DOCSLIB.ORG

Mutations in the HIV-1 Envelope Glycoprotein Can Broadly Rescue Blocks at Multiple Steps in the Virus Replication Cycle

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mutations in the HIV-1 Envelope Glycoprotein Can Broadly Rescue Blocks at Multiple Steps in the Virus Replication Cycle Mutations in the HIV-1 envelope glycoprotein can broadly rescue blocks at multiple steps in the virus replication cycle Rachel Van Duynea, Lillian S. Kuoa,1, Phuong Phama, Ken Fujiia,2, and Eric O. Freeda,3 aVirus–Cell Interaction Section, HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702 Edited by Joseph G. Sodroski, Dana-Farber Cancer Institute, Boston, MA, and accepted by Editorial Board Member Stephen P. Goff March 19, 2019 (received for review November 29, 2018) The p6 domain of HIV-1 Gag contains highly conserved peptide efficient mode of viral propagation than cell-free infection, is motifs that recruit host machinery to sites of virus assembly, thereby initiated by interactions between Env expressed on the surface of promoting particle release from the infected cell. We previously the infected cell and CD4 on the surface of the target cell, in the – reported that mutations in the YPXnL motif of p6, which binds the absence of cell cell fusion, inducing the formation of a virological host protein Alix, severely impair HIV-1 replication. Propagation of synapse (VS) (27). Alternatively, when cell-surface HIV-1 Env en- the p6–Alix binding site mutants in the Jurkat T cell line led to the gages CD4 on target cells, cell fusion can occur, resulting in the emergence of viral revertants containing compensatory mutations formation of multinucleated cells, or syncytia. Several studies have not in Gag but in Vpu and the envelope (Env) glycoprotein subunits demonstrated the importance of cell-to-cell transmission in vitro in gp120 and gp41. The Env compensatory mutants replicate in Jurkat overcoming barriers to cell-free infection, including target cell T cells and primary human peripheral blood mononuclear cells, de- infectability, virus stability, and defects in virus production (28–30). spite exhibiting severe defects in cell-free particle infectivity and Env- Additionally, cell-to-cell transmission can allow HIV-1 spread in the mediated fusogenicity. Remarkably, the Env compensatory mutants presence of broadly neutralizing antibodies (bNabs) (31). Finally, can also rescue a replication-delayed integrase (IN) mutant, and ex- cell-to-cell transmission of HIV-1 has been shown to be less sensitive hibit reduced sensitivity to the IN inhibitor Dolutegravir (DTG), dem- to antiretrovirals (ARVs) compared with cell-free transmission (29, onstrating that they confer a global replication advantage. In 32–35). The ability of the virus to evade blocks to infection may in addition, confirming the ability of Env mutants to confer escape from part be attributed to a higher multiplicity of infection (MOI) during DTG, we performed de novo selection for DTG resistance and ob- cell-to-cell vs. cell-free infection, allowing for a higher percentage of served resistance mutations in Env. These results identify amino acid cells to be infected with more than one virus (36). These findings substitutions in Env that confer broad escape from defects in virus raise the intriguing possibility that HIV-1 could potentially escape replication imposed by either mutations in the HIV-1 genome or by an the inhibitory activity of antiviral agents through the acquisition of – antiretroviral inhibitor. We attribute this phenotype to the ability of mutations in Env that promote highly efficient cell cell transmission. the Env mutants to mediate highly efficient cell-to-cell transmission, We have previously shown that mutations in the Alix binding resulting in an increase in the multiplicity of infection. These findings site of p6 induce relatively minor defects in Gag processing, virus have broad implications for our understanding of Env function and release, and cell-free particle infectivity, but impose significant the evolution of HIV-1 drug resistance. delays in replication kinetics in physiologically relevant cell types drug resistance | cell–cell transmission | Dolutegravir | virological synapse Significance he assembly of HIV type 1 (HIV-1) particles is driven by the HIV-1 adapts over time to bypass blocks imposed by genetic Texpression of the viral Gag polyprotein precursor, Pr55Gag, lesions in the viral genome, typically by acquiring compensa- which contains several major structural domains required for tory mutations in the defective gene itself. Here we report that virus-like particle production, including the p6 domain that HIV-1 can evade replication blocks by acquiring mutations in promotes membrane scission to release budding virions (1–4). the envelope (Env) glycoprotein that enhance cell-to-cell HIV-1 p6 encodes two highly conserved peptide motifs, known as transmission. We identified mutations in Env that arose in “late domains,” that recruit components of the cellular endosomal the presence of the antiretroviral inhibitor Dolutegravir, thereby circumventing restriction. These data, which demon- sorting complexes required for transport (ESCRT) machinery to – sites of virus assembly (5–7). The physiological function of the strate that mutations in Env can provide escape from an anti HIV-1 drug in vitro, could have broad implications for HIV-1 drug ESCRT apparatus is to drive membrane-scission reactions that resistance and viral transmission. occur in a variety of cellular contexts, including the biogenesis of – multivesicular bodies and cytokinesis (5 7). The Pro-Thr/Ser-Ala- Author contributions: R.V.D., L.S.K., K.F., and E.O.F. designed research; R.V.D., L.S.K., P.P., Pro (PT/SAP) motif of p6 interacts directly with the ESCRT-I and K.F. performed research; R.V.D., L.S.K., P.P., and K.F. analyzed data; and R.V.D. and subunit Tsg101 (8–14); the Tyr-Pro-Xn-Leu (YPXnL, where X is E.O.F. wrote the paper. any residue and n = 1–3 amino acids) motif of p6 binds the ESCRT- The authors declare no conflict of interest. associated protein Alix (15–21). While the requirement for the p6– This article is a PNAS Direct Submission. J.G.S. is a guest editor invited by the Tsg101 interaction in HIV-1 release is well established, the physi- Editorial Board. ological role of p6–Alix binding is less well defined. Published under the PNAS license. Expression of Gag alone is sufficient for the formation of 1Present address: Division of AIDS, National Institute of Allergy and Infectious Diseases, virus-like particles, but the incorporation of the HIV-1 envelope Bethesda, MD 20892. (Env) glycoprotein complex is required for the generation of 2Present address: Neurovirology Project, Tokyo Metropolitan Institute of Medical Science, infectious particles. Env expression on the membranes of both 156-8506 Tokyo, Japan. free virions and infected cells promotes viral spread. Productive 3To whom correspondence should be addressed. Email: [email protected]. viral transmission from infected to uninfected cells can occur via This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. two pathways: cell-free infection or cell-to-cell transmission (22– 1073/pnas.1820333116/-/DCSupplemental. 26). The latter pathway, which is thought to be a more rapid and Published online April 11, 2019. 9040–9049 | PNAS | April 30, 2019 | vol. 116 | no. 18 www.pnas.org/cgi/doi/10.1073/pnas.1820333116 Downloaded by guest on October 1, 2021 (37). To further characterize the significance of p6–Alix interac- structed pNL4-3 p6/Env and p6/Vpu mutant clones and evalu- tions, we selected for viral revertants that alleviate the replication ated their replication kinetics, in parallel with WT and the defects imposed by a panel of mutations in the p6 YPXnLmotif. original p6-mutant clones, in Jurkat cells. The Vpu-inactivating We identified second-site compensatory changes in both Vpu and mutations partially rescued the replication-defective p6-Y36S/L44R Env that rescue replication defects imposed by the mutations in p6. and p6-L41A mutants (SI Appendix,Fig.S1C and D). The p6-Y36A The three Env compensatory mutations that arose can rescue virus replication defect was largely rescued by both Env-P81S and Env- replication despite exhibiting severe defects in cell-free particle in- A556T (Fig. 2A). Similarly, the replication defects of both p6-Y36S/ fectivity. Strikingly, these Env mutations also provide a replication L44H and p6-L41R were rescued by Env-A556T, with reverse- advantage in the context of an integrase (IN) mutant and in the transcriptase (RT) peaks occurringatornearthedayofpeakRT presence of the IN strand-transfer inhibitor (INSTI) Dolutegravir for the WT (Fig. 2 B and D, respectively). In contrast, the replication (DTG). De novo selection in the presence of DTG led to the defective p6-Y36A substitution was not rescued by Env-A327T (SI acquisition of at least one additional Env mutation that confers Appendix,Fig.S1B) and the p6-Y36S/L44R mutations were not cell-line–independent resistance to DTG in vitro. We attribute rescued by Env-I744V or Env-R786K (SI Appendix,Fig.S1C). The the decreased DTG sensititivity of the Env mutants to their ability delay in replication exhibited by p6-L41A was rescued by Env-Y61H, to efficiently transmit viral material in a cell-associated manner, but not by Env-R166I (Fig. 2C and SI Appendix,Fig.S1D). Because resulting in an increased MOI during spreading infections. the Env mutations R166I, A327T, I744V, and R786K did not con- tribute to rescue of the p6 mutants, they were not analyzed further. Results Similarly, considering that Vpu mutations often arise during propa- p6–Alix-Binding Site Mutants Acquire Second-Site Mutations in Vpu gation of replication-defective HIV-1 mutants in Jurkat cells, we and Env. To further characterize the role of the p6–Alix in- elected to focus on the Env compensatory mutations. teraction in HIV-1 replication, we propagated the p6 mutants (Fig. 1A, Left) in culture to select for viral revertants. The Jurkat T cell Env Compensatory Mutants Display Highly Efficient Replication line was transfected with pNL4-3 WT and p6 mutant proviral clones Kinetics in Jurkat T Cells and Peripheral Blood Mononuclear Cells and virus replication was monitored over time.
Load more

Recommended publications
  • The Nef-Infectivity Enigma: Mechanisms of Enhanced Lentiviral Infection Jolien Vermeire§, Griet Vanbillemont§, Wojciech Witkowski and Bruno Verhasselt*
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central 474 Current HIV Research, 2011, 9, 474-489 The Nef-Infectivity Enigma: Mechanisms of Enhanced Lentiviral Infection Jolien Vermeire§, Griet Vanbillemont§, Wojciech Witkowski and Bruno Verhasselt* Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University, Belgium Abstract: The Nef protein is an essential factor for lentiviral pathogenesis in humans and other simians. Despite a multitude of functions attributed to this protein, the exact role of Nef in disease progression remains unclear. One of its most intriguing functions is the ability of Nef to enhance the infectivity of viral particles. In this review we will discuss current insights in the mechanism of this well-known, yet poorly understood Nef effect. We will elaborate on effects of Nef, on both virion biogenesis and the early stage of the cellular infection, that might be involved in infectivity enhancement. In addition, we provide an overview of different HIV-1 Nef domains important for optimal infectivity and briefly discuss some possible sources of the frequent discrepancies in the field. Hereby we aim to contribute to a better understanding of this highly conserved and therapeutically attractive Nef function. Keywords: Nef, HIV, infectivity, viral replication, mutation analysis, envelope protein, cholesterol, proteasome. 1. INTRODUCTION 2. THE MULTIFACETED NEF PROTEIN Despite the globally declining number of new human As early as 1991, infections of rhesus monkeys with nef- immunodeficiency virus (HIV) infections [1] and the hopeful deleted simian immunodeficiency virus (SIV) revealed a observation that cure from HIV infection does not seem dramatic reduction of viral loads and disease progression in impossible [2], the HIV pandemic still remains a very absence of nef [6].
    [Show full text]
  • HIV-1 Rev Downregulates Tat Expression and Viral Replication Via Modulation of NAD(P)H:Quinine Oxidoreductase 1 (NQO1)
    ARTICLE Received 25 Jul 2014 | Accepted 22 Apr 2015 | Published 10 Jun 2015 DOI: 10.1038/ncomms8244 HIV-1 Rev downregulates Tat expression and viral replication via modulation of NAD(P)H:quinine oxidoreductase 1 (NQO1) Sneh Lata1,*, Amjad Ali2,*, Vikas Sood1,2,w, Rameez Raja2 & Akhil C. Banerjea2 HIV-1 gene expression and replication largely depend on the regulatory proteins Tat and Rev, but it is unclear how the intracellular levels of these viral proteins are regulated after infection. Here we report that HIV-1 Rev causes specific degradation of cytoplasmic Tat, which results in inhibition of HIV-1 replication. The nuclear export signal (NES) region of Rev is crucial for this activity but is not involved in direct interactions with Tat. Rev reduces the levels of ubiquitinated forms of Tat, which have previously been reported to be important for its transcriptional properties. Tat is stabilized in the presence of NAD(P)H:quinine oxidoreductase 1 (NQO1), and potent degradation of Tat is induced by dicoumarol, an NQO1 inhibitor. Furthermore, Rev causes specific reduction in the levels of endogenous NQO1. Thus, we propose that Rev is able to induce degradation of Tat indirectly by downregulating NQO1 levels. Our findings have implications in HIV-1 gene expression and latency. 1 Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi 110095, India. 2 Laboratory of Virology, National Institute of Immunology, New Delhi 110067, India. * These authors contributed equally to this work. w Present address: Translational Health Science and Technology Institute, Faridabad, Haryana 121004, India. Correspondence and requests for materials should be addressed to A.C.B.
    [Show full text]
  • Opportunistic Intruders: How Viruses Orchestrate ER Functions to Infect Cells
    REVIEWS Opportunistic intruders: how viruses orchestrate ER functions to infect cells Madhu Sudhan Ravindran*, Parikshit Bagchi*, Corey Nathaniel Cunningham and Billy Tsai Abstract | Viruses subvert the functions of their host cells to replicate and form new viral progeny. The endoplasmic reticulum (ER) has been identified as a central organelle that governs the intracellular interplay between viruses and hosts. In this Review, we analyse how viruses from vastly different families converge on this unique intracellular organelle during infection, co‑opting some of the endogenous functions of the ER to promote distinct steps of the viral life cycle from entry and replication to assembly and egress. The ER can act as the common denominator during infection for diverse virus families, thereby providing a shared principle that underlies the apparent complexity of relationships between viruses and host cells. As a plethora of information illuminating the molecular and cellular basis of virus–ER interactions has become available, these insights may lead to the development of crucial therapeutic agents. Morphogenesis Viruses have evolved sophisticated strategies to establish The ER is a membranous system consisting of the The process by which a virus infection. Some viruses bind to cellular receptors and outer nuclear envelope that is contiguous with an intri‑ particle changes its shape and initiate entry, whereas others hijack cellular factors that cate network of tubules and sheets1, which are shaped by structure. disassemble the virus particle to facilitate entry. After resident factors in the ER2–4. The morphology of the ER SEC61 translocation delivering the viral genetic material into the host cell and is highly dynamic and experiences constant structural channel the translation of the viral genes, the resulting proteins rearrangements, enabling the ER to carry out a myriad An endoplasmic reticulum either become part of a new virus particle (or particles) of functions5.
    [Show full text]
  • Filoviral Immune Evasion Mechanisms
    Viruses 2011, 3, 1634-1649; doi:10.3390/v3091634 OPEN ACCESS viruses ISSN 1999-4915 www.mdpi.com/journal/viruses Review Filoviral Immune Evasion Mechanisms Parameshwaran Ramanan 1,3, Reed S. Shabman 2, Craig S. Brown 1,4, Gaya K. Amarasinghe 1,*, Christopher F. Basler 2,* and Daisy W. Leung 1,* 1 Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA 2 Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029, USA 3 Biochemistry Graduate Program, Iowa State University, Ames, IA 50011, USA 4 Biochemistry Undergraduate Program, Iowa State University, Ames, IA 50011, USA * Authors to whom correspondence should be addressed; [email protected] (G.K.A); [email protected] (C.F.B); [email protected] (D.W.L.). Received: 11 August 2011 / Accepted: 15 August 2011 / Published: 7 September 2011 Abstract: The Filoviridae family of viruses, which includes the genera Ebolavirus (EBOV) and Marburgvirus (MARV), causes severe and often times lethal hemorrhagic fever in humans. Filoviral infections are associated with ineffective innate antiviral responses as a result of virally encoded immune antagonists, which render the host incapable of mounting effective innate or adaptive immune responses. The Type I interferon (IFN) response is critical for establishing an antiviral state in the host cell and subsequent activation of the adaptive immune responses. Several filoviral encoded components target Type I IFN responses, and this innate immune suppression is important for viral replication and pathogenesis. For example, EBOV VP35 inhibits the phosphorylation of IRF-3/7 by the TBK-1/IKKε kinases in addition to sequestering viral RNA from detection by RIG-I like receptors.
    [Show full text]
  • A Novel Ebola Virus VP40 Matrix Protein-Based Screening for Identification of Novel Candidate Medical Countermeasures
    viruses Communication A Novel Ebola Virus VP40 Matrix Protein-Based Screening for Identification of Novel Candidate Medical Countermeasures Ryan P. Bennett 1,† , Courtney L. Finch 2,† , Elena N. Postnikova 2 , Ryan A. Stewart 1, Yingyun Cai 2 , Shuiqing Yu 2 , Janie Liang 2, Julie Dyall 2 , Jason D. Salter 1 , Harold C. Smith 1,* and Jens H. Kuhn 2,* 1 OyaGen, Inc., 77 Ridgeland Road, Rochester, NY 14623, USA; [email protected] (R.P.B.); [email protected] (R.A.S.); [email protected] (J.D.S.) 2 NIH/NIAID/DCR/Integrated Research Facility at Fort Detrick (IRF-Frederick), Frederick, MD 21702, USA; courtney.fi[email protected] (C.L.F.); [email protected] (E.N.P.); [email protected] (Y.C.); [email protected] (S.Y.); [email protected] (J.L.); [email protected] (J.D.) * Correspondence: [email protected] (H.C.S.); [email protected] (J.H.K.); Tel.: +1-585-697-4351 (H.C.S.); +1-301-631-7245 (J.H.K.) † These authors contributed equally to this work. Abstract: Filoviruses, such as Ebola virus and Marburg virus, are of significant human health concern. From 2013 to 2016, Ebola virus caused 11,323 fatalities in Western Africa. Since 2018, two Ebola virus disease outbreaks in the Democratic Republic of the Congo resulted in 2354 fatalities. Although there is progress in medical countermeasure (MCM) development (in particular, vaccines and antibody- based therapeutics), the need for efficacious small-molecule therapeutics remains unmet. Here we describe a novel high-throughput screening assay to identify inhibitors of Ebola virus VP40 matrix protein association with viral particle assembly sites on the interior of the host cell plasma membrane.
    [Show full text]
  • Progressive Multifocal Leukoencephalopathy and the Spectrum of JC Virus-​Related Disease
    REVIEWS Progressive multifocal leukoencephalopathy and the spectrum of JC virus- related disease Irene Cortese 1 ✉ , Daniel S. Reich 2 and Avindra Nath3 Abstract | Progressive multifocal leukoencephalopathy (PML) is a devastating CNS infection caused by JC virus (JCV), a polyomavirus that commonly establishes persistent, asymptomatic infection in the general population. Emerging evidence that PML can be ameliorated with novel immunotherapeutic approaches calls for reassessment of PML pathophysiology and clinical course. PML results from JCV reactivation in the setting of impaired cellular immunity, and no antiviral therapies are available, so survival depends on reversal of the underlying immunosuppression. Antiretroviral therapies greatly reduce the risk of HIV-related PML, but many modern treatments for cancers, organ transplantation and chronic inflammatory disease cause immunosuppression that can be difficult to reverse. These treatments — most notably natalizumab for multiple sclerosis — have led to a surge of iatrogenic PML. The spectrum of presentations of JCV- related disease has evolved over time and may challenge current diagnostic criteria. Immunotherapeutic interventions, such as use of checkpoint inhibitors and adoptive T cell transfer, have shown promise but caution is needed in the management of immune reconstitution inflammatory syndrome, an exuberant immune response that can contribute to morbidity and death. Many people who survive PML are left with neurological sequelae and some with persistent, low-level viral replication in the CNS. As the number of people who survive PML increases, this lack of viral clearance could create challenges in the subsequent management of some underlying diseases. Progressive multifocal leukoencephalopathy (PML) is for multiple sclerosis. Taken together, HIV, lymphopro- a rare, debilitating and often fatal disease of the CNS liferative disease and multiple sclerosis account for the caused by JC virus (JCV).
    [Show full text]
  • Inhibition of Tat-Mediated Transactivation and HIV Replication with Tat Mutant and Repressor Domain Fusion Proteins
    Gene Therapy (1998) 5, 946–954 1998 Stockton Press All rights reserved 0969-7128/98 $12.00 http://www.stockton-press.co.uk/gt Inhibition of Tat-mediated transactivation and HIV replication with Tat mutant and repressor domain fusion proteins C Fraisier1,2, DA Abraham1, M van Oijen1, V Cunliffe3, A Irvine3, R Craig3 and EA Dzierzak1,2 1National Institute for Medical Research, Division of Eukaryotic Molecular Genetics, London, UK; 2Erasmus University Rotterdam, Department of Cell Biology and Genetics, Rotterdam, The Netherlands; and 3Therexsys Ltd, The Science Park, University of Keele, Keele, UK Strategies to inhibit the spread of HIV infection consist of moter. This fusion mutant was also examined for its a number of specific molecular approaches. Since viral capacity to block both Tat-mediated transactivation and production is dependent upon Tat-mediated transactivation HIV replication. We show that three mutants Tat⌬53, of the HIV promoter through the Tat activating region Tat⌬58 and Tat⌬53/Eng result in a transdominant pheno- (TAR), tat antisense RNA, anti-tat ribozymes, TAR decoys type inhibiting wild-type Tat-mediated transactivation, and and dominant negative Tat mutant proteins have been sug- that the inhibiting potential is increased by the presence of gested as therapeutic inhibitors. We produced and tested the entire basic domain or the fusion of a repressor several Tat mutant proteins, including a newly generated domain. However, only the transdominant mutants Tat⌬58 form Tat⌬58, for the ability to inhibit Tat-mediated trans- and Tat⌬53/Eng significantly inhibit HIV-1 replication after activation and HIV production. In addition, we generated a infection of transfected T cell lines.
    [Show full text]
  • Feline Origin of Rotavirus Strain, Tunisia
    Article DOI: http://dx.doi.org/10.3201/eid1904.121383 Feline Origin of Rotavirus Strain, Tunisia Technical Appendix Table 1. Primers used for amplification and sequencing of the whole genome of group A rotavirus strain RVA/human-wt/TUN/17237/2008/G6P[9] from Tunisia, 2008 Gene Primer name Primer sequence VP1 Gen_VP1Fb 5 -GGC TAT TAA AGC TRT ACA ATG GGG AAG -3 Gen_VP1Rb 5 -GGT CAC ATC TAA GCG YTC TAA TCT TG -3 MG6_VP1_447F 5 -TGC AGT TAT GTT CTG GTT GG -3 Hosokawa_VP1_2587R 5 -ACG CTG ATA TTT GCG CAC -3 LAP_VP1_1200F 5 -GCT GTC AAT GTC ATC AGC -3 Gen_VP1_2417R 5 -GCT ATY TCA TCA GCT ATT CCY G -3 30-96_VP1_3163F 5 -GGA TCA TGG ATA AGC TTG TTC TG -3 26097_VP1_269R 5 -GCG TTA TAC TTA TCA TAC GAA TAC G -3 VP2 Gen_VP2Fc 5 -GGC TAT TAA AGG YTC AAT GGC GTA CAG -3 Gen_VP2Rbc 5 -GTC ATA TCT CCA CAR TGG GGT TGG -3 26097_VP2_458F 5 -AGT TGC GTA ATA GAT GGT ATT GG -3 B1711_VP2_2112R 5 -GCA ATT TTA TCT GAG GCA CG -3 NCDV_VP2_1868F 5 -AGG ATT AAT GAT GCA GTG GC -3 LAP_VP2_2543F 5 -GAC ATC AAA TCT TAC CTT CAC TG -3 260-97_VP2_345R 5 -GAC TCT TTT GGT TCG AAA GTA GG -3 FR5_VP2_23F 5 -TAC AGG AAA CGT GGA GCG -3 260-97_VP2_744R 5 -GTACTCTTTGTCTCATTTCCGC -3 Gen_VP2_2739Ra 5 -TAC AAC TCG TTC ATG ATG CG -3 VP3 Gen_VP3_24F 5 -TGY GTT TTA CCT CTG ATG GTG-3 Gen_VP3_2584R 5 -TGA CYA GTG TGT TAA GTT TYT AGC-3 NCDV_VP3_2026R 5 -CAT GCG TAA ATC AAC TCT ATC GG -3 MG6_VP3_488F 5 -GCA GCT ACA GAT GATGAT GC -3 B10925_VP3_2416F 5 -ACA ATC GAG AAT GTT CAT CCC -3 TUN1_VP3_167R 5 -TTT CTA CTG CAG CTA TGC CAG-3 VP4 LAP_VP4_788F 5 -CCT TGT GGA AAG AAA TGC-3 VP4_2348-2368Re 5
    [Show full text]
  • Investigation of Proton Conductance in the Matrix 2 Protein of the Influenza Virus by Solution NMR Spectroscopy © Daniel Turman
    Investigation of proton conductance in the matrix 2 protein of the influenza virus by solution NMR spectroscopy © Daniel Turman Emmanuel College Class 0[2012 Abstract The Influenza Matrix 2 (M2) protein is a homo-tetrameric integral membrane protein that forms a proton selective transmembrane channell Its recognized function is to equilibrate pH across the viral envelope following endocytosis and across the trans-golgi membrane during viral maturation2 Its function is vital for viral infection and proliferation but the mechanism and selectivity of proton conductance is not well understood. Mutagenesis studies have identified histidine 37 as the pH sensing element and tryptophan 41 as the gating selectivity filter3 This study uses solution nuclear magnetic resonance spectroscopy and an M2 transmembrane protein construct to elucidate key interactions between the aromatic residues believed to confer proton selectivity and pH dependent conduction of M2 in the low pH open and high pH closed states. PH dependent 13 C_1 H HSQC-Trosy experiments were completed in the pH range of 8.0 - 4.0 and the 13 C, and 13 Co2 chemical shift perturbations of histidine 37 revealed multiple saturation points. The protonation states of histidine 37 suggest a shuttling mechanism for proton conduction. Introduction Influenza is a pathogenic virus that has reached pandemic status four times in the twentieth century. The latest pandemic occurred in 2009 from the influenza A HINI strain (figure 1t The World Health Organization (WHO) commented in July of 2009, "this outbreak is unstoppable." Following this event, significant research has been allocated to understand all aspects of the influenza virus in an effort to produce effective vaccines and medications to prevent and control another pandemic.
    [Show full text]
  • Lentivirus and Lentiviral Vectors Fact Sheet
    Lentivirus and Lentiviral Vectors Family: Retroviridae Genus: Lentivirus Enveloped Size: ~ 80 - 120 nm in diameter Genome: Two copies of positive-sense ssRNA inside a conical capsid Risk Group: 2 Lentivirus Characteristics Lentivirus (lente-, latin for “slow”) is a group of retroviruses characterized for a long incubation period. They are classified into five serogroups according to the vertebrate hosts they infect: bovine, equine, feline, ovine/caprine and primate. Some examples of lentiviruses are Human (HIV), Simian (SIV) and Feline (FIV) Immunodeficiency Viruses. Lentiviruses can deliver large amounts of genetic information into the DNA of host cells and can integrate in both dividing and non- dividing cells. The viral genome is passed onto daughter cells during division, making it one of the most efficient gene delivery vectors. Most lentiviral vectors are based on the Human Immunodeficiency Virus (HIV), which will be used as a model of lentiviral vector in this fact sheet. Structure of the HIV Virus The structure of HIV is different from that of other retroviruses. HIV is roughly spherical with a diameter of ~120 nm. HIV is composed of two copies of positive ssRNA that code for nine genes enclosed by a conical capsid containing 2,000 copies of the p24 protein. The ssRNA is tightly bound to nucleocapsid proteins, p7, and enzymes needed for the development of the virion: reverse transcriptase (RT), proteases (PR), ribonuclease and integrase (IN). A matrix composed of p17 surrounds the capsid ensuring the integrity of the virion. This, in turn, is surrounded by an envelope composed of two layers of phospholipids taken from the membrane of a human cell when a newly formed virus particle buds from the cell.
    [Show full text]
  • HIV-1: to Splice Or Not to Splice, That Is the Question
    viruses Review HIV-1: To Splice or Not to Splice, That Is the Question Ann Emery 1 and Ronald Swanstrom 1,2,3,* 1 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA; [email protected] 2 Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA 3 Center for AIDS Research, University of North Carolina, Chapel Hill, NC 27599, USA * Correspondence: [email protected] Abstract: The transcription of the HIV-1 provirus results in only one type of transcript—full length genomic RNA. To make the mRNA transcripts for the accessory proteins Tat and Rev, the genomic RNA must completely splice. The mRNA transcripts for Vif, Vpr, and Env must undergo splicing but not completely. Genomic RNA (which also functions as mRNA for the Gag and Gag/Pro/Pol precursor polyproteins) must not splice at all. HIV-1 can tolerate a surprising range in the relative abundance of individual transcript types, and a surprising amount of aberrant and even odd splicing; however, it must not over-splice, which results in the loss of full-length genomic RNA and has a dramatic fitness cost. Cells typically do not tolerate unspliced/incompletely spliced transcripts, so HIV-1 must circumvent this cell policing mechanism to allow some splicing while suppressing most. Splicing is controlled by RNA secondary structure, cis-acting regulatory sequences which bind splicing factors, and the viral protein Rev. There is still much work to be done to clarify the combinatorial effects of these splicing regulators. These control mechanisms represent attractive targets to induce over-splicing as an antiviral strategy.
    [Show full text]
  • Respiratory Syncytial Virus and Coronaviruses
    Chapter 3 Structural and Functional Aspects of Viroporins in Human Respiratory Viruses: Respiratory Syncytial Virus and Coronaviruses Wahyu Surya, Montserrat Samsó and Jaume Torres Additional information is available at the end of the chapter http://dx.doi.org/10.5772/53957 1. Introduction Viroporins are an increasingly recognized class of small viral membrane proteins (~60-120 amino acids) which oligomerize to produce hydrophilic pores at the membranes of virus- infected cells [1]. The existence of ‘viroporins’ was proposed more than 30 years ago after observing enhanced membrane permeability in infected cells [2]. These proteins form oligomers of defined size, and can act as proton or ion channels, and in general enhancing membrane permeability in the host [3]. Even though viroporins are not essential for the rep‐ lication of viruses, their absence results in attenuated or weakened viruses or changes in tropism (organ localization) and therefore diminished pathological effects [4, 5]. In addition to having one – sometimes two – α-helical transmembrane (TM) domain(s), viro‐ porins usually contain additional extramembrane regions that are able to make contacts with viral or host proteins. Indeed, the network of interactions of viroporins with other viral or cellular proteins is key to understand the regulation of viral protein trafficking through the vesicle system, viral morphogenesis and pathogenicity. In general, viroporins participate in the entry or release of viral particles into or out of cells, and membrane permeabilization may be a desirable functionality for the virus. Indeed, sev‐ eral viral proteins that are not viroporins are known to affect membrane permeabilization, e.g., A38L protein of vaccinia virus, a 33-kDa glycoprotein that allows Ca2+ influx and indu‐ ces necrosis in infected cells [6].
    [Show full text]