 1Emory University School of Medicine and Atlanta VA proposed as a master pathway of astrocyte activation in Medical Center; 2Atlanta VA Medical Center; 3Arizona State response to different neural injuries. We showed previous- University; 4Emory University; 5Emory University and the ly that HIVtat activates the neuropathogenic and Atlanta VA Medical Center; 6Meiogen immunopathogenic MSRV and Syncytin-1 elements of the HERV-W family of endogenous retroviruses, in hu- Infection with HIV commonly causes HIV associated man macrophages and in astrocytes. We hypothesized that neurocognitive disorders (HAND) despite life- these elements could contribute to the pathogenic phe- prolonging combined antiretroviral therapy (cART). nomena leading to HIV-related neurodegeneration. The There is substantial evidence that interferon-alpha mechanism of HERV-Ws stimulation by Tat is an indirect plays a role in cognitive impairment in HAND. We one, through interaction with Toll-like Receptor-4 have previously shown that a decoy protein that se- (TLR4), without internalization, and induction of TNF- questers type I interferons, B18R, ameliorates HIV in- alpha. It was shown that HIVtat interacts with the 1–6 duced histopathology. We tested if co-administration of EGF-like repeats of Notch proteins, with a possible link B18R with cART provides enhanced protection from with AIDS pathogenesis. Therefore, we exposed the HIV encephalitis in comparison to B18R or cART PHFA, SVG and U87MG human astrocyte cells to treatments alone in a mouse model of HAND. After HIVtat, EGF and other stimuli, and the expression of 10 days of treatment mice were sacrificed and brains MSRV and Syncytin-1 HERV-W transcripts was evaluated extracted and frozen for tissue sectioning. The data by real time RT-PCR. The results indicate that EGF coun- suggests that B18R simultaneously administered with teracts the effects of HIVtat on the HERV-Ws, by inter- cART reduced encephalitis markers more than either fering with the induction of TNF-alpha by HIVtat. The of the two treatments administered alone. In a separate EGF-mediated counteraction of HIVtat effects is set of experiments, B18R was tested for its ability to preventedbypre-exposuretoantibodyagainsttheEGF-R. reverse behavioral abnormalities of HAND mice using an Object Recognition Test (ORT). The ORT was administered to HAND mice and controls before and af- P160 ter treatment with B18R. Prior to B18R treatment JC polyomavirus expression and bell-shaped regulation HAND mice were behaviorally impaired on the ORT. of its SF2/ASF suppressor in multiple sclerosis patients Then HAND mice received either B18R or saline sub- under therapy with Natalizumab cutaneously twice a day for 5 days prior to repeat ORT. Performance was significantly improved in Elena Uleri, Gabriele Ibba, Claudia Piu, Maurizio Caocci, B18R treated HAND mice compared to saline treated Caterina Serra, Antonina Dolei mice. Pathological studies in these ORT, B18R treated (corresponding author: [email protected]) and untreated HAND mouse groups are ongoing. Because of its ability to act in concert with cART as Department of Biomedical Sciences, University of Sassari well as reverse behavioral abnormalities in HAND mice, B18R is a potential therapeutic agent for a Natalizumab is effective against multiple sclerosis (MS), but Phase I trial in HAND patients. rarely associated with progressive multifocal S78 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 leukoencephalopathy (PML), fatal brain disease due to the for the first time that HIV-infected HIV+CD14+CD16+ JCV polyomavirus. The SF2/ASF (splicing factor2/ monocytes preferentially transmigrate across the BBB in com- alternative splicing factor) inhibits JCV in glial cells. parison to uninfected but HIV-exposed HIVexpCD14+ We wondered about SF2/ASF modulation in the blood CD16+ monocytes, both by p24 FACS and HIV DNA. We of Natalizumab-treated patients, and if this could influ- also show that this is mediated, in part, due to increased ence JCV reactivation. A 2-year study was performed on junctional proteins JAM-A and ALCAM on HIV+CD14+ blood cells from Natalizumab-treated and Fingolimod- CD16+ monocytes. We also showed that JAM-A and treated MS patients. A bell-shaped SF2/ASF regulation ALCAM blocking antibodies inhibit transmigration across was observed only under Natalizumab: SF2/ASF was up- the BBB of HIV+CD14+CD16+ monocytes. In addition, regulated, during the first year, only in JCV DNA- CCR2, the only known CCL2 receptor on monocytes, is positive patients, or with high anti-JCV antibody, with increased on CD14+CD16+ monocytes from HIV-infected enrichment of SF2/ASF-negative B-cells. The expression individuals with HAND, but not on those with impairment of the JCV T-Ag protein in circulating B cells was in- due to other causes. Using neuroimaging, we showed that versely related to SF2/ASF protein expression. The in- increased CCR2 on CD14+CD16+ monocytes correlates crease of SF2/ASF-negative B-cells, parallel to JCV ac- with decreased neuronal activity in the basal ganglia. tivation, during the second year of therapy with Additionally, using HIV DNA analysis we found that in- Natalizumab, but not with Fingolimod, may help creased CCR2 correlates with increased peripheral PBMC explaining the increased risk of PML with Natalizumab, HIV DNA. This provides strong evidence that CCR2 iden- and not with Fingolimod. tifies individuals with cognitive impairment due to HIV, and that CCR2 may be a HAND biomarker. To examine CCR2 in addition to JAM-A and ALCAM as a therapeutic P161 target, we will use Cenicriviroc to prevent transmigration JAM-A and ALCAM Mediate Preferential of HIV+CD14+CD16+ monocytes into the CNS. It is Transmigration Across the BBB of HIV-infected HIV + hoped that these strategies will result in decreased neuronal CD14+CD16+ Monocytes: Continued Viral Seeding damage and inflammation, and, HAND. of the CNS and HAND.

Mike Veenstra1, Bruce Shiramizu2, Lucio Gama3, Kathryn P162 Anastos4, Matilde Inglese5,DesireeByrd5,SusanMorgello5, Characterization of Zika virus infection in Human Joan Berman1 Astrocytes (corresponding author: [email protected]) Courtney Veilleux, Eliseo Eugenin 1Department of Pathology, Albert Einstein College of (corresponding author: [email protected]) Medicine; 2John A Burns School of Medicine, University of Hawaii at Manoa; 3Department of Molecular and Public Health Research Institute (PHRI), Rutgers the State Comparative Pathobiology, Johns Hopkins University; University of New Jersey, Newark, NJ, USA; Department of 4Department of Medicine, Albert Einstein College of Microbiology, Biochemistry, and Molecular Genetics, Medicine; 5Department of Neurology, Mount Sinai School Rutgers the State University of New Jersey, Newark, NJ, USA of Medicine Zika viral infection is correlated with an increased rate of HIV associated neurocognitive disorders (HAND) affect microcephaly in children born of mothers infected during >50 % of HIV-infected individuals. HAND is mediated by pregnancy and neurocognitive disorders in adults. Although entry of infected CD14+CD16+ monocytes into the CNS that documented as a mild febrile illness in less than two dozen establish and reseed CNS viral reservoirs, even when people cases prior to 2003, the Brazilian Zika outbreak strain spread have undetectable viral loads. CD14+CD16+ monocytes to over 40 counties and territories since its suspected origin in transmigrate across the blood–brain barrier (BBB) into the Brazil in 2015 and has infected over 7300 in the US and US CNS in response to chemokines, including CCL2. Viral res- territories since June 2015. Additionally, 479 pregnancies ervoirs release early viral proteins and cytokines, even during within the continental US and 493 pregnancies in US terri- active cART, that mediate persistent neuroinflammation and tories have had evidence of Zika infection during pregnancy. neuronal damage leading to HIV neuropathogenesis. To de- Microcephaly as a result of Zika has been identified in more velop therapeutics that can prevent viral (re)seeding of the than a dozen US births. The pathogenesis of Zika and its CNS and subsequent HAND, we examined the mechanism effects on the developing brain are unknown. Previous re- of entry of these HIV-infected cells into the CNS. We show search in our laboratory has demonstrated that astrocytes play J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S79 a significant role in perpetuating viral infection within the In uninfected animals NR treatment had no significant brain. Our laboratory has identified astrocytes as key viral impact on the KTR relative to pretreatment values. In reservoirs and mediators of apoptosis in CNS cells during infected animals, however, NR treatment appeared to in- viral infection. Thus, our objective is to characterize crease KTR at 100 mg/kg dose, and decrease CD4/CD8 whether Zika infection in human fetal astrocytes alters ratio at the highest dose (400 mg/kg). The regulation of survival or glial and neuronal cells. We hypothesize that the kynurenine pathway appears to be complex, yet an Zika infection of human fetal astrocytes causes apoptosis attractive target for HIV therapeutics. Understanding the that contributes to the devastating CNS consequences of mechanism by which HIV modulates the KYN/TRP path- this virus. Our results indicate that Zika induces apoptosis way could provide new therapeutic targets for the treat- of glial and neuronal cells. Altogether, these results illus- ment of HIV and HAND. trate a distinct mechanism of Zika infection in human fetal astrocytes. Understanding infection in primary CNS resident cells is critical in understanding the pathogenesis P164 of Zika infection in the developing brain. A novel murine model of enterovirus-71 (EV-A71) -induced neurogenic pulmonary edema

P163 Carla Bianca Luena Victorio1, Yishi Xu1, Qimei Ng1, Beng Modulation of Tryptophan degradation via Hooi Chua2, Sylvie Alonso3, Vincent Chow3,KawBing the kynurenine pathway during HIV and SIV infection Chua1 (corresponding author: [email protected]) Stephani Velasquez1, Wendeline Wagner2,MarkG.Lewis2, Jay Rappaport1 1Temasek Lifesciences Laboratory; 2Curtin University; (corresponding author: [email protected]) 3Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore 1Department of Neuroscience, Lewis Katz School of Medicine Temple University; 2Bioqual Enterovirus 71 (EV-A71) is now the most common neuro- trophic enterovirus following the near eradication of poliovi- Tryptophan (TRP) is an essential amino acid and precursor for ruses. It causes Hand, Foot, and Mouth Disease (HFMD) - a the de novo synthesis of nicotinamide adenine dinucleotide self-limiting febrile illness - and occasional fatal neurological (NAD), as well as serotonin and melatonin, by two different disease in children. Current animal models of EV-A71 infec- pathways. TRP is catabolized by the kynurenine (KYN) path- tion are not clinically authentic. They fail to replicate the full way to produce NAD and the methoxyindole pathway to gen- spectrum of human diseaseäóîspecifically that of neurogenic erate serotonin and melatonin. Increased KYN/TRP ratios pulmonary edema (NPE), the major cause of EV-A71 infec- have been associated with the progression of acquired im- tion-related child mortalityäóîand lack key pathological fea- mune deficiency syndrome (AIDS) as determined by associ- tures observed in fatal human cases. In order to more fully ation with lower CD4/CD8 ratios, reduced T cell recovery understand the neuro-pathogenesis of severe EV-A71 infec- after initiation or cART, and increased mortality risk. tion, especially with respect to NPE, our group generated nov- Activation of the KYN pathway as well as the neurotoxic el mouse-cell-adapted (MCA-EV-A71) viral strains that pro- metabolite quinolinic acid has been implicated in the patho- ductively infected both primate and rodent cell lines (Victorio genesis of HIV associated neurocognitive disorders (HAND). et al., 2014) and utilize the physiologically expressed viral We identified an increase in the KYN/TRP ratios in the SIV receptor expressed in rodents (mSCARB2) to infect murine macaque model with a positive correlation with viral load and cells (Victorio et al., 2016a). We subsequently used these soluble CD163 in plasma, with the latter suggesting the role of strains to infect 1-week-old BALB/c mice. The infected mice macrophage activation in this process. In an effort to modulate not only exhibited severe, acute lethal disease, but also in- the K/T pathway, and decreased tryptophan catabolism, we duced a spectrum of neurological deficits, including that of performed a dose escalation study with the NAD salvage path- NPE. We noted signs of respiratory distress and upon necrop- way precursor, nicotinamde riboside (NR), in SIV infected sy, gross pathological observations confirmed pulmonary ede- macaques. NR treatment significantly reduced the mean fluo- ma. Pathological findings in the lung tissues include focal rescence intensity and percent frequency of CD14+/CD16+ haemorrhagic lesions and accumulation of proteinaceous fluid monocytes as determined by flow cytometry, in both SIV in the alveoli; while CNS tissue sections revealed massive infected and uninfected animals. This monocyte subset has neuronal destruction and accumulation of viral antigens in been implicated in the pathogenesis of HIV infection, the brainstemäóîmost notably in the medulla oblongata HAND, as well as other HIV associated comorbidities. (Victorio et al., 2016b). We also observed high levels of serum S80 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 catecholamines, confirming NPE. Thus, we have developed samples from clinically well-defined HIV+ patients (n =45), an authentic mouse model of EV-A71 infection that re- including both symptomatic and asymptomatic individuals, capitulated the wide spectrum of clinical signs observed and representing a range of CNS disease severities, HIV-1 in patients as well as the underlying pathological fea- infection was measured by DNAScope. We observed an in- tures observed in fatal cases (Wong et al., 2008). This crease in HIV-1 staining that correlated with increased model could aid in the further understanding of the disease severity. Significantly, HIV-1 DNA was detected pathogenesis of EV71-induced neurological diseases in both macrophages/microglia (CD68) and astrocytes and in the development of novel therapies and (GFAP) in virally suppressed patients. Further, HIV-1 prophylactics. Envelope V3 sequences isolated by laser capture micro- dissection from macrophages/microglia of 1 patient, demonstrated compartmentalization relative to V3 sequences P165 isolated from PBMCs from the same patient. DNAScope Detection of HIV-1 DNA in brain tissue of virally coupled with laser capture micro-dissection, is a powerful suppressed patients supports the existence of a persistent technique that allows for the detection, quantification and CNS viral reservoir characterisation of HIV-1 infected cells within the CNS and allows for the characterization of the CNS viral res- Emma L. Wanicek1, Lachlan R. Gray2, Wan-Jung Cheng1, ervoir. This study provides strong evidence that HIV-1 Jacob D. Estes3,SharonR.Lewin4,PaulR.Gorry5,Melissa DNA can persist within the CNS of virally suppressed J. Churchill6 patients and supports the existence of a CNS viral reser- (corresponding author: [email protected]) voir with important implications for cure research and the development of cure strategies. 1Centre for Biomedical Research, Burnet Institute, Melbourne, VIC; 2Centre for Biomedical Research, Burnet Institute, Melbourne, VIC 2. Department of Infectious P166 Diseases, Alfred Hospital and Monash University, Investigating the role of Tetherin-dependent Melbourne, VIC; 3AIDS and Cancer Virus Program, pro-inflammatory microparticle release Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD; 4Department of Emily Weber, Meera Singh, Vir Singh, Joe Jackson, Nicole Infectious Diseases, Alfred Hospital and Monash University, Stirpe, Sanjay Maggirwar Melbourne, VIC 2. The Peter Doherty Institute for Infection (corresponding author: EmilyA_Weber@URMC. and Immunity, The University of Melbourne and Royal Rochester.edu) Melbourne Hospital, Melbourne, VIC; 5Centre for Biomedical Research, Burnet Institute, Melbourne, VIC 2. Department of Microbiology and Immunology, University of Department of Infectious Diseases, Alfred Hospital and Rochester School of Medicine and Dentistry Monash University, Melbourne, VIC 3. School of Health and Biomedical Sciences, RMIT University, Melbourne, Despite the emergence of combined antiretroviral therapy VIC 4. Department o; 6Centre for Biomedical Research, (cART) that is able to suppress the human immunodeficiency Burnet Institute, Melbourne, VIC 2. Department of virus (HIV) to undetectable levels in plasma, HIV-positive Medicine, Monash University, Melbourne, VIC 3. individuals still face an array of co-morbidities, including an Department of Microbiology, Monash University, Clayton, increased risk of neurocognitive disorders. Studies have sug- VIC gested that chronic inflammation resulting from HIV infection may be due to cytokines/chemokines and viral proteins secret- Combination antiretroviral therapy (cART) has transformed ed by activated/infected cells as well as cART administration. patient care and disease outcomes for millions of HIV-1 pa- In particular, small vesicles known as microparticles (MPs) tients globally. However, cART alone is unable to eradicate secreted by various activated cell types, such as monocytes, HIV-1 due to the persistence of numerous viral reservoirs often contain pro-inflammatory signals, which can contribute throughout the body. The central nervous system (CNS) is a to neurodegenerative diseases. Increased inflammatory MP potential viral reservoir but is difficult to study due to limita- levels have been reported in cART treated HIV-infected indi- tions in access to brain tissue. Here, we have applied a novel viduals. Recent data from our lab suggests that the host re- and highly sensitive (single copy sensitivity) in situ hybridi- striction factor Tetherin (BST-2/CD317) can sequester and zation technique called DNAScope, to detect and visualize inhibit the release of MPs from monocytes. Further, mono- HIV-1 DNA in the brain tissue from HIV-infected individuals cytes from HIV-infected individuals have reduced levels of on suppressive cART. Using a comprehensive bank of tissue Tetherin, and plasma samples from these individuals show J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S81 increased levels of monocyte-derived MPs. Exposure to the macrophages. To evaluate EAR, human monocyte derived HIV viral protein, Tat, can lead to the down regulation of macrophages were infected with HIV. By immunoblot, HIV surface levels of Tetherin on monocytes and an increased suppressed HO-1 levels, in macrophages, while concur- release of monocyte-derived MPs from healthy individ- rent treatment with SDG increased HO-1 levels. This uals. Further, MP sequestration is observed on the surface was paralleled by increased translocation of nrf2 protein of monocytes that over express a degradation-resistant from the cytoplasm to the nucleus and decreased nuclear mutant of Tetherin, in which the sequence that is used expression of HO-1’s negative regulator, Bach1. for Vpu-mediated ubiquation is mutated. Additional data Prolonged HO-1 deficiency in macrophages has been neg- from mice harboring a degradation-resistant form of atively correlated with increased HIV replication in hu- Tetherin shows decreased levels of MPs and increased man macrophages. HO-1 levels were decreased in macro- blood brain barrier function. Information gleaned from phages 12-day post infection. To determine if SDG can these studies will reveal the underlying mechanism of reverse prolonged HO-1 deficits, macrophages were Tetherin-mediated MP release and its role in neurological pretreated with SDG for 1 h prior to infection and disease along with other co-morbidities evident in HIV- replenished every 3 days. SDG treatment increased HO- infected individuals. Furthermore, this work is important 1 protein levels after 12 days post infection. Given these in enhancing our understanding of how this novel molec- data, SDG increases endogenous antioxidants pathways ular mechanism of Tetherin-dependent MP release relates and may be a possible adjunctive therapeutic for HIV to HIV infected individuals, as well as other MP associ- associated neurocognitive disorders. ated diseases.

P168 P167 Insulin Receptor Secretion is not associated Effects of flaxseed lignin, Secoisolariciresinol diglucose, with abnormal glucose metabolism in HIV-seropositive on endogenous antioxidant pathway in HIV-infected women macrophages Valerie Wojna1, Miriam Matos2, Rosa J. Rodriguez-Benitez2, Kimberly Williams, Andrea Guerrero, Shelley Pu, Kelly Raissa Menendez-Delmestre2, Avindra Nath3, Yamil Gerena4 Jordan-Sciutto (corresponding author: [email protected]) (corresponding author: [email protected]) 1Neurology Division, NeuroAIDS Program, University of University of Pennsylvania Puerto Rico, Medical Sciences Campus; 2NeuroAIDS Program, University of Puerto Rico, Medical Sciences Macrophages and microglia play pivotal roles in pathogen- Campus; 3Section of Infections of the Nervous System, esis HIV associated neurocognitive disorders. During acute NINDS, NIH; 4Department of Pharmacology, NeuroAIDS infection, activated and/or infected monocytes infiltrate the Program, University of Puerto Rico, Medical Sciences central nervous system (CNS) and differentiate into Campus perivascular macrophages. HIV can then spread to microglia, creating an active, protected reservoir in the CNS. The Background: Previously we found that high levels of soluble ensuing inflammatory macrophage/microglia activation and insulin receptor (sIR) in the plasma of HIV-seropositive wom- secretion of a myriad of toxic factors cause damage to neu- en were associated with HIV-associated neurocogntivie disor- rons, which are not infected. Thus, current work strives to ders (HAND). Although we found evidence of cellular insulin develop strategies to suppress M/M-driven inflammation and resistance it is not clear if sIR is associated with peripheral oxidative stress. Numerous studies utilizing exogenous anti- insulin resistance as determined by the homeostasis model inflammatory and antioxidants to mitigate disease progres- assessment-estimated insulin resistance (HOMA-IR) index. sion have been unsuccessful; however, targeting endogenous In this study we investigated if sIR is associated with antioxidant pathways may be more advantageous. A cellular HOMA-IR. Methods: 50 HIV-seropositive women and 18 mechanism that mitigates oxidative stress and inflammation controls without history of diabetes were tested for oral glu- is the endogenous antioxidant response (EAR) pathway, cose tolerance test (including fasting blood sugar [FBS] and which upregulates key antioxidant enzymes, including heme insulin levels), glycosylated hemoglobin (HgA1c), HOMA- oxygenase 1 (HO-1), to reduce free radicals and suppress IR, and plasma sIR full-length. Participants were also evalu- tissue damage. In this study, we investigated the role of a ated for metabolic syndrome with blood pressure, anthropo- flaxseed lignin, Secoisolariciresinol Diglucose (SDG), on metric measures, and lipid profile. HAND was determined viral replication and oxidative stress in HIV-infected following the HAND nosology criteria. Results: No S82 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 significant differences were observed between controls and bioluminescence imaging showed a significant reduction of HIV-seropositive women stratified by HAND (normal cogni- EcoHIV infection in the neck lymph nodes and whole body. tion [18], asymptomatic neurocognitive impairment To demonstrate if Cas9/sgRNAs excise HIV provirus in la- [ANI,13], and symptomatic neurocognitive impairment tently infected cells in vivo, we administrated quadruplex [SNI, 19]) regarding age, BMI, HgA1c, insulin and sgRNA/saCas9 AAV-DJ/8 (2x10e12 GC/mouse, i.v.) in FBS, and HOMA-IR. An association was observed be- humanized Bone marrow-Liver-Thymus (BLT) mice with tween plasma sIR levels and HAND (p =0.003), where intravaginal inoculation of HIV-1NL-BaL-eLuc. These women with ANI presented with higher levels. No HIV-infected BLT mice showed significant vaginal infec- Spearman’s correlation were observed between plasma tion during the first 3 weeks of inoculation but no observed sIR levels and age, BMI, HIV RNA copies/mL, CD4 cell systemic HIV dissemination for at least 60 days prior to count, use of protease inhibitors, HGA1c, FBS, insulin AAV/saCas9 treatment. Successful excision of HIV-1 pro- levels, and HOMA-IR. Conclusion: Plasma sIR is not as- virus was detected by PCR genotyping in spleen, lung, sociated with standard measures of peripheral insulin re- heart, colon and brain as well as human thymic organoid sistance such as HOMA-IR. It is possible that the pres- on the left kidney as early as 2 weeks after AAV injection. ence of plasma sIR levels and its association with HAND In conclusion, in vivo excision of HIV proviral DNA in is related to a chronic subclinical cellular insulin resis- solid tissues/organs can be achieved via a single intrave- tance. Supported by R21MH095524, R01NS099036, nous injection of AAV-DJ/8 carrying quadruplex sgRNA/ U54MD007587, G12MD007600 saCas9. This is an important step moving forward to the clinical application of the CRISPR/Cas9 gene editing strategy. Supported by NIH grants to KK/WH (R01NS087971), P169 KK (P30MH092177) and WY (R21MH100949). In vivo excision of HIV provirus by SaCas9 and multiplex sgRNAs in pre-clinical animal models P170 Chaoran Yin1,TingZhang1,XiyingQu1, Yonggang Zhang1, Evidence that Dysfunctional Endolysosomal Biogenesis is Raj Putatunda1, Fang Li1, Huaqing Zhao1,ShenDai1,Xuebin Associated with Cognitive Impairment in HIV-Infected Qin1, Xianming Mo2, Jennifer Gordon1, Won-bin Young3, Individuals; Agonists of TRPML1 May Restore Kamel Khalili1, Wenhui Hu1 Lysosomal Function Through Luminal Acidification (corresponding author: [email protected]) Seung-Wan Yoo1, Joshua Lisinicchia2, Jacqueline Lovett1, 1Department of Neuroscience, Center for Neurovirology and Benjamin B Gelman2, Norman J Haughey3 The Comprehensive NeuroAIDS Center, Temple University (corresponding author: [email protected]) Lewis Katz School of Medicine; 2Laboratory of Stem Cell Biology, State Key Laboratory of Biotherapy, West China 1Department of Neurology, Richard T Johnson Division of Hospital, West China Medical School, Sichuan University; Neuroimmunology and Neurological Infections, Johns 3Department of Radiology, University of Pittsburgh School Hopkins University School of Medicine, Baltimore, of Medicine, Pittsburgh Maryland; 2Department of Pathology, Neuroscience and Cell Biology, University of Texas Medical Branch Galveston, Cas9/gRNA-mediated genome editing provides a promising Texas; 3Department of Neurology, Richard T Johnson cure for HIV-1/AIDS by excising the proviral DNA from host Division of Neuroimmunology and Neurological Infections, genome. Here, we demonstrate the feasibility of excising the Johns Hopkins University School of Medicine, Baltimore, HIV-1 proviral genome in experimental animal models using Maryland saCas9 along with 2–4 multiplex sgRNAs in a single AAV vector. Both duplex and quadruplex sgRNAs/saCas9 can be Dysfunction of endolysosomal systems in HIV-Associated efficiently packaged into AAV-DJ or AAV-DJ/8 with high titer Neurocognitive Disorders (HAND) has been implicated by dis- at 10e13-14 genome copy (GC)/ml. The quadruplex sgRNA/ turbances in transluminal ion gradients, and intraluminal depo- saCas9 vector outperformed the duplex one in cleaving the sition of proteins and lipids. Here, we determined if the integrated HIV-1 genome in cultured neural stem/progenitor Coordinated Lysosomal Expression And Regulation cells and various organs of HIV Tg26 transgenic mice. (CLEAR) gene network is impaired by HIV infection using Excision of HIV-1 proviral DNA by quadruplex AAV-DJ/8 tissues from the National NeuroAIDS Tissue Consortium (i.v.) was demonstrated by PCR genotyping in liver, lung, (NNTC; mini gauntlet sample set). Frontal neocortices were brain and spleen of the mice inoculated (i.v.) with chimeric obtained from HIV+ cognitively normal (CN; n = 41), EcoHIV carrying a firefly luciferase reporter (eLuc). Live subsyndromic (SS; n = 9), Minor Cognitive Motor Disorder J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 S83

(MCMD; n = 36), HIV Associated Dementia (HAD; n = 29), known to change with age and drug use. As the HIV and HIV- controls (n= 68). All HIV infected subjects showed infected population is steadily aging, the effects of age alterations in the expression of multiple genes coding for hy- andpastdrugusemayconfound attempts to identify drolytic enzymes and luminal acidification compared to HIV-. specific effects of serostatus on WM microstructure. In A master regulator of lysosomal biogenesis, transcription factor a cross-sectional study, we examined the effects of EB, was increased, and genes related to luminal acidification serostatus, age and past drug use on WM microstruc- (chloride channel 7 and transient receptor potential mucolipin- ture. Methods: Participants included 15 seropositive and 1; TRPML1) were decreased in MCMD and HAD compared to 20 seronegative men, ages 54–77, with 12 reporting CN. These data suggest that HIV infection is associated with a past drug use. Seropositive participants were all treated dysregulation of the CLEAR network in neocortex, and cogni- with anti-retroviral therapies. Mixed effects models were tive impairment is specifically associated with the induction of used to explore effects of serostatus, race, age, and drug lysosomal biogenesis and a reduced ability for luminal acidifi- use on executive function, learning/memory, attention/ cation. Based on these human data we next used a rodent model working memory, verbal fluency, processing speed, and of HIV-associated endolysosomal dysfunction (gp120/APP/ motor performance domains. WM microstructure mea- PS1 mice) in which the CLEAR network is impaired and lyso- sures included fractional anisotropy (FA) and apparent somes are engorged with Abeta and sphingomyelin to deter- diffusion coefficient (ADC). Results: Seropositive partic- mine if luminal acidification could rescue lysosomal function. ipants exhibited bihemispheric increases in ADC A 26 day intraventricular infusion of the TRPML1 agonist ML- (p < 0.05 FWE-corrected) and FA (p <0.05 FWE- SA1 by mini osmotic pump reduced cortical Abeta, corrected). Independent effects of age and past drug sphingomyelin, and restored CLEAR network gene expression use on FA and ADC were seen (p <0.05 FWE- to Wt levels. These findings suggest that therapeutic approaches corrected). Processing speed decreased with age to enhance lysosomal function may protect the CNS in the (p < 0.01). Motor performance decreased with age, setting of HIV infection. showing effects of race and an interaction between serostatus and age (all p < 0.01). Executive function, verbal fluency, attention/working memory, and learning/ P171 memory did not show effects of serostatus, age, race or Drug Use and Age Effects on White Matter drug use. Conclusions: In a sample of cognitively intact Microstructure and Behavior in Treated HIV Infection older participants, we observed separate effects of serostatus, age and past drug use on two measures of Thomas Zeffiro1,ErinO’Connor2, Melissa Murray3, WM microstructure and associated slowing of move- Lawrence Kingsley3, James Becker3 ment execution. The relatively wide variation in pub- (corresponding author: [email protected]) lished studies of HIV infection effects on white-matter microstructure might reflect differences among experi- 1Temple University; 2Lewis Katz School of Medicine at mental samples related to age or past drug use. Temple University; 3University of Pittsburgh Moreover, WM microstructural changes following HIV infection, while associated with slow motor perfor- Purpose: Although diffusion tensor imaging (DTI) studies mance, are not invariably associated with cognitive have documented white matter (WM) microarchitecture changes assessed across multiple domains. changes following HIV infection, WM properties are also S84 J. Neurovirol. (2016) 22 (Suppl 1):S1–S89 Author Index

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