Elucidating the Mechanisms of HIV-1 Antiviral Activity by SERINC5

Master’s Thesis

Khaled Moumneh

Division of Experimental Medicine McGill University, Montreal Date of submission: April 2017 Supervisor: Dr. Chen Liang

A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science.

©Khaled Moumneh, 2017 Abstract (English) The serine incorporators (SERINC) are a highly conserved transmembrane protein family in eukaryotes that are known to play an important role in stimulating lipid biosynthesis in a variety of cells. As their name suggests, they activate phosphatidylserine synthase and palmitoyltransferase via the incorporation of the amino acid serine to drive the synthesis of phosphatidylserine and sphingolipids, respectively. In 2015, two groups independently discovered that in the absence of HIV-1 Nef, SERINC5 and to a lesser extent SERINC3 incorporated into the virion and prevented proper viral pore expansion thus preventing viral core deposition and decreasing infectivity. These findings identified SERINC5 as a host restriction factor and finally solved the mystery behind the mechanism of Nef-mediated up-regulation of HIV-1 infectivity. Little is known about the SERINC5 protein itself and the specifics of its downregulation of HIV-1 and its downregulation by Nef. In our study, SERINC5 post-translational modification by ubiquitin was explored via co-immunoprecipitation. We discovered that SERINC5 is ubiquitinated, and that this ubiquitination most likely does not correspond to its counteraction either by Nef or proteasomal degradation. Next, we set out to find a Nef binding motif on SERINC5 through the use of a novel CD4-SERINC5 chimera internalization assay. Lastly, the SERINC5 region or motif required for antiviral activity against HIV-1 was investigated through the use of two sets of SERINC5 and non-antiviral SERINC1 region-swapped chimeras. Through these experiments, a central region of SERINC5 encompassing amino acids 176-311 was found to be required for its anti-HIV-1 activity. These observations pave the way for future studies to find specific motifs required for SERINC5-HIV-1 and SERINC5-Nef interaction and elucidate a more detailed antiviral mechanism.

 Abstract (French) Les incorporateurs de sérine (SERINC) constituent une famille de protéines transmembranaires hautement conservée chez les eucaryotes et qui sont connues pour jouer un rôle important dans la stimulation de la biosynthèse des lipides dans de nombreux types cellulaires. Comme leur nom le suggère, ils activent la phosphatidylsérine synthase et la palmitoyltransférase via l’incorporation de l’acide aminé sérine afin de faciliter la synthèse de la phosphatidylsérine et des sphingolipides, respectivement. En 2015, deux groupes ont indépendamment découvert qu’en l’absence de Nef du VIH-1, SERINC5 et, dans une moindre mesure, SERINC3, sont alors incorporés dans le virion et préviennent l’expansion adéquate des pores viraux, permettant donc de prévenir la déposition du noyau viral et réduire l’infectiosité. Ces résultats ont identifié SERINC5 comme étant un facteur de restriction de l’hôte et a finalement résolu le mystère se cachant derrière le mécanisme de la régulation positive de l’infectiosité du VIH-1 médiée par Nef. Peu de choses sont connues à propos de la protéine SERINC5 elle-même et les spécificités de sa régulation négative du VIH-1 et de sa régulation négative par Nef. Dans notre étude, des modifications post-traductionnelles de SERINC5 par de l’ubiquitine ont été explorées via co-immunoprécipitation. Nous avons découvert que SERINC5 était en effet ubiquitinée, et que cette ubiquitination ne correspond probablement pas à sa régulation négative par Nef ou à la dégradation protéasomale. Nous avons ensuite recherché un motif de liaison de Nef sur SERINC5 grâce à l’utilisation d’un nouveau test d’internalisation de CD4-SERINC5 chimérique. Finalement, le motif ou la région SERINC5 requise pour l’activité antivirale contre le VIH-1 a été examinée en utilisant deux ensembles de protéines chimériques par échange de régions entre SERINC5 et la protéine non-antivirale SERINC1. À travers ces expériences, une région centrale de SERINC5, englobant les acides aminés 176 et 311, a été découverte comme étant requise pour ses activités anti-VIH-1.

Ces observations permettent de préparer la voie pour de futures études ayant pour but de trouver des motifs spécifiques requis pour l’interaction entre SERINC5-HIV-1 et SERINC5-Nef et élucider un mécanisme antiviral plus détaillé.

 Table of Contents

ABSTRACT (ENGLISH) ...... 1 ABSTRACT (FRENCH) ...... 2 TABLE OF CONTENTS ...... 3 ACKNOWLEDGEMENTS ...... 5 ABBREVIATIONS ...... 6 CHAPTER 1: INTRODUCTION ...... 10 1.1 HISTORY AND EPIDEMIOLOGY OF HIV-1 ...... 10 1.1.1 Discovery ...... 10 1.1.2 Epidemiology ...... 10 1.1.3 Classification and Evolution of HIV ...... 11 1.2 HIV-1 PATHOGENESIS ...... 15 1.2.1 HIV-1 Transmission ...... 15 1.2.2 Disease Progression ...... 15 1.3 HIV-1 TREATMENTS ...... 17 1.4 VIROLOGY ...... 21 1.4.1 HIV-1 Genome and Accessory Proteins ...... 21 1.4.2 HIV-1 Particle Structure ...... 23 1.4.3 HIV Replication ...... 24 1.4.3.1 Virion Attachment and Fusion ...... 24 1.4.3.2 Reverse transcription and integration ...... 25 1.4.3.3 Sequential Production of Viral Proteins ...... 28 1.4.4.4 Viral Particle Assembly, Release, and Maturation ...... 30 1.5 HOST RESTRICTION AGAINST HIV-I ...... 32 1.5.1 Properties of Host Restriction Factors ...... 32 1.5.2 APOBEC3G ...... 33 1.5.3 TRIM5α ...... 35 1.5.4 Tetherin ...... 36 1.5.5 SAMHD1 ...... 38 1.5.6 SLFN11 ...... 39 1.5.7 MxB ...... 40 1.5.8 IFITM ...... 40 1.5.9 Recently Identified Restriction Factors ...... 41 1.5.10 Nef and SERINC5 ...... 43 1.6 PROJECT OBJECTIVES ...... 45 CHAPTER 2: METHODS ...... 47

2.1 CELL LINES AND CULTURING SYSTEM ...... 47 2.2 PLASMIDS AND CHIMERIC CONSTRUCTS ...... 47 2.3 TRANSFECTION ...... 50 2.4 CO-IMMUNOPRECIPITATION ...... 50 2.5 FLOW CYTOMETRY ...... 51 2.6 LUCIFERASE ASSAY ...... 52

 2.7 RT ASSAY ...... 52 2.8 WESTERN BLOT ...... 52 CHAPTER 3: RESULTS ...... 54 3.1 UBIQUITINATION ...... 54 3.1.1 SERINC5 is Ubiquitinated ...... 54 3.1.2 SERINC5 ubiquitination does not involve the proteasome ...... 55 3.1.3 Determination of SERINC5 polyubiquitination ...... 57 3.1.4 Nef does not increase SERINC5 ubiquitination ...... 58 3.2 NEF INTERACTION WITH SERINC5 ...... 60 3.2.1. Chimeric design ...... 60 3.2.2 Experimental viability of the CD4-SERINC5 internalization assay ...... 61 3.3 ELUCIDATION OF SERINC5 DOMAINS NECESSARY FOR INTERACTION WITH HIV-1 ...... 64 3.3.1 Primary chimeric design ...... 64 3.3.2 SERINC5 downregulates HIV-1 in a dose-dependent manner ...... 66 3.3.3 Central region of SERINC5 is necessary for antiviral activity ...... 67 3.3.4 A second set of chimeras indicate that Amino acids 208-311 are required for SERINC5 antiviral activity ...... 70 CHAPTER 4: DISCUSSION ...... 73 REFERENCES ...... 77

Acknowledgements I would first and foremost like to thank my supervisor, Dr. Chen Liang for the opportunity to work in his lab and study in the department of Experimental Medicine. His patience and support throughout my project was greatly appreciated and helped me to develop a more scientific and methodical way of thinking. Secondly, I would also like to thank my advisory committee members, Dr. Anne Gatignol, Dr. Rongtuan Lin, and Dr. Miltiadis Paliouras for attending my thesis committee meeting and providing me with valuable insight on my project. I would also like to thank all the lab members, especially Saina Beitari for her patience and guidance especially with teaching me basic lab skills. Thanks to our lab technician, Qinghua Pan for her help with the RT assays and Zhen Wang for her guidance with regards to cloning. Lastly, I would also like to thank Yimeng Wang for the company and all the interesting conversations. Lastly, I would like to thank all my friends and family for supporting me through this great journey. I would also like to give a special thanks to my best friend, Charlotte Mascret for her emotional support, great company, and her assistance with the French translation of my abstract.

Abbreviations

µg Microgram µl Microliter 6HB 6-helix bundle AIDS Acquired immunodeficiency syndrome ALIX ALG-interacting protein X ALLN Calpain Inhibitor I APC Antigen presenting cell APOBEC3G Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 G ARV Antiretroviral agent BFA Brefeldin A CA/p24 Capsid CCR5 C-C chemokine receptor type 5 CDC Centers for Disease Control CDK9 Cyclin-dependent kinase 9 CIP Calf intestinal alkaline phosphatase Crm1 Exportin 1 CTD Carboxy-terminal domain CTL CD8+ cytotoxic T lymphocyte CXCR4 C-X-C chemokine receptor type 4 CycT1 Cyclin T1 CypA Cyclophilin A DCAF1 DDB1-Cul4A-associated-factor-1 DMEM Dulbecco’s Modified Eagle Medium dNTP Deoxynucleotide triphosphates dsDNA Double-stranded deoxyribonucleic acid DSIF DRB sensitivity-inducing factor EDTA Ethylenediaminetetraacetic acid EIAV Equine infectious anemia virus Env Envelope

ERAD Endoplasmic reticulum-associated protein degradation ERmanI ER-associated α-mannosidase I ESCRT Endosomal sorting complex required for transport FDA Federal Drug Administration of the United States Gag Group-specific antigen GBP5 Guanylate-binding protein 5 GPI Glycophosphatidylinositol HAART Highly active antiretroviral therapy HIV-1 Human immunodeficiency virus type 1 HIV-2 Human immunodeficiency virus type 2 HRP Horseradish peroxidase HTLV Human T-lymphotropic virus IFITM Interferon induced transmembrane protein IFN Interferon IN Integrase ISG Interferon stimulated gene kD Kilodalton LTR Long terminal repeat MA/p17 Matrix MARCH8 Membrane-associated RING-CH 8 MDM Monocyte-derived macrophage MHCII Major histocompatibility complex II mL Milliliter MLV Murine leukemia virus MxB/Mx2 Myxovirus resistance protein 2 NC/p9 Nucleocapsid NEDD4 Neural precursor cell expressed developmentally down-regulated protein 4 Nef Negative regulatory factor NELF Negative elongation factor NES Nuclear export signal ng Nanogram

NLS Nuclear localization signal NPC Nuclear pore complex P/S Penicillin/Streptomycin PBS Dulbecco’s Phosphate Buffered Saline pbs Primer binding site PEI Polyethylenimine PFA Paraformaldehyde

PI(4,5)P2 Phosphatidylinositol 4,5-bisphosophate PIC pre-integration complex PNK Polynucleotide kinase Pol Polymerase ppt Polypurine tract sequence PR Protease pTEFb Positive transcription elongation factor b R Direct repeat Rev Regulator of expression of virion proteins RING Really interesting new gene RRE Rev-response element RT Reverse transcriptase RTC Reverse transcription complex SAMHD1 SAM domain and HD domain containing protein 1 SDS Sodium dodecyl sulfate SERINC Serine Incorporator SIV Simian immunodeficiency virus SLFN11 Schlafen 11 SP1 Spacer 1 region ssRNA Single Stranded ribonucleic acid TAR Transactivation-response region Tat Transactivator of Transcription TBS Tris buffer saline TM Transmembrane/Transmembrane domain

TNFα Tumor necrosis factor-alpha TRIM 

Tripartite-motif containing cytosolic protein 
TSG101 Tumor susceptibility gene 101 TSPO Mitochondrial translocator protein UNAIDS United Nations Program on HIV/AIDS UTR Untranslated region Vif Viral infectivity Factor Vpr Viral protein R VPS4 Vacuolar protein sorting 4 Vpu Viral protein unique Vpx Viral protein X

 Chapter 1: Introduction 1.1 History and Epidemiology of HIV-1 1.1.1 Discovery Human immunodeficiency virus type 1 (HIV-1) is the major viral cause of the acquired immunodeficiency syndrome (AIDS) global pandemic. In 1981, homosexual men and intravenous drug users were admitted in the United States displaying symptoms of opportunistic infections such as pneumocystis carinii pneumonia, mucosal candidiasis, and later, Kaposi’s sarcoma. All of these ailments are rare in individuals with intact immunity thus leading to the declaration by the US Centers for Disease Control (CDC) of an outbreak of an immunodeficiency disease of unknown cause1,2. Due to the life-style choices of the patients, the condition was initially called gay compromise syndrome3. Later in 1982, upon finding an increase of non-homosexual occurrences, the condition was given its more appropriate current name, AIDS4. In 1983, Dr. Luc Montagnier’s group isolated a new virus in a pre-AIDS patient and named it lymphadenopathy associated virus5. Later that year, Dr. Robert Gallo’s group also found a new virus in a similar patient to Dr. Montagnier’s, but observed that it was similar to the previously discovered Human T-lymphotropic virus (HTLV) thus naming the AIDS causing virus, HTLV-III6. In the end, the International Committee of the Taxonomy of Viruses declared in 1986 that the new virus with many forms officially be named human immunodeficiency virus7. 1.1.2 Epidemiology Based on the report by the United Nations Program on HIV/AIDS (UNAID), an estimated 36.7 million people are living with AIDS as of the end of 2015. Of those infected, 2.1 million were newly infected that year while 1.1 million had died due to AIDS related complications. While there have been many advances in terms of antiretroviral therapies and awareness to prevent spread, the increase in lifespan of those infected contributes to the consistently high statistic of those currently living with HIV/AIDS. At the same time, however, the number of newly infected children has decreased by 50% since 2010 partially due to an increase to around 77% of pregnant mothers with HIV/AIDS utilizing antiretroviral therapies to prevent transmission to their newborn children. Compared to the peak of AIDS-related deaths in 2005, the number of deaths per year has decreased from 2 million to 1.1 million8,9.


The disease is most prevalent in Sub-Saharan Africa, especially in lower income populations where around 70% of those affected with HIV/AIDS can be accounted for. In contrast, the Middle East and North Africa have remained the least affected region at .6% infected. The primary transmission mode in Sub-Saharan Africa remains to be heterosexual sex and poor health conditions while in Eastern Europe, the Pacific, and Asia, the primary mode of transmission is drug-related infections via reused needles, unprotected intercourse especially among sex workers, and sex between men8,9. These risk factors are especially prevalent in countries with lower socioeconomic statuses which accounted for around 45% or 15 million of all infections in 2010. Even in high income countries such as North America and Western/Central Europe, the number of people living with HIV has continued to grow to 2.3 million in 2009, a significant increase from 1.8 million in 2001. In these countries, the primary mode of transmission tends to be unprotected sex between men10. With the advent of new antiretroviral therapies and increased understanding of the mechanisms governing HIV, treatments which can suppress and maintain a low viral load have made their way into the mainstream. UNAIDS is determined to end the AIDS pandemic by 2030. However, without a cure, infected individuals will continue acting as viral reservoirs and spreading the infection. As such, international sustained operation to advance research, increase awareness to prevent spread, and increase access to treatment is imperative to reach this goal8. 1.1.3 Classification and Evolution of HIV HIV-1 is a member the Retroviridae family which are positive-sense single stranded ribonucleic acid (ssRNA) viruses5,6. The main characteristic among retroviruses is the ability to reverse transcribe the ssRNA viruses into double-stranded deoxyribonucleic acid (dsDNA) sequences for integration into the host DNA and subsequent expression11-13. HIV-1 is a complex retrovirus with additional regulatory and accessory proteins thus placing it in the lentivirus genus along with other similar immunodeficiency viruses such as simian immunodeficiency virus (SIV)14,15. HIV-I is divided into four distinct lineages based on different cross-species zoonotic transmission events: group M (main), group N (non-M and non-O), group O (outlier), and group P (putative)16. Group M was the first to be identified and represents the majority pandemic form of the virus17. This group is further subdivided into nine clades: A, B, C, D, F, G, H, J, and K18. Each of these clades differ in their sequences and geographic distribution. Clade A for instance


is the dominant form in West Africa while Clade D is mostly found in Central Africa which is widely considered to be the epicenter of HIV-I due to the variety of strains present (Figure 1)19- 21. Group N was discovered in 1998 and is less prevalent than groups M and O with only 13 documented cases in Cameroon as of 201022. Group O which is also less prevalent than group M, was discovered in 1990 and represents a total of 1% of all HIV infections23,24. It is mainly found in Cameroon and neighboring countries25,26. Like group M, group O also has enough sequence diversity to be further subdivided into 5 different clades: I-V27. Lastly, group P was discovered in 2009 in a Cameroonian woman living in France and later, another Cameroonian individual28,29.

Figure 1: Global Distribution of HIV-1 (adapted from Hemelaar 2012)20,21. The prevalence of HIV-1 subtypes and recombinants from 2004 are indicated by the colored pie charts. The global percentages are shown in the box on the right while the legend for the different subtypes is on the left. HIV-1 group M Clade C is the most prevalent globally while the most diverse area is Central Africa which includes Cameroon.

Based on phylogenic cluster analyses (Figure 2), the origins of HIV groups M and N have been attributed to SIV from the Central African species of chimpanzee, Pan troglodytes


troglodytes (SIVcpzPtt) which itself potentially emerged from recombination between SIV strains from red-capped mangabeys and greater spot-nosed monkeys30,31. More specifically, group M and N represent HIV emerging from south-eastern and south-central Cameroon, respectively32. Due to a lack of data, groups O and P most likely originated from SIV of western lowland gorillas, Gorilla gorilla gorilla (SIVgor)33. On the other hand, SIVgor probably originated from a transmission event from SIVcpz in their current distributions especially in the case of group O34. In 1986, a virus more closely related to SIV than HIV-I was isolated from a patient in West Africa and was later classified as HIV-235. Like HIV-I, HIV-2 is a zoonotic virus, but instead of originating from SIVcpz, it originated from SIV infected sooty mangabeys, Cercocebus torquatus atys (SIVsmm)36. HIV-2 is divided into epidemic groups (A and B) which are responsible for the epidemics in West Africa where they are primarily confined, and the rare non-epidemic groups (C-H)37-39. As of 2005, only one individual for each non-epidemic group has been identifying leading some to believe that these are dead-end transmission events thus failing to spread among the human population30,40,41. Besides these differences, HIV-2 has a lower rate of progression towards AIDS, lower viral loads, and therefore, lower overall transmission rates42.



Figure 2: Phylogenetic classification of HIV-1, HIV-2, and SIV (adapted from Bush et al. 2014)43. Tables show time to most recent common ancestor (MRCS), oldest available sequence (Seq Age) and its country of origin (Country), methods in which CD4 is downregulated (CD4 DR), and method of tetherin antagonism (Tetherin). As can be seen, SIVgor is closely related to group O and P of HIV-1 and is thought to have given rise to them through cross-species transmission. On the other hand, HIV-1 is more related to SIVcpz.


Based on the most recent common ancestor predictions, the main form of HIV (group M) is thought to have originated between 1799 and 1900 thus making it the oldest predicted form of HIV21,44. The exact mechanism of how humans could have contracted these strains of HIV is unknown, but based on transmission dynamics, mucous membrane or cutaneous exposure to the blood or fluids of chimpanzees could have created four independent transmission events30,45. 1.2 HIV-1 Pathogenesis 1.2.1 HIV-1 Transmission HIV-1 is transmitted via direct viral exposure at mucosal surfaces and percutaneously via damaged tissue. The virus itself is found in semen, pre-seminal, rectal and vaginal fluids, blood, and breast milk of infected individuals. As such, HIV-1 is most commonly transmitted through unprotected sexual intercourse (anal and vaginal), needle sharing during intravenous drug use, blood transfusion, and organ transplantation. In terms of sexual intercourse, the rate of infection via anal intercourse is higher than vaginal intercourse at 1 in 3 vs 1 in 10, respectively. HIV can also be transmitted vertically via breast feeding and labor. Rates of infection via blood transfusion, organ transplant, and vertical transmission have been drastically decreased recently due to extensive screening procedures and the advent of effective antiretroviral treatments46,47. Other body fluids such as saliva, sweat, and tears do not harbor HIV-1. Thus, close contact with those individuals be it eating food that they had prepared, shaking hands, or using the same washroom does not pose a risk for infection granted that both the infected and uninfected individuals have intact skin to prevent any unwanted contact with infected fluid. Lastly, although HIV-1 is present in the blood, the virus cannot be transmitted via insect vectors such as mosquitos or ticks48. 1.2.2 Disease Progression The onset of an HIV-1 infection is often instigated by a single virion49. Once established the infection progresses through four main stages: eclipse phase, acute phase, chronic infection or clinical latency, and AIDS (Figure 3)50. Based on factors such as if the exposed individual is under the effect of antiretroviral therapies or even if viral targets are naturally mutated, the progression of the virus can be hindered. The eclipse phase occurs 1-2 weeks after the initial exposure event and is characterized by unhindered viral replication and spread. This stage is asymptomatic with no detectable levels of viremia either. Next, the acute phase occurs around 2- 4 weeks after exposure and is marked by a peak of viral load at 107 copies of HIV-1 viral


RNA/mL50. Due to the high viremia, an immune response in the form of activated CD4+ T cells, CD8+ T cells, and antibodies against viral proteins is present, which results in flu-like symptoms in at least 50% of cases46,51. As HIV targets mainly CD4+ T cells, the increased immune response is likely a partial reason why the viremia becomes so high. Towards the end of the phase however, the levels of viremia decline almost 100-fold as the body brings the infection under control and activated CD4+ T cells are exhausted thus leading to a decline of these lymphocytes50,52. For the next 1-20 years, HIV goes into a period of asymptomatic chronic infection where the levels of viremia slowly increase from their depleted status from around 1 to 100,000 copies of HIV-1 viral RNA/mL. Even though this phase is asymptomatic, CD4+ T cells are slowly being infected and depleted. This depletion goes on until the AIDS phase where the levels of CD4+ T cells decline to 200 or fewer cells/µl at which point immune control of the HIV-1 infection is lost thus allowing for further depletion of lymphocytes and increased viremia50,53. The affected individual will oftentimes die of HIV-related opportunistic infections and diseases such as tuberculosis and Kaposi’s sarcoma1,2,52. There is also a population of infected individuals who maintain high levels of CD4+ T cells above 500 cell/µl without antiretroviral therapy and do not progress to AIDS. These individuals are known as long-term non-progressors and are often found to have defective or less capable HIV-1 viral factors54. In addition, HIV-1 can evade the blood-brain barrier by latently hiding within peripheral monocytes and actively producing virus only when the cell differentiates into a macrophage especially in the case that it is called to replace a macrophage in the brain. This in turn causes inflammatory factors such as tumor necrosis factor-alpha (TNFα) to attract more monocytes thus compounding the effect leading to HIV-associated neurological disorders such as dementia55,56. If left untreated, death is the most probable outcome due to opportunistic infections thus making an HIV-1 infection one of the most lethal diseases with a mortality rate of over 95%50.



Figure 3: HIV-1 pathological progression (adapted from Maartens et al. 2014)53. HIV-1 infection starts with the eclipse phase where for the first two weeks, no detectable levels of viremia are present. For the next year or so, the infection is in its acute phase where HIV-1 is replicating unhindered initially therefore depleting CD4+ T cells. Towards the end of this phase, the adaptive immunity begins to counter the viremia level and regenerate the T cells. Once viremia is stabilized at a low level, HIV-1 slowly replicates asymptomatically and slowly depletes CD4+ T cells for the next decade or so until a CD4+ T cell count of below 200 is achieved. At this point, the host develops AIDS and is at risk for many opportunistic infections. In addition, as HIV-1 replicates, many mutations and strains accumulate to counter host factors or treatments thus contributing to the extreme difficulty of treating this chronic infection.

1.3 HIV-1 Treatments After the discovery of HIV-1, in 1984 Margaret Heckler, the US secretary of health and human services, declared that a vaccine would be available for testing within the next 2 years. Unfortunately, this was a very optimistic estimate and due to the lack of a proper neutralizing


antibody response to the particular strain of the patient and other complications owing to the high variability and escape mutation rate of HIV-1, a vaccine has been largely unsuccessful57. Currently, vaccination methods that help induce CD8+ cytotoxic T lymphocyte responses against infected cells are being investigated58. It was not until 1987, that the first antiretroviral drug was approved by the Federal Drug Administration of the United States (FDA) targeting a vital part of HIV-1, reverse transcriptase. Since then, the FDA has approved a total of 44 antiretroviral drugs that fall into one of 8 classes (Table 1)59. In the early 1990’s, single and dual therapy drugs were used60. However, due to the error-prone nature of HIV-1 reverse transcription, for every replication cycle, up to 10 new mutations may be introduced61. This high mutation rate along with a selective force from the antiretroviral drug leads to rapid drug resistance development62,63. To counteract this, the administration of a cocktail of multiple antiretroviral agents (ARVs) in a combination called highly active antiretroviral therapy (HAART) started to be used in 199664,65. In this method, at least three different mutations are required to develop resistance against all three drugs. As a result, the combination is often chosen in a manner that may push for mutations that will allow other ARVs to function even more effectively66,67. In this way, viral replication may be suppressed to undetectable levels for decades. No matter the treatment however, HIV remains a chronic disease and will eventually develop a resistance to the drugs used therefore increasing the importance of further investigation into new ARVs and resistance testing when a patient no longer responds to HAART60. Lastly, alternative methods have recently begun to appear in the field of genome editing where the idea is to use a sequence targeted nucleosome such as the CRISPR/CAS9 system to target and delete or mutate a vital part of HIV- 168.

Drug Class Mechanism Generic Name Nucleoside Reverse Prevents elongation during •
Abacavir (1998) Transcriptase Inhibitors reverse transcription by using •
Didanosine (1991 and (NRTIs) deoxyribonucleosides lacking 2000) a 3’-OH. When transcribing •
Emtricitabine (2003) DNA, a 3’ monophosphate is •
Lamivudine (1995) added thus terminating the •
Stavudine (1994) chain69.


•
Tenofovir disoproxil fumarate (2001) •
Zidovudine (1987) Non-Nucleoside Reverse Prevents completion of •
Efavirenz (1998) Transcriptase Inhibitors reverse transcription by •
Etravirine (2008) (NNRTIs) binding the NNRTI binding •
Nevirapine (1996 and pocket of reverse 2011) transcriptase thus inducing a •
Rilpivirine (2011) conformational change, inactivating the enzyme70. Protease Inhibitors (PIs) Competitive active site •
Atazanavir (2003) inhibitors for HIV-1 against •
Darunavir (2006) HIV-1 protease. They •
Fosamprenavir (2003) prevent proteolytic cleavage •
Indinavir (1996) of structural and enzymatic •
Nelfinavir (1997) proteins therefore preventing •
Ritonavir (1996) proper maturation and •
Saquinavir (1995) function71. •
Tipranavir (2005) Fusion Inhibitors Synthetic peptide that mimics •
Enfuvirtide (2003) a part of HIV-1 gp41 and prevents proper formation of the six-helix bundle required for fusion of the virion to the target cell72. Entry Inhibitors Binds CCR5 inducing a •
Maraviroc (2007) conformational change thus preventing interaction with HIV-1 gp120 and preventing entry73.


Integrase Inhibitors Prevents integrase binding to •
Dolutegravir (2013) viral DNA by binding to •
Elvitegravir (2014) catalytic site and inducing a •
Raltegravir (2007) conformational change74. Pharmacokinetic Enhancers Enhances activity of other •
Cobicistat (2014) ARVs when taken in tandem75. Combination HIV Combination of other ARVs •
Abacavir and Medicines in a manner that prevents fast lamivudine (2004) escape mutation development •
Avacavir, and increases drug- dolutegravir, and advantageous mutation lamivudine (2014) 60 development . •
Abacavir, lamivudine, and zidovudine (2000) •
Atazanavir and cobicstat (2015) •
Darunavir and cobicstat (2015) •
Efavirenz, emtricitabine, and tenofovir disoproxil fumarate (2006) •
Emtricitabine, rilpivirine, and tenofovir alafenamide (2016) •
Emtricitabine, rilpivirine, and tenofovir disoproxil fumarate (2011)


•
Emtricitabine and tenofovir alafenamide (2016) •
Emtricitabine and tenofovir disoproxil fmarate (2004) •
Lamivudine and zidovudine (1997) •
Lopinavir and ritonavir (2000)

Table 1: Classes of HIV antiretroviral drugs approved by the FDA and their mechanisms. 44 ARVs including their combinations have been approved by the FDA as of 2016. Additional approval years indicate a new release or slight modification of the previous drug. Adapted from the NIH website59.

1.4 Virology 1.4.1 HIV-1 Genome and Accessory Proteins The HIV-1 genome is encoded in a 9.7kb single stranded RNA flanked by two long terminal repeats (LTR). These LTRs are indispensable for driving the expression of the HIV gene using host machinery. The whole gene encodes nine open reading frames which include three polyproteins: group-specific antigen (Gag), polymerase (Pol), and envelope (Env), four accessory proteins: viral infectivity factor (Vif), viral protein unique (Vpu), viral protein R (Vpr) and negative regulatory factor (Nef), and two regulatory proteins genes: transactivator of transcription (Tat) and regulator of expression of virion proteins (Rev) (Figure 4)14,76,77. The Gag structural gene gives rise to a 55 kilodalton (kD) protein, p55 which is myristoylated on its N terminus. This modification allows for its association to the cytoplasm and subsequent budding within the virus. P55 is cleaved by virally packaged protease and gives rise to four structural proteins: matrix (p17 or MA), capsid (p24 or CA), nucleocapsid (p9 or NC), and p6. P17 is involved in nuclear transport of the viral genome via a karyophilic signal, stabilizing the viral particle, and transport of viral RNA to the budding site. P24 forms the


conical core of the virion and recruits cyclophilin A (CypA) which is essential for viral infectivity. P7 recruits and packages viral RNA in the virion as well as participating in reverse transcription. Finally, the p6 polypeptide facilitates packaging of the viral accessory protein Vpr as well as viral release from the plasma membrane14,76. The pol gene lacks an initiation codon and is instead translated as the Gag-Pol precursor fusion protein, Pr160Gag-Pol due to a ribosomal frame-shifting event triggered by a cis-acting RNA motif during the translation of Gag. This fusion protein is then cleaved by viral protease into three viral enzymes: protease (PR), reverse transcriptase (RT), and Integrase (IN). PR is an aspartyl protease that is required to form Pol during the maturation process described above. RT is responsible for reverse transcription of viral RNA into viral dsDNA which is then inserted into host genomic DNA using IN78. Env is a 160kD protein which is translated as the precursor, gp160 and undergoes multiple glycosylation events on its way to the plasma membrane. On the way gp160 is cleaved by a cellular protease into the surface and transmembrane moieties, gp120 (SU) and gp41 (TM). Gp120 facilitates attachment with target cells while gp41 allows for proper fusion of the viral particle thus facilitating viral infection76. Besides structural proteins and enzymes, HIV-1 also contains the regulatory proteins Tat and Rev which facilitate viral genomic expression and export, and the accessory proteins Vif, Vpu, Vpr, and Nef which act to facilitate viral infectivity and counteract host defenses76. Additionally, HIV-2 contains viral protein X (Vpx) rather than Vpu79.


Figure 4: HIV-1 genome schematic (adapted from Nkeze et al. 2015)80. The full HIV-1 genome is around 9.7kb in length flanked by two LTRs. The genome encodes a total of nine proteins including three polyproteins: Gag, Pol, and Env, four accessory proteins: Vif, Vpr, Vpu, and Nef, and two regulatory proteins: Tat and Rev. The polyproteins are further processed into MA, CA, NC, and p6 (Gag), PR, RT, IN (Pol), SU and TM (Env).

1.4.2 HIV-1 Particle Structure After HIV-1 infects the cell and integrates into the genome, viral proteins and viral genomic RNA start to be produced to form a viral particle that will then go on to infect other cells. HIV is a spherical enveloped virus with a diameter of around 120nm (Figure 5)81. The envelope for the virion originates from a host lipid bilayer and is studded with spikes of heterodimeric gp120 surface and gp41 transmembrane stem heterodimers which are joined by the stems non-covalently into trimers. The inner surface of the virus is lined with MA to stabilize the particle while CA creates the conical inner structure to house the two copies of tightly wound viral RNA protected from nucleases by NC, and all other viral proteins82-84.

Figure 5: HIV-1 viral particle structure (adapted from Shum et al. 2013)85. The HIV-1 particle consists of a plasma membrane derived lipid layer which is studded with trimeric gp120 and gp41. Matrix lines the inside of the lipid layer and protects the conical capsid core which in


turn protects other viral components. As the lipid outer layer is host-derived, embedded host proteins will still be present and may act to inhibit viral activity.

1.4.3 HIV Replication 1.4.3.1 Virion Attachment and Fusion The first step in the HIV-1 lifecycle is virion attachment and entry. This step can be subdivided into four sub-steps: virion binding to target cell, CD4 binding, co-receptor binding, and membrane fusion (Figure 6)86. First the virions must bind their target CD4+ T cells. Although this step is not necessary for proper viral entry, there is evidence that it augments viral infectivity in vitro. In this case, Env on the virion can interact with either negatively charged cell surface heparan sulfate proteoglycans or more specific proteins such as α4β7-integrin to come into proximity with its primary receptor target, CD487-89. The surface Env glycoprotein, gp120 consists of 5 conserved domains (C1-5) for CD4 binding and 5 variable loop domains (V1-5) for co-receptor binding and immune evasion. The conserved domains of gp120 bind the CD4 receptor thus causing a conformational change in the viral Env and exposing the hidden V3 domain for co-receptor binding86. There are two surface co-receptors that HIV-1 can use for entry: C-C chemokine receptor type 5 (CCR5) and C-X-C chemokine receptor type 4 (CXCR4). HIV can be classified based on its co-receptor binding: CCR5 binding (R5 HIV), CXCR4 binding (X4 HIV), and both (R5X4 HIV)90. In the beginning of the infection, R5 HIV predominates while a co-receptor switch occurs later to increase the versatility of the virus91. Binding the co-receptor causes another conformational change to expose the hydrophobic N- terminal of gp41 which inserts into the host cell membrane and acts as a hinge to bring the amino-terminal helical region (N-HR) and carboxy-terminal helical region (C-HR) of the gp41 fusion peptide together thus forming a 6-helix bundle (6HB)92,93. This 6HB brings the viral and host membranes into close contact to form and stabilize a fusion pore through which the viral capsid core is deposited86.



Figure 6: HIV-1 virion attachment and entry (adapted from Wilen et al. 2012)86. An overview of the viral binding and entry is shown. First, gp120 interacts with CD4 on the target cell which allows for the binding of the V3 loop to a host coreceptors, either CCR5 or CXCR4. This interaction triggers the insertion of gp41 into the host membrane and formation of the 6HB which drives membrane fusion and viral core deposition.

1.4.3.2 Reverse transcription and integration The next step is to convert the single-stranded viral RNA into an integratable dsDNA with the use of the reverse transcription complex (RTC) (Figure 7). This RTC consists of the viral RNA, NC, and RT. Reverse transcription is initiated when the lys3 tRNA binds the primer binding site (pbs), which in the case of HIV-1, is located 180bp away from the 5’ end of the viral RNA. This leads to the reverse transcription of a negative-sense strand as the template is a positive-sense RNA. At the same time, RT, which also has RNase H activity degrades the RNA template that it is reverse-transcribed from94-96. Like DNA polymerase, RT can only transcribe 5’ to 3’, therefore it drops off at the 5’ end along with the partially transcribed negative sense ssDNA. The ssDNA hybridizes with the 3’ end as it contains a direct repeat (R) and acts as a primer to reinitiate reverse transcription97,98. This second round continues while the RNA is degraded except for a polypurine tract sequence (ppt) which is resistant to RNase H. HIV-1 has one dispensable ppt sequence in the middle of the RNA to increase efficiency and a necessary one towards the 3’ end to initiate the third round of reverse transcription for positive sense DNA formation99,100. Once RT copies the first 18 nucleotides of the tRNA, RNase H cleaves the tRNA except for the ribonucleotide, adenosine101,102. The transcribed portion of the plus-strand DNA is then transferred to the 3’end of the minus-strand where it binds to the corresponding pbs due to


the 18nt of copied tRNA sequence. Lastly, the plus- and minus- strands extend in both directions using each other as templates to complete the formation of the double-stranded proviral DNA94. Additionally, the error-prone process of reverse transcription allows for recombination with other strands of viral RNA thus leading to rapid generation of variants with differing resistances and advantages94,103.

Figure 7: Overview of HIV-1 reverse transcription (adapted from Hu et al. 2012)94. HIV-1 RNA genome is labeled in light blue while reverse transcribed DNA is labeled in dark blue. Dotted blue lines indicate RNA degradation via RNase H. Lys3 tRNA binds the pbs near the 5’ end (A). RT transcribes the RNA from 5’-3’ (B). Minus strand disengages, transfers to the opposite end and hybridizes with the R sequence thus allowing continued minus-strand DNA synthesis (C). minus-strand elongation continues and ppt resists degradation by RNase H (D). Ppt initiates plus-strand transcription which elongates until passing the first 18nt of lys3 tRNA (E) at which point RNase H cleaves the tRNA sequence on the minus-strand DNA leaving an


adenosine and the plus-strand is transferred to the 5’ end to hybridize with the minus-strand pbs (F). Elongation continues to completion (G).

At the same time, a process known as uncoating occurs where the RTC is formed and finally converted into the pre-integration complex (PIC)104. There are two prevailing hypotheses for this process, cytoplasmic uncoating and uncoating near the nuclear pore complex (NPC). In cytoplasmic uncoating, CA is slowly but not completely lost during RT. However, the purpose of the CA coat is to protect the viral genome from host DNA sensors and restriction factors105,106. As a result, the second model points towards an intact coat until the NPC is reached at which point the CA is lost in order to facilitate the movement of the otherwise 50-60nm diameter intact RTC through the 39nm diameter NPC107,108. Once reverse transcription and uncoating are complete, integrase binds to the LTRs on both ends of the proviral DNA thus completing the PIC. This PIC contains viral proteins as well including NC, IN, and Vpr, the last of which contains a nuclear localization signals (NLS) which is integral in infecting non-dividing cells. Still in the cytoplasm, integration initiates (Figure 8) with 3’ end processing where the last 2 nucleotides from both plus- and minus- strands are removed thus leaving CA-3’. Once inside the nucleus, factors such as LEDGF/P75 tether and target IN to active transcription sites such as euchromatin where HIV is preferentially inserted109,110. The next step, is for IN to remove 5bp from the cellular DNA to create 5’ overhangs that will allow subsequent insertion of the viral DNA. these 3’ overhangs attack the phosphodiester bonds on opposite strands of the target DNA separated by a 5bp gap using IN thus resulting in a covalently bonded intermediate. Integration is finally complete once the gaps between the viral and cellular DNA strands are filled by host DNA repair enzymes110-112. 113


Figure 8: Overview of HIV-1 integration (adapted from Van Maele et al. 2006)113. Integration begins with 3’ end processing in the cytoplasm which leaves CA-3’ overhangs. The PIC is imported through the NPC and is directed towards the target DNA by certain host factors such as LEDGF/P75. With the help of IN, DNA ends are inserted into the target with each end separated by 5bp (DNA-strand transfer). Gaps are repaired by host machinery thus completing integration.

1.4.3.3 Sequential Production of Viral Proteins After insertion, the HIV provirus may lie dormant and act as a reservoir or actively be transcribed depending on the epigenetic control of the region114. Cellular transcription factors such as NF-κB can activate the provirus by binding to the LTR promoter region. This induces a high enough drive for viral transcription, but not full viral production115. To reach this stage, the regulatory proteins Tat and Rev must be synthesized. Tat is one of the first viral products during this early low-level of transcription, and binds to the transactivation-response region (TAR) of the early viral RNA in the nucleus116. This binding is accompanied by the recruitment of positive transcription elongation factor b (pTEFb) which consists of cyclin T1 (CycT1) and cyclin-dependent kinase 9 (CDK9). These factors act together to deactivate negative elongation factor (NELF) and remove the Spt5 subunit of DRB sensitivity-inducing factor (DSIF) through phosphorylation thus removing a blockage and creating a positive elongation factor, respectively. In addition, the carboxy-terminal domain (CTD) of RNA polymerase II is phosphorylated thus leading to enhanced transcription117-119. The HIV primary transcript has four different splice donor sites and eight different splice acceptor sites thus leading to over 40 alternative splice


variations which can be broadly categorized as unspliced (~9kb), incompletely spliced (~4kb), and fully-spliced mRNA (~1.8kb). Generally, primary intron containing RNA is retained in the nucleus, as such, only fully-spliced mRNA can initially exit and go on to be translated. This constitutes the first batch of viral factors including Tat, Rev, and Nef (Figure 9)120. As Tat increases the levels of transcription, Rev interacts with the Rev-response element (RRE) on the unspliced and incompletely spliced mRNA121,122. Using a leucine-rich nuclear export signal (NES), Rev binds to the karyopherin family member, exportin 1 (Crm1) which assists in transport into the cytoplasm123,124. Rev then re-enters the nucleus using importin- β. Now in the cytoplasm, the unspliced mRNA is used as the viral genome and can be translated into the Gag- Pol polyprotein while incompletely spliced mRNAs lead to Env, Vif, Vpu, Vpr, and Tat proteins (Figure 9)120,125.

Figure 9: Early and late HIV-1 Transcripts (adapted from Tazi et al. 2010)126. After integration and activation by cellular transcription factors, HIV-1 is transcribed and fully spliced to produce Rev, Tat, and Nef. Tat further drives transcription by interacting with the TAR and Rev binds incompletely and unspliced viral RNA in order to export them out of the nucleus without further modification.


1.4.4.4 Viral Particle Assembly, Release, and Maturation Now in the cytoplasm, the incompletely spliced mRNA can be translated on the rough endoplasmic reticulum into Env and Vpu as they are integral membrane protein. After translation, gp160 travels through the golgi apparatus via the secretory pathway where it is glycosylated and processed into trimeric complexes with gp120 and gp41 subunits with the protease, furin. This mature Env is then sent to the plasma membrane via vesicular transport. The other HIV-1 viral proteins are translated in cytosolic polysomes, the most important of which is Gag which has a major role in orchestrating the assembly and budding of the virion (Figure 10)127. Plasma membrane-specific lipid phosphatidylinositol 4,5-bisphosophate

(PI(4,5)P2) is abundant on the inner-leaflet of the plasma membrane in lipid-raft rich domains. Viral production is dependent on such factors as sphingolipids and cholesterol and their composition also helps form a detergent resistant membrane for the virion. PI(4,5)P2 plays an important role in targeting Gag and small Gag oligomers via their MA domains which subsequently exposes an N-terminal myristoyl group to be inserted into the plasma membrane thus anchoring the forming virion securely128-130. This interaction with MA also promotes the recruitment of Env to these lipid-raft rich regions of the plasma membrane131,132. Along the way, NC binds to dimerized unspliced copies of the HIV-1 genome through the interaction with its zinc-finger domains and the packaging signal sequence (Ψ) at the 5’ untranslated region (UTR) of the dimeric RNA133. In addition, one copy of tRNALys3 binds to each pbs of both viral RNAs via Watson-Crick base pairing to facilitate reverse transcription in the future and Gag-Pol is incorporated via CA interaction127,134. Once at the plasma membrane, Gag oligomerization is stabilized by CA and spacer 1 region (SP1) lateral interactions to form an immature lattice135,136. This immature lattice consists of hexameric subunits with interspersed non-hexameric defects and holes which allow for proper curvature formation137. These irregular defects end up becoming one of twelve pentameric defects in the mature virion138. As the immature lattice assembles and the components of the virion are present, the viral particle begins the process of budding. In the case of HIV-1, this budding and subsequent release is driven by host endosomal sorting complex required for transport (ESCRT) which is recruited by the p6 subunit of Gag. P6 contains two late domains: PTAP and YPXL which interact with tumor susceptibility gene 101 (TSG101) and ALG-interacting protein X (ALIX), respectively. TSG101 recruits ESCRT-I and ALIX is an adaptor protein involved in ESCRT-III


membrane remodeling and recruitment. Neural precursor cell expressed developmentally down- regulated protein 4 (NEDD4), an E3 ubiquitin ligase, is recruited by the CA domain of Gag and is also involved in the recruitment of ESCRT via the ubiquitination of Gag139-141. Once ESCRT- III and ESCRT-I are recruited, the virion is pinched off in a process of membrane fission catalyzed by the AAA ATPase, vacuolar protein sorting 4 (VPS4)142. Finally, viral maturation occurs with the cleavage of Gag and Gag-Pol with PR at 10 different sites producing MA, NC, p6, PR, RT, and CA which further goes on to become a fullerene-like conical capsid core143,144.


Figure 10: HIV-1 virion development and budding (adapted from Freed 2015)145. Env is synthesized on the rough ER and goes through the secretory system until ending up at the plasma membrane in future virion budding sites. Gag and Gag-Pol fusion proteins are synthesized from full-length viral RNA and multimerize on their way to the lipid raft rich regions of the plasma membrane. NC of Gag recruits two copies of full length viral RNA and lys3 tRNA. The components come together forming a viral bud. P6 recruits ESCRT-I and -III which allows for the completion of membrane fission. PR cleaves polyproteins thus completing maturation and the formation of the HIV-1 virion.

1.5 Host Restriction against HIV-I 1.5.1 Properties of Host Restriction Factors As part of the innate defense against viruses, the host produces factors such as interferons (IFNs) which further activate downstream JAK/STAT pathways leading to an initial immune response and activation of interferon stimulated genes (ISGs). Restriction factors are an important part of this category and represent a major defense against HIV-1 for which four accessory proteins have evolved to counteract this response146. All HIV-1 host restriction factors have a variety of functions and mechanisms, but they do share four core characteristics. First, restriction factors must directly and dominantly cause a significant decrease in HIV-1 infectivity. Second, an equally powerful counter must have been evolved by HIV-1. Third, due to the direct interactions between host factors and viral accessory proteins, there are often signs of rapid evolution through positive selection indicating a genetic arms race of sorts. Fourth, the restriction factors are closely tied to innate immunity and are either ISG-inducible or constitutively expressed. In addition, specialized against host restriction factors in humans, HIV- 1 is poorly equipped to infect non-human cells thus suppressing cross-species transmission. Since the classification of HIV-1 in 1983, investigation into viral accessory proteins has led to the discovery of multiple host restriction factors and a better understanding of viral mechanisms (Figure 11)146,147.



Figure 11: Overview of host restriction factors against HIV-1 (adapted from Barré- Sinoussi et al. 2013)148. Over the past 30 years many endogenous host restriction factors against HIV-1 have been identified targeting different parts of the viral lifecycle. SERINC3/5 and IFITM2/3 target viral fusion, TRIM5α targets uncoating, APOBEC3G and SAMHD1 target reverse transcription and are counteracted by Vif and Vpx (HIV-2), respectively, MxB prevents viral entry and potentially integration, SLFN11 inhibits viral RNA translation, and Tetherin, which is counteracted by Vpu prevents viral release. In addition, the recently discovered 90K, GBP5, MARCH8, and ERManI have been found to interfere with proper Env maturation, modification, and incorporation. Research into these host restriction factors has illuminated many different targets and pathways that have aided in the design of ARVs (green boxes).

1.5.2 APOBEC3G In an effort to investigate HIV-1 Vif, a viral accessory protein required for replication in non-permissive cells such as CD4+ T cells and monocyte-derived macrophages (MDMs), a cDNA subtraction-based screening identified Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 G (APOBEC3G or A3G) as a factor sufficient in inhibiting viral replication in Vif-deficient HIV146,149. A3G is a part of the APOBEC3 single-stranded DNA deaminase family of seven members including three single-domain deaminases A3A, A3C, A3H and four double-


domain deaminases, A3B, A3D, A3F, and A3G. These deaminases are expressed widely across human tissues and particularly in hematopoietic cells. A3G targets the reverse transcription step of the viral lifecycle and as such must be integrated into the virion for subsequent action during the next infection event150,151. During viral assembly, A3G interacts with NC in an RNA- dependent manner and less specifically with viral RNA to incorporate into the viral core. As the NC interaction is more important than that with the RNA, A3G is able to incorporate into HIV-1 even with high genomic variability152. A3G has two zinc-coordinating deaminase domains (Z domains), a catalytic C-terminal Z domain, and a non-catalytic N-terminal Z domain focused on integration. Once RT is initiated in the next target cell, A3G uses its C-terminal Z domain to catalyze the deamination of cytosines (C) on the transcribed negative-sense viral ssDNA to the ribonucleotide, uridine (U) which are then transcribed to adenine (A) rather than guanine (G) on the positive-sense strand. The result is a massive G to A hypermutation in the viral genome thus introducing many nonsense and missense mutations. This effect is further aided due to the preference of A3G to mutate the second C of a CC sequence thus resulting in a GG to GA mutation and therefore a higher frequency of nonsense mutations (Figure 12)153-156. The result is a viral DNA that is degraded by endogenous host machinery in the cytosol or produces non- viable viral products once integrated. A3G is also known to restrict reverse transcription in a deaminase independent manner by preventing tRNAlys3 binding to the pbs, causing termination of minus-strand synthesis, and impeding elongation of HIV-1 DNA157. Lastly, the similarities between A3G and certain APOBEC3 members such as A3B, A3D, A4F and A3H have led to evidence of HIV-1 antiviral activity as well, albeit to varying degrees146,153. To counter this restriction factor, HIV-1 Vif evolved a function which induces APOBEC3G degradation via a ubiquitin-dependent degradation pathway. Briefly, Vif recruits a cullin-5 based E3 ubiquitin ligase complex composed of elongin B, elongin C and Rbx-1 which all act to polyubiquitinate A3G thus leading to proteasomal degradation158. Surprisingly, even though Vif has evolved a function to specifically counter this restriction factor, a small amount of A3G action can introduce a beneficial low incidence of hypermuation that may confer rapid advantages on the virus159.



Figure 12: Mechanism of APOBEC3G antiviral activity and downregulation by Vif (adapted from Harris et al. 2012)147. In the absence of Vif, APOBEC3G is integrated into the virion thus causing G to A hypermuation and reducing viral viability. Vif counters this by triggering ubiquitination and proteasomal degradation of APOBEC3G.

1.5.3 TRIM5α In 2004, TRIM5α was identified through a screen of rhesus macaque genes that could restrict HIV-1 infection when expressed in human cells. As one of 70 members of the tripartite- motif (TRIM) containing cytosolic proteins160, TRIM5α has a TRIM domain composed of an N- terminal really interesting new gene (RING), B-box type 2, and coiled-coil domains as well as a C-terminal B30.2 or PRYSPRY domain. The protein itself is expressed ubiquitously at low levels and can be upregulated by interferon161. A major characteristic of TRIM5α is its inability to inhibit viruses of the same host species. For example, human TRIM5α inhibits Murine Leukemia Virus (MLV) as well as Equine Infectious anemia virus (EIAV), but is inactive against


HIV-1162-164. In contrast, the rhesus macaque ortholog of TRIM5α is effective in antagonizing HIV-1, but not the SIV strain affecting macaques160. Human TRIM5α has also been reported to mildly inhibit HIV-2 thus providing a potential explanation into why HIV-2 has a lower transmission rate165. Overall, the function of TRIM5α seems to be preventing cross-species infection of viruses162. The mechanism of TRIM5α viral inhibition is not well characterized, but it is believed that interaction with CA after insertion leads to premature uncoating and failure to complete viral cDNA synthesis. Using the PRYSPRY domain, TRIM5α binds to CA in a multivalent manner166,167. TRIM5α monomers were found to interact weakly with CA and as such it was found that the B-box and coiled-coil domains acted to multimerize the protein and create a complimentary lattice that could strongly interact with the capsid protein lattice168. Furthermore, the RING domain acts as an E3-ubiquitin ligase and the TRIM5α-HIV-1 complex was found to be associated with proteasomes indicating a potential degradation mechanism169. However, it was later found that even in the presence of proteasome inhibitors or deficient ubiquitin activating enzymes, HIV-1 was still inhibited. Another potential mechanism of blocking nuclear import motifs in order to prevent nuclear entry has been proposed170. Additionally, TRIM5α has been found to activate innate immunity by promoting AP-1 and NF-κB signaling171. Lastly, HIV- 1 does not have any accessory protein to counteract TRIM5α and as such, SIV must have successfully mutated CA to avoid inhibition during the original cross-species transmission event172. 1.5.4 Tetherin Bone marrow stromal antigen 2 or tetherin was first discovered in 2008 through a microarray screening of interferon inducible membrane associated proteins in cells that display inefficient or severely decreased virion release of Vpu deficient HIV-1 virions173. Tetherin is an IFNα stimulated type-II single-pass transmembrane protein that inhibits the release of virions by tethering them to the surface of the cell after budding. Structurally, tetherin encodes a transmembrane anchor close to its N-terminus, a glycophosphatidylinositol (GPI) at its C- terminus, and an extracellular domain that links the two terminuses in a coiled-coil174. In addition, tetherin is generally found as a homodimer linked by three disulfide bridges between cysteins at positions 53, 63, and 91175. Tetherin is unique in its non-specificity as it can target viruses from retroviruses, filoviruses, arenaviruses and herpesviruses. Even when major parts of


the restriction factor are replaced with sequences that produce a similar structure, it is still able to produce an antiviral activity thus leading to the conclusion that tetherin recognizes and binds to the host membrane that viruses such as HIV-1 use176,177. The mechanism of this downregulation involves insertion of either the C-terminus GPI, or transmembrane domain into the virion membrane and the other end into the host cell membrane as an anchor. Furthermore, tetherin was found to preferentially insert its N-terminus into the host membrane and could even insert both ends of one tetherin from the homodimer into one membrane and interact with another dimer via coiled-coil interactions to strengthen the anchoring146,178. The same method of tetherin homodimer to homodimer linking also links virions together (Figure 13). The result is that these virions accumulate at the surface and are eventually degraded once internalized via receptor- mediated endocytosis179. Tetherin also acts as a viral sensor for innate immunity: once the accumulation of tetherin causes detachment from the actin cytoskeleton and exposure of two tyrosine residues on the cytoplasmic tail a signal cascade is activated that upregulates NF-κB and therefore, proinflammatory C-X-C motif chemokine 10 (CXCL10), IL-6 and type I IFN180. To counteract this non-specific tethering, HIV-1 has evolved Vpu which is a type I transmembrane accessory protein that interacts with tetherin via its transmembrane domain to downregulate it through two main pathways. Although the exact mechanisms are not clear, Vpu is able to sequester tetherin in the trans-golgi network and signal for the internalization and degradation of tetherin through proteasome-mediated degradation which involves ubiquitination by SKP1-CUL-F box E3 ubiquitin ligase complex activated by β-TrCP2 on the cytoplasmic tail of Vpu181. Finally, many SIV strains do not encode Vpu and instead have evolved Nef and in some cases, even the viral envelope to counteract tetherin182,183. Additionally, due to the broad antiviral activity of tetherin, other viruses have evolved countermeasures such as herpes simplex virus 1 glycoprotein M, EIAV envelope, and influenza neuraminidase184,185.



Figure 13: Models of tetherin and virion interaction (adapted from Perez-Caballero et al. 2009)176. In model 1, two tetherin dimers (red proteins) anchor the virion as a dimer with both its GPI anchors (black) in the host membrane. Virions are linked together in a similar manner forming a chain. Model 2 depicts the opposite scenario while model 3 shows each subunit of one tetherin dimer inserting both of its anchors into either the host membrane or virion. Lastly, model 4 depicts the same scenario, but includes a second tetherin dimer interacting with the first.

1.5.5 SAMHD1 SAM domain and HD domain containing protein 1 (SAMHD1) was first discovered using a mass-spectroscopy-pull-down approach on Vpx in infected MDMs and other myeloid lineage quiescent cells which are not normally infectable by HIV-1186. Furthermore, Vpx is only found in HIV-2 and most SIV strains187. Structurally, SAMHD1 consists of an N-terminal sterile alpha motif (SAM) which is used for protein-protein interactions, a nuclear localization signal, and a C-terminal histidine-aspartic (HD) domain which is used for oligomerization and nucleic acid degradation147. When SAMHD1 was knocked out from mice MDMs, HIV-1 was able to infect these cells when it normally could not. This led to the conclusion that Vpx counteracted this restriction factor186,188. In addition, Vpr, which Vpx is duplicated from, is also known to cause gap 2 phase (G2) arrest and improve infection of MDMs, but to a lesser extent when


compared to Vpx189. SAMHD1 is a dGTP-regulated triphosphohydrolase that removes the phosphate from deoxynucleotide triphosphates (dNTP) thus leading to their depletion. dGTP must bind to an allosteric site which allows for tetramer formation and subsequent enzyme activation. Viral reverse transcription requires this pool of dNTPs to synthesize the cDNA, therefore, SAMHD1 blocks infection at the reverse transcription phase190,191. An additional model has also been proposed where SAMHD1 acts as an RNase that will bind viral genomic RNA-cDNA hybrids during reverse transcription and degrade them once a threonine residue near the C-terminal is phosphorylated192. Vpx is able to counteract SAMHD1 by promoting its proteasomal degradation. Briefly, Vpx binds SAMHD1 through its N-terminus and recognizes the C-terminal tail. It then tightly associates with the nuclear protein, DDB1-Cul4A-associated-factor-1 (DCAF1), and then binds to SAMHD1 through its N-terminus. Once bound, Vpx recognizes the SAMHD1 C-terminal tail and utilizes DCAF1, which is a substrate for Cul4A E3 ubiquitin ligase, to recruit the complex and polyubiquitinate SAMHD1 for proteasomal degradation188. In addition, the C-terminal tail of SAMHD1 is highly divergent across vertebrates therefore making them Vpx neutralization resistant when faced with different strains of virus193,194.

1.5.6 SLFN11 Schlafen (SLFN) genes encode a family of proteins that have 6 family members in humans and are upregulated during an HIV-1 infection especially in response to type I IFN. In 2012, a group discovered that when IFN was introduced, SLFN11 was the most prominent family member induced195. Structurally, SLFN11 has a conserved N-terminus ATPase Associated with diverse cellular Activities (AAA) domain and a RNA helicase-like motif. The mechanism of HIV-1 downregulation by SLFN11 is poorly understood, but it is believed that SLFN11 acts on a late stage of viral RNA translation thus preventing the synthesis of crucial viral structural and accessory proteins. More specifically, SLFN11 binds crucial tRNA using the viral codon-bias towards A/T nucleotides. Thus, maturation of these usually rare or HIV-1 introduced tRNAs are inhibited via posttranscriptional processing inhibition or deacylation upregulation. Another possibility is that the tRNAs are simply sequestered196.


1.5.7 MxB By observing gene expression through transcriptional profiling in cells pre-treated with IFN-α, myxovirus resistance protein 2(Mx2 or MxB) was discovered to have a clear antiviral effect against HIV-1197. MxB is a large GTPase that belongs to the dynamin superfamily. It consists of an N-terminal GTPase domain and NLS which are dispensable for antiviral activity, and a C-terminal stalk connected by three bundle signaling elements (BSE)198. MxB is found both in the cytoplasm and the heterochromatin region below the nuclear envelope due to the NLS. MxB also contains an N-terminal tri-arginine motif which has been found to interact with CypA on the HIV-I capsid199-201. Although the exact mechanism of action is unknown, MxB is thought to restrict HIV-1 uncoating therefore preventing nuclear entry of the PIC. However, MxB only recognizes complete CA assemblies and as such adopts an antiparallel dimer configuration, driven by the C-terminal stalk, which is required for the interaction202. MxB may also serve a function in preventing viral cDNA integration. HIV-1 does not have a specific accessory protein to counter this restriction factor, but instead introduces an escape mutation at alanine 88 to prevent binding of CypA and therefore, MxB restriction197. 1.5.8 IFITM Interferon induced transmembrane proteins 1-3 (IFITM1-3) were discovered to have antiviral activity against HIV-1 through an shRNA knockdown screening of ISGs in SupT1 cells203. IFITMs are part of the recently classified dispanin protein family and are characterized by C- and N-terminal variable regions, two-membrane associated regions, and a conserved intracellular loop. These restriction factors are ubiquitously expressed in different cells at a basal level, but are localized in different areas depending on the cell type and the IFITM204,205. The N- terminal domain is used for different subcellular localizations and, in the case of IFITM3, has been linked to its antiviral activity while the C-terminal domain is distinct in IFITM1 and is involved in its anti-viral function206,207. IFITM2 and 3 are known to inhibit viral entry in both producer and target cells. Their efficacy however, depends on the viral strain. For example, IFITM1 displays strong antiviral activity against the BH10 strain of HIV-1, but has no antiviral function in the NL43 strain207. IFITM2 and 3 are incorporated into nascent virion envelopes during the viral formation thus reducing the viability of the virus through various mechanisms. More specifically, the processing of Env is perturbed, and the turnover of gp160 is increased thus resulting in fewer


incorporated gp120. All in all, IFITM2 and 3 affect the ability for HIV-1 to create a proper virion thus preventing fusion at the target cell208. IFITM1 is thought to act after during viral protein synthesis and is thought to target viral double-stranded RNAs with a RRE or TAR, which HIV-1 mRNA contain. The result is a reduction in the expression of Gag, Vif, and Nef which will reduce the viability of the virus significantly209. Lastly, although HIV-1 does not have a specific factor to counteract these restriction factors, some strains have developed escape mutations in the Env or Vpu region208. 1.5.9 Recently Identified Restriction Factors Host restriction factors continue to be a well-researched topic and recently 90K, GBP5, ERMANI, and MARCH8 have been identified. 90K was identified as a host restriction factor that interferes with HIV Env maturation and incorporation in 2013. 90K or LGAL3SBP is an IFN-inducible secretory glycoprotein that is a member of the scavenger receptor cysteine-rich (SRCR) domain-containing proteins. This protein is generally found secreted in body fluids ranging from blood to breast milk and was often used as a serological indicator of AIDS progression. Although the exact mechanism of antiviral activity and potential viral countermeasures have not been elucidated, two central-binding domains, BTB/POZ and IVR have been found to be crucial for antiviral activity. In addition, a potential direct interaction with Env has been hypothesized due the 90K N-terminal glycosylated signal peptide thus shuttling the protein through a similar pathway as HIV-1 Env210. Furthermore, presence of elevated 90K in breast milk has been positively correlated with reduced mother to child HIV transmission211,212. In 2015, guanylate-binding protein 5 (GBP5), a member of the dynamin superfamily of GTPases, was identified using a genome-wide screen of human genes with evolutionarily and molecularly similar signals to other known host restriction factors213. GBP5 is localized to the golgi apparatus in viral producing cells and variably in macrophages. The mechanism of action has not been fully elucidated, but clear interference with the processing of HIV-1 Env, specifically the N-linked glycosylations, reduces the incorporation of gp120 and decreases the viral infectivity. To counter this effect, Vpu, which is translated as part of gp160 is mutated at its start codon thus resulting in immediate Env expression and decreased sensitivity to GBP5. However, this mutation also increases Vpu-deficient HIV strains which tend to cause more inflammation as Vpu normally inhibits NF-κB activity to suppress inflammatory cytokines.


Furthermore, this increase in inflammation has been hypothesized to be involved in HIV- associated neurological disorders214. In the same year, an IFN-inducible membrane-associated RING-CH 8 (MARCH8) was discovered to have antiviral activity against HIV and other enveloped viruses by blocking Env incorporation into virions thus decreasing infectivity215. MARCH8 is part of the recently discovered RING-finger E3 ubiquitin ligase family and is endogenously expressed in MDM’s and dendritic cells or terminally differentiated myeloid cells. In addition, IFNα has been shown to slightly elevate MARCH8 expression in macrophages. Structurally, MARCH8 is a membrane associated protein with two or more potential transmembrane domains and a C4HC3 RING finger domain in its N-terminal cytoplasmic domain, which was found to interact with an E2 enzyme. Furthermore, MARCH8 was previously found to downregulate a variety of host transmembrane proteins including major histocompatibility complex II (MHCII) and IL-1. In terms of HIV-1 Env, MARCH8 has been hypothesized to recognize the three-dimensional structure of Env, rather than a motif, from the surface of the cell and sequester it away from the virion thus decreasing overall infectivity of virions produced. Currently, a viral countermeasure has not been found216,217. Lastly, Zhou et al. previously reported that the mitochondrial translocator protein (TSPO) induced HIV-1 Env degradation via the endoplasmic reticulum-associated protein degradation (ERAD) pathway218. As such they found a host protein involved in ERAD responsible for this degradation was the ER-associated α-mannosidase of the glycoside hydrolase family 47 (GH47), ERManI. ERManI contains a cytoplasmic and transmembrane domain as well as a luminal catalytic domain219. This catalytic domain is involved in the first mannose trimming step of N- linked glycosylation and in the degradation of misfolded protein via the ubiquitin/proteasome pathway after the misfolded protein is retro-translocated to the cytoplasm220. In non-permissive cell lines, Env was found to be degraded via this pathway and after multiple CRISPR/CAS9 knockout and mutational studies, the catalytic domain of ERManI was found to be necessary for its antiviral activity. As Env folding is dependent on the formation of many disulfide bonds and N-terminal glycosylations, the mitochondria must produce reactive oxygen species and deliver them via the mitochondrial-associated ER membrane, which consists of anion channels. The overexpression of TSPO is thought to negatively interact with these channels thus reducing the available reactive oxygen species and therefore inhibit proper modification of Env. The result is


detection of misfolding by ERManI and activation of ERAD. As with many other newly identified host restriction factors, a fully-supported specific mechanism and viral countermeasure has yet to be fully determined221. 1.5.10 Nef and SERINC5 The HIV-1 accessory protein Nef is a 27kDa versatile myristoylated protein that has many roles in viral pathogenesis and infectivity222. First, Nef can promote the internalization of many important receptors and immune molecules on the cell surface. For example, CD4 is shuttled into clathrin coated vesicles for endocytosis via binding to CD4 and adaptor protein 2 (AP-2) for lysosomal degradation. This prevents superinfection by HIV-1, increases viral release, and overall infectivity223,224. Furthermore, the downregulation of other costimulatory molecules such as CD28 prevents communication between T cells and antigen presenting cells (APCs) thus delaying an immunological response225. CD8+ cytotoxic T lymphocyte (CTL) lysis of infected cells is also avoided via downregulation of MHC-I by Nef activation of clathrin coated endocytosis and subsequent degradation226. Nef also interacts with several kinases thus interfering with many signaling pathways and increasing the production of NF-κB thus driving the stable promotion of HIV-1 transcripts via the LTR promoter region227. Another important function of Nef is the upregulation of Fas ligand and other similar factors to induce bystander cell death and further decrease a potential immune response against the infected cell228,229. Lastly, in 1994, Nef was reported to increase viral infectivity thus contributing to high viral loads via an unknown mechanism230. 21 years later, two groups independently discovered that Nef downregulates SERINC5 and to a lesser extent SERINC3 to increase viral infectivity by up to 100-fold231,232. These proteins are part of the homologous serine incorporator family (SERINC). This family is highly conserved across eukaryotes and consists of five members of multi-pass transmembrane proteins with at least 10 transmembrane domains (TM) that have been shown to be expressed in, neurons, lymphocytes, macrophages, and other peripheral blood mononuclear cells. SERINCs have been proposed to facilitate the synthesis of phospholipids such as phosphatidylserine and sphingolipids, that require the non-essential amino acid, serine. As the name suggests, SERINCs are thought to act as a carrier of serine, which is a polar and hydrophilic amino acid produced in the cell by 3-phosphoglycerate dehydrogenase, into the lipid bilayer membrane. In addition, SERINCs are predicted to act as a scaffold for two enzymes, phosphatidylserine synthase and


palmitoyltransferase which are involved in phosphatidylserine and sphingolipids synthesis, respectively233,234. In terms of HIV-1, SERINC5 was found to incorporate into forming virions at the plasma membrane. This process was further enhanced due to HIV-1 virion preference for formation at lipid raft regions where SERINCs are normally found. Once the mature HIV-1 virion attempts to enter the target cell, SERINC5 prevents proper widening of the fusion pore or its formation to begin with thus inhibiting release of the HIV-1 capsid core235. The result is a significant decrease of infectivity in Nef-deficient HIV-1 (Figure 14). Nef was found to counteract SERINC by downregulating it into Rab7-positive late endosomes through a clathrin- mediated pathway231,232. Furthermore, the V3 loop of the Env glycoprotein of certain HIV-1 strains was found to be important in downregulating SERINC5 as well236.

Figure 14: HVI-1 antiviral activity by SERINC5 and counteraction by Nef (adapted from Aiken et al. 2015)237. When HIV-1 expresses Nef properly, SERINC5 and SERINC3 are downregulated from the surface of the infected cell producing the virion into RAB7-positive late endosomes thus allowing unperturbed viral delivery to the target cell (A). When Nef is defective

or not expressed, host plasma membrane containing SERINC5 will be integrate into the forming virion membrane thus impairing cytoplasmic delivery of the capsid core at the target cell and reducing overall infectivity.

Unlike most host restriction factors, SERINC5 is not known to be IFN inducible and has been found to not have had an evolutionary arms race with its antiviral countermeasures, Nef and Env238. Rather Nef has been hypothesized to have evolved its current function due to the accumulation of SERINC5 in HIV-1 strains with weaker entry capabilities thus overcoming the Env countermeasure, changing its conformation to expose gp41 domains, and opening the HIV-1 virion to inhibition by broadly neutralizing antibodies235,236. 1.6 Project Objectives SERINC5 has recently been shown to reduce HIV-1 infectivity in the absence of Nef. Currently, the underlying mechanism behind this antiviral effect has been attributed to the reduced expandability of the viral fusion pore upon entry thus preventing the proper delivery of the viral core and stopping the HIV-1 infection from occurring in the target cell. This mechanism however, is relatively superficial and there is still much that is not known about SERINC5 as a protein and as a host restriction factor. To learn more about these two subjects, this work is focused on three main objectives231,232. First, the ubiquitination of SERINC5 was investigated. There is no current evidence confirming the presence of ubiquitination, however, we hypothesize that there is a potential for the downregulation by Nef to involve a ubiquitination method similar to the counteraction of tetherin by Vpu. Ubiquitin itself has a mass of 8.5kDa and has a variety of uses from regulating proteasomal and lysosomal degradation to regulating protein interactions, localization, and in some cases, stability239. These functions are triggered once a mono- or poly-ubiquitin chain is established on certain lysine residues or the N-terminus of a protein post-translationally240. Furthermore, ubiquitination is facilitated by three types of ubiquitin enzymes: ubiquitin- activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligase enzymes (E3s)241-243. Once a basis for ubiquitination was established via co-immunoprecipitation experiments, the effect of various proteasomal and lysosomal inhibitor drugs as well as ubiquitin mutants were tested to establish a better understanding of the purpose of this post-translational modification with regards to the antiviral activity of SERINC5.

Secondly, a potential interaction between Nef and the intracellular domains of SERINC5 was investigated using a chimeric-CD4/SERINC5 internalization assay. Lastly, the domains of SERINC5 that are required for its antiviral activity against Nef-deficient HIV-1 was explored. In this case, chimeras between the non-antiviral SERINC1 and antiviral SERINC5 with a broad followed by a narrower exchange of domains were generated, and evidence for high antiviral activity in the central third was established.

Chapter 2: Methods 2.1 Cell Lines and Culturing System HEK 293T cells, or primary human embryonic kidney cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher) supplemented with 10% Fetal Bovine Serum (FBS) (Thermo Fisher) and 1% Penicillin/Streptomycin (P/S) (Thermo Fisher). TZM-bl luciferase reporter cells which are a HeLA derived cell line which has an HIV-LTR driven firefly luciferase reporter gene were also maintained in the same medium. Both the HEK 293T (Cat # 103) and TZM-bl (Cat# 8129) cells were obtained from the NIH AIDS Reagent program. All cells were grown in carbon dioxide incubators with a CO2 concentration of 5% and a temperature of 37 °C. 2.2 Plasmids and Chimeric Constructs pQCXIP and pQCXIH were purchased from Clontech. Ha-tagged pUb, pK63, pK48, and pUbKO plasmids were kindly provided by Dr. Rongutuan Lin. N-terminal FLAG labeled IFITM3 cloned into pQCXIP was kindly provided by Yimeng Wang. pNL4-3 was obtained from the NIH AIDS Reagent Program. CD4 and CD4-5 (Nef binding-KO) in pQCXIH, Nef, HIV-1 (G2A) mutant, N-terminal FLAG tagged SERINC5, and SERINC1 in pQCXIP were kindly provided by Saina Beitari. CD4-5 was previously generated by removing the C-terminal tail harboring the Nef- binding site via PCR. All primers were generated through Invitrogen (Figure 15A). Next, primers were designed each with one half of the 6 intracellular domains of SERINC5 (hereafter referred to as IC1-6). Each pair of primers were flanked by Bsi-WI and Bam-HI restriction sites (NEB) which corresponded to a restriction site flanking the CD4-5 terminus and the multiple cloning site, respectively. Intracellular regions of SERINC5 were amplified via PCR, digested along with CD4-5 with Bam-HI and Bsi-WI (NEB), and ligated using T4 DNA ligase (Invitrogen) for 1 hour. IC2, 4, and 6 of SERINC5 contained 12 amino acids or below and as such, oligonucleotides were designed and ordered from Invitrogen in a similar fashion to the primers, but without a gap in between the pairs. These oligonucleotides were phosphorylated with T4 Polynucleotide Kinase (PNK) (NEB), annealed together using T4 DNA ligase (NEB), and finally, ligated into the CD4-5 plasmid as the others were. Six SERINC5/SERINC1 chimeras interchanging areas encompassing 3-4 transmembrane domains were designed and synthesized by NeoBioLab. Designs were assisted by

transmembrane domain prediction using TMHMM Server v. 2.0 (DTU Center for Biological Sequence Analysis) following the recommended protocol. Mlu-I and Eco-RI (NEB) restriction sites were added into the design at the 5’ and 3’ end, respectively. Mlu-I and Eco-RI (NEB) are also present in the multiple cloning site for pQCXIP and were used to digest both the synthesized gene and vector to ligate the insert gene with T4 DNA ligase (NEB). A second pair of SERINC5/SERINC1 chimeras were constructed using the C2 chimera previously synthesized. Two regions of the C2 chimeras, one between amino acid 144 (the beginning of the previously interchanged sequence) and an inserted AleI restriction site (NEB) corresponding to SERINC5 as well as between the same AleI restriction site (NEB) and amino acid 311 (the end of the previously interchanged sequence) was amplified via PCR (Figure 15B). These inserts were then digested along with FLAG-tagged SERINC5 with Mlu-I and AleI or AleI and Eco-RI (NEB). The inserts were phosphorylated with T4 PNK and the vector dephosphorylated with calf intestinal alkaline phosphatase (CIP) (NEB) followed by ligation with T4 DNA ligase (Invitrogen).

Figure 15: Primer design. (A) Primers for CD4-SERINC5 chimera and CD4-5 construction (provided by Saina Beitari). Primers designed for IC2-4 are meant to be annealed together and inserted. (B) Primers for SERINC5/SERINC1 C2 chimera construction. Red text indicates restriction sites, blue text indicates SERINC5 sequence, green text indicates SERINC1 or CD4 sequence, brown text indicates FLAG tag, and black text indicates a protection sequence. All primers were designed by hand and confirmed to fit the proper 40-60% GC content and a melting temperature between 65-75°C. These values were confirmed with the online Multiple Primer Analyzer (Thermo Fisher).

All clones were transformed into DH-5α competent cells (Invitrogen). The transformed bacteria were plated in ampicillin-positive agar plates (BioShop) and were allowed to grow at 37oC for 16 hours. The successful clones were selected out by the indication of ampicillin resistance in their viral plasmids. These colonies were mini-prepped using E.Z.N.A. Plasmid Mini Kit (Omega bio-tek) and the plasmids were sent to be sequenced at MCLAB. Plasmids


indicating a successful sequence were maxi-prepped using E.Z.N.A. Plasmid Maxi Kit (OMEGA Bio-tek) and re-checked with MCLAB. All temperatures and concentrations used were those that were directed by the respective product manuals. 2.3 Transfection For co-transfections involving ubiquitin, 4x106 HEK 293T cells were seeded into each 10cm cell culture dish and allowed to grow in 6mL antibiotic free 10% FBS DMEM medium 20 hours prior to transfection. 4µg of total DNA, 250µl Opti-Modified Eagle Medium (opti-MEM) (Invitrogen), and 12µl of polyethylenimine (PEI) were mixed and incubated at room temperature for 15 minutes at which point they were added to the 10cm dish of cells. The media was changed gently 6 hours later.

For all other co-transfections, .5x106 HEK 293T cells were seeded into each well of a 6- well cell culture plate and allowed to grow in 2mL antibiotic free 10% FBS DMEM 20 hours prior to transfection. 1µg total DNA, 100µl opti-MEM (Invitrogen), and 3µl PEI were used with the same procedure as mentioned before.

2.4 Co-Immunoprecipitation 6 hours before harvesting, 1µM MG132, 20µM Calpain Inhibitor I (ALLN), 1µM Brefeldin A (BFA), or 10µM Bafilomycin A1 (Sigma-Aldrich) were added in some experiments to inhibit proteasomal or lysosomal degradation therefore testing whether SERINC5 is degraded via those pathways. Supernatant from 10cm plates were discarded and 5mL ice-cold Dulbecco’s Phosphate Buffered Saline (PBS) (Gibco) containing 2% FBS was used to remove cells with scraping. Cells were centrifuged at 1300rpm for 3 minutes to remove the supernatant followed by two more washes with ice-cold PBS 2% FBS. Cells were then incubated with 500µl lysis buffer on ice for 40 minutes. The lysis buffer was made using 50mM Tris HCl pH 8.0, 100mM NaCl, 1mM Ethylenediaminetetraacetic acid (EDTA), 1% triton x-100 (Sigma-Aldrich), and protease inhibitor (Roche). Lysate was centrifuged at 13,200rpm for 15 minutes at 4°C. 400µl of lysate supernatant was then divided equally into two tubes of 200µl, one for immunoprecipitation and the other for the input western blot. The rest of this procedure is done in a 4°C cold-room. First, 30µl of affinity gel with FLAG antibody (Sigma-Aldrich) was prepared for each sample. The beads were washed with 1mL of lysis buffer without protease inhibitor, rotated for 1 minute, centrifuged at 6000rpm for 3 minutes, and allowed to rest on ice


for 5 minutes before removing the supernatant. Meanwhile, the protein load was normalized to the concentration of the lowest concentrated sample by using a Bradford protein assay. To perform this test, 200µl 1x BioRad protein assay dye reagent was mixed with 10µl protein sample in a plastic cuvette followed by protein concentration measurement by a spectrophotometer at 595nm. Each sample was properly diluted with lysis buffer to a total volume of 200µl which was then added to the beads and left to rotate overnight. The next day, the mixture was centrifuged at 2500rpm for 5 minutes and the supernatant removed. The beads were then washed twice with lysis buffer without protease inhibitor, rotated for 10 minutes followed by centrifugation at 2500rpm for 3 minutes. After removing the supernatant, the beads were washed twice with 1mL Tris buffer saline (TBS) consisting of 50mM Tris HCl (pH 7.4) and 150mM NaCl. The conjugated protein was then eluted with 50µl 3xFlag peptide elusion solution consisting of 3% 3XFlag peptide in TBS for 30 minutes on ice. Lastly, the supernatant containing eluted protein was collected after centrifugation at 6000rpm for 6 minutes. Due to low sample detection in some experiments, 4X loading buffer was added directly to the beads to elute all proteins. All samples were analyzed via western blot. 2.5 Flow Cytometry Cells requiring further analysis via flow cytometry were gently removed with PBS containing 2% FBS and 1mM EDTA. Cells were washed twice with 500µl of PBS 2% FBS and centrifuged at 1300rpm for 3 minutes to remove the supernatant. For cells infected with virus, cells were incubated with 2% paraformaldehyde (PFA) PBS 2% FBS mixture on ice for 30 minutes to fix cells. For surface staining only, 50µl of mouse monoclonal anti-human CD4 antibody conjugated to phycoerythrin (PE) fluorochrome (Sigma-Aldrich) diluted 1:5000 in PBS containing 2% FBS was added for 1 hour on ice in the dark. If the cells were not being intracellular stained as well, the surface stained cells were washed three times with 500µl PBS containing 2% FBS and centrifuged at 1300rpm for 3 minutes to remove the supernatant. For intracellular staining, fixed cells were washed once more with 500µl PBS 2% FBS and then twice with 500µl 1x IC permeabilization buffer (Invitrogen). 50µl Mouse monoclonal anti-Flag (Sigma-Aldrich) diluted 1:5000 in 1x permeabilization buffer was added to the fixed and permeabilized cells for 1 hour on ice in the dark. Cells were then washed three times with 1x permeabilization buffer. For all samples, after adding antibody, all work was done away from direct light. All cells (surface and/or intracellular stained) were resuspended in 200µl of PBS


containing 2% FBS for analysis by BD FACSCalibur flow cytometer (BD Biosciences). Flow cytometer data was analyzed with the FlowJo program. 2.6 Luciferase Assay 1.2x106 TZM-bl cells were seeded into each well of a 24-well plate 20 hours prior to infection in .5mL DMEM 10% FBS 1% P/S (Thermo Fisher). 100µl of virus containing supernatant from viral producing cells 48 hours post-transfection was transferred to the corresponding wells to infect the TZM-bl cells. Cells were supplemented with an additional .5mL DMEM 10% FBS 1% P/S. 48 hours post-infection, supernatant was removed and cells were washed with .5mL plain PBS. Cells were then lysed with 100µl 1x passive lysis buffer (Promega). After 30 minutes, 10µl of cell lysate was mixed with 40µl of luciferase substrate (Promega) and the luciferase activity was measured using Glomax 20/20 luminometer. 2.7 RT Assay 10µl of the collected supernatant containing viruses were added to a 96-well plate. 40µl of the reaction cocktail containing: Triton-X, poly-adenosine RNA template, and tritium labeled deoxy tritium nucleotide triphosphate (dTTP) (Perkin Elmer) was added to each sample in the o plate. The mixture was then incubated for 3 hours at 37 C and 5% CO2 for reverse transcription to proceed. Reverse transcription was stopped and the synthesized nucleotides precipitated by adding 150µl of cold 10% trichloroacetic acid (TCA) (Millipore), and incubating at 4oC for 30 minutes. A MultiScreen filter plate (Millipore) was moistened with 200µl 10% TCA and the solvent from each sample was removed via vacuuming through the filter. In this way, the precipitated tritium labeled DNA was trapped in the filter membrane and then washed twice with 200µl 10% TCA, and once with 200µl 95% ethanol. Each membrane was transferred into scintillation vials (Diamed) and 3ml of liquid scintillation cocktail (MP Biomedicals) was added. The amount of radioactivity released from each membrane was measured by detecting β-decay using the Wallac 1410 Liquid Scintillation Counter (PerkinElmer). The data was analyzed in excel and allowed the quantification of absolute infectivity as it measured the amount of virus loaded for each sample in the luciferase assay. 2.8 Western Blot 48 hours post-transfection, the supernatant for the 293T cells in 6-well plates was removed and 300µl Cytobuster Protein Extraction Reagent (EMD Millipore Novagen) with protease inhibitor (1 tablet per 10mL) (Roche) was added. After 5 minutes, cells were pipetted


up and down to release them from the plate and immediately moved into 1.5mL Eppendorf tubes on ice. If the cells were also to be used for flow cytometry, detachment following the aforementioned flow cytometry protocol was used and the cells were split into separate tubes after detachment. After 45 minutes, the lysate was centrifuged at 13,200rpm at 4 oC for 15 minutes to separate the cell debris. 100µl of protein supernatant (either from co- immunoprecipitation or the viral producing samples) was then denatured by mixing with 33.3µl 4x protein loading buffer followed by 5 minutes of boiling. Due to the difficulty of SERINC5 detection, samples were split in half prior to boiling and reserved to be processed unboiled. The protein samples were then loaded onto 1% sodium dodecyl sulfate (SDS) (BioShop) and 10% polyacrylamide gel (BioShop) for the detection of p24, SERINC5 (Flag-tag), and tubulin. When detecting gp120, 1% SDS and 8% polyacrylamide gel was used instead. Proteins were separated by electrophoresis at 90V for 30 minutes and then 120V for the next 1-2 hours. This was followed by a transfer onto polyvinylidene difluoride membrane (Roche) at either 30V overnight or 80V for 2 hours on ice. The membrane was then blocked with 5% skim milk dissolved in 1x PBS with 0.1% Tween 20 (PBST) (BioShop) for 1-hour rocking, followed by incubation with 1:5000 diluted primary antibodies for 2 hours at room temperature accompanied with rocking. The primary antibodies include: sheep α-gp120 single chain antibody (NIH AIDS Reagent program), polyclonal rabbit α-p24 antibody (Sigma-Aldrich), monoclonal mouse α-flag antibody (Sigma-Aldrich), rabbit polyclonal anti-HA antibody (Sigma-Aldrich), mouse monoclonal anti-β-Tubulin antibody (Santa Cruz Biotechnology), and mouse monoclonal anti- HIV-1 Nef antibody (Abcam). The membranes were then washed five times with PBST. Next, the membrane was further incubated with 1:10000 diluted secondary antibodies for 1 hour at room temperature with rocking followed by four washes with PBST. The secondary antibodies include: horseradish peroxidase (HRP)-linked donkey anti-rabbit IgG (GE Healthcare), HRP- linked sheep anti-mouse IgG (GE Healthcare), and HRP-Rabbit Anti-Sheep IgG (Invitrogen). Lastly, the membranes were visualized with Western Lightning Plus-ECL Enhanced Chemiluminescence Substrate (PerkinElmer) and recorded on X-ray film (Carestream) at varying exposure times.


Chapter 3: Results 3.1 Ubiquitination 3.1.1 SERINC5 is Ubiquitinated Aside from the SERINC family role in facilitating phospholipid biosynthesis, not much is known about the proteins themselves. We chose to focus on ubiquitination as it has a role in trafficking and in the case of certain restriction factors such as tetherin, another transmembrane domain protein, is the mechanism used for the downregulation of its antiviral activity. We co- transfected 293T cells with 3µg FLAG-tagged SERINC5 and 1µg HA-tagged ubiquitin followed by co-immunoprecipitation and immunoblotting for detection. FLAG-tagged IFITM3, which has been found to be downregulated by ubiquitination to limit its antiviral effect against the influenza virus, was used as a positive control244. In addition, 1µM MG132, a 26S proteasome complex inhibitor, which is involved in ubiquitin-conjugated protein degradation, was added after transfection to half of the cells to see if there was any chance that we could increase the detection of ubiquitinated SERINC5 (Figure 16)245. The presence of a band in the SERINC5+Ub lane of the IP anti-HA immunoblot suggests that SERINC5 is indeed ubiquitinated. In addition, the lack of an increase in SERINC5 detection (anti-FLAG immunoblot) and the minor increase in its ubiquitination (IP anti-HA immunoblot) while in the presence of MG132 suggests that SERINC5 is most likely not degraded by the 26S proteasome complex.

Figure 16: SERINC5-ubiquitin co-immunoprecipitation. Representative anti-FLAG (detecting SERINC5 and IFITM3), anti-HA (detecting ubiquitin), and tubulin input and immunoprecipitation (IP) immunoblots using FLAG co-immunoprecipitation. Two faint bands for SERINC5+Ub and SERINC5+Ub+MG132 can be seen on the HA-IP immunoblot thus indicating SERINC5 ubiquitination. Three major bands can be seen on the HA-IP immunoblot for IFITM3+Ub due to its known polyubiquitination. Tubulin was used as a loading control. Experiments were replicated twice (n=2).

3.1.2 SERINC5 ubiquitination does not involve the proteasome Next, we tested three additional inhibitors to MG132: bafilomycin A1, calpain inhibitor I (ALLN), and brefeldin A (BFA). Bafilomycin A1 is a macrolide antibiotic isolated from

Streptomyces griseus and inhibits protein degradation in lysosomes by downregulated vacuolar- type H+-ATPase thus preventing proper acidification of lysosomes246,247. Similar to MG132, the peptide aldehyde ALLN also inhibits 26S by blocking the chymotrypsin-like activity of the proteasome248,249. Lastly, BFA is a fungal metabolite that disrupts the structure and function of the golgi, impairing transport between endosomes and lysosomes250. These inhibitors were added 6 hours before cell-harvesting of 293T cells co-transfected with 3µg Flag-tagged SERINC5 or Flag-tagged IFITM3, and 1µg HA-tagged ubiquitin (Figure 17). As can be seen on the IP anti- FLAG and IP anti-HA immunoblots, SERINC5 retention and ubiquitination was not affected to a significant degree by any of the inhibitors.

Figure 17: Ubiquitination of SERINC5 under the effect of proteasomal and lysosomal inhibitors in 293T cells. Representative anti-FLAG (detecting SERINC5 and IFITM3), HA

(detecting ubiquitin), and tubulin immunoblots for the input and IP samples of FLAG co- immunoprecipitation after treatment of cells with MG132, BFA, ALLN, or Bafilomycin A1 (BafA1) 6 hours prior to cell harvesting. The non-specific band in the FLAG-IP immunoblot is due to the light chain of the anti-FLAG antibody eluting out with the loading buffer. Prior attempts using the FLAG elution peptide produced faint results. Tubulin was used as a loading control. Experiments were replicated twice (n=2).

3.1.3 Determination of SERINC5 polyubiquitination With the knowledge of SERINC5 ubiquitination, we set out to determine if SERINC5 is polyubiquitinated. Polyubiquitination chain linkages generally occur at the lysine 48 and lysine 63 residues (K48 and K63) of ubiquitin and are most often involved in proteasomal and lysosomal degradation, respectively251,252. To investigate the potential polyubiquitination of SERINC5, 3µg of FLAG-SERINC5 or FLAG-IFITM3 control, 1µg HA-ubiquitin or its HA- tagged mutants: Ub-K63, Ub-K48, and Ub-KO were transfected into 293T cells. These ubiquitin mutants consist of lysine to arginine mutations at all lysines except for the 48th or 63rd one therefore making it possible for only that specific polyubiquitination chain to be established. Ub-KO does not include any lysine residues and as such is not capable of polyubiquitination253. After transfection, these proteins were co-immunoprecipitated and detected via immunoblot (Figure 18). As can be seen in the IP anti-HA immunoblot, SERINC5 seems to be polyubiquitinated with K63 ubiquitin linkages due to the stronger signal in the S5+Ub-K63 vs S5+Ub-K48 lane. Furthermore, the signal in the S5+Ub-K48 lane was about as weak as the one in the S5+Ub-KO lane thus indicating a lack of ubiquitination at the 48th lysine residue. In addition, SERINC5 retention as indicated in the IP anti-FLAG immunoblots did not change depending on the ubiquitination state.

Figure 18: Evidence of potential SERINC5 polyubiquitination at the lysine 63. Representative anti-FLAG (detecting SERINC5 and IFITM3), anti-HA (detecting ubiquitin and its mutations), and anti-tubulin input and IP immunoblots of FLAG co-immunoprecipitation. The non-specific bands in the FLAG-IP immunoblot are due to the light chain of the anti-FLAG antibody eluting out with the loading buffer. Bands appear smeared in the HA-blots due to the presence of multiple ubiquitination linkages on SERINC5 or IFITM3. Tubulin was used as a loading control. All experiments were replicated three times (n=3).

3.1.4 Nef does not increase SERINC5 ubiquitination Lastly, we investigated whether Nef increased the ubiquitination of SERINC5. MG132 was also added to half of the samples to conclude on whether SERINC5-Nef interaction resulted in proteasomal degradation. For this experiment, 1µg non-tagged NL43 Nef, 1µg ubiquitin, and 3µg FLAG-tagged SERINC5 were co-transfected into HEK 293T cells, co-immunoprecipitated,

and detected via western blot (Figure 19). As can be seen in the IP anti-FLAG immunoblot, the amount of retained and therefore, non-degraded SERINC5 was decreased in the presence of Nef, but rescued by MG132. Furthermore, the degree of SERINC5 ubiquitination as shown by the IP HA-immunoblot especially in the presence of MG132 and Nef was significantly less than ubiquitination without the protease inhibitor.

Figure 19: Effect of Nef on SERINC5 ubiquitination. (A) Representative anti-FLAG (detecting SERINC5 and IFITM3), anti-HA (detecting ubiquitin), and anti-tubulin input and IP


immunoblots of FLAG co-immunoprecipitation. The non-specific bands in the FLAG-IP immunoblot are due to the light chain of the anti-FLAG antibody eluting out with the loading buffer. Bands appear smeared in the HA-blots due to the presence of multiple ubiquitination linkages on SERINC5 or IFITM3. FLAG-input is absent due to poor transfer quality. Tubulin was used as a loading control and in (B), Nef was used as a control to detect if Nef was properly expressing.

3.2 Nef interaction with SERINC5 3.2.1. Chimeric design As previously described, CD4 interaction is of integral importance for both an immune response and HIV-1 infection. After HIV-1 has infected a cell, Nef causes the internalization of the transmembrane CD4 through an interaction requiring the last 29 amino acids of the N- terminus of CD4254. Using PCR and restriction digests, we removed this region and cloned the new Nef-mediated pulldown deficient CD4 (CD4-5) into pQCXIH to drive its overexpression. To test whether SERINC5 interacted with Nef directly, we first determined the domain structure organization using the TMHMM server and designed chimeras where one of the six intracellular domains of SERINC5 was added in place of the Nef-interaction site on CD4 (Figure 20). These chimeras were designed to be used alongside HIV-1 Nef and would in theory be internalized if Nef interacted with one of the CD4-SERINC5 chimeras via the SERINC5 intracellular domain tail.



Figure 20: CD4-SERINC5 chimera designs. (A) TMHMM domain prediction for SERINC5 was used to create a domain map for SERINC5 highlighting its intracellular domains (B). (C) Each intracellular domain was then used to replace the Nef binding site on the cytoplasmic tail thus creating 6 chimeras (IC1-IC6).

3.2.2 Experimental viability of the CD4-SERINC5 internalization assay Before using these chimeras, an optimized dosage of Nef needed to be determined. To do this, a titration of Nef at dosages of 50-500ng along with 50ng CD4 were co-transfected into


HEK 293T cells and the measurement of surface CD4 was done via flow cytometry (Figure 21). From this experiment, 50ng Nef, which was the smallest dosage, was determined to be sufficient to induce the internalization of CD4 without saturating the decrease in the signal.

Figure 21: Titration of Nef; CD4 internalization assay. (A) flow cytometry data for 50, 100, 200, and 500ng Nef cotransfected with 50ng wildtype CD4 measuring the surface levels of CD4 increasing on the X- axis as well as cellular granularity (SSC) on the Y- axis which is used for cell quantification selection. Flow cytometry data is summarized in (B). Experiment was replicated twice (n=2).

Next, 50ng of the intracellular chimera and Nef plasmids were co-transfected into HEK 293T cells, harvested, surface stained for CD4, and analyzed via flow cytometry (Figure 22A-D). At the time of these initial experiments, selecting the proper clone for IC2 proved to be difficult and as such was not included. Regardless, in some experiments, IC1 was internalized to a greater degree compared to IC5 when introduced to Nef (Figure 22A+B). However, this data was not reciprocated in other repeated experiments with the rest of the chimeras (except IC2)


where IC5 and to a lesser extent, IC4 were internalized to a greater degree than the rest once Nef was introduced (Figure 22C+D).

Figure 22: CD4-SERINC5 chimera internalization assays reveal unstable trends. (A-B) flow cytometry as well as summarized data for internalization assay of 50ng IC1 and IC5 chimeras and wildtype CD4 cotransfected with and without 50ng Nef. (C-D) Flow cytometry as well as summarized data for internalization assay of IC1, 3-6 with and without Nef. Each experiment was replicated twice (n=2).

These discrepancies led to our investigation into the viability of the internalization assay. We postulated that the chimeras may not have been expressing properly and as such transfected the chimeras alone at dosages of 50, 100, 200, and 500ng (Figure 23). As can be seen in the


summary graphic, all the chimeras were properly expressed throughout all dosages therefore indicating a fundamental and fatal flaw for the CD4-SERINC5 internalization assay.

Figure 23: CD4-SERINC5 expression titration reveals proper expression regardless of dosage. (A-B) 50, 100, 200, and 500 ng of CD4-SERINC5 chimeras and wildtype CD4 were co- transfected into HEK 293T cells. Flow cytometry after surface staining of CD4 as well as the summarized data are shown. Experiment was replicated twice (n=2).

3.3 Elucidation of SERINC5 domains necessary for interaction with HIV-1 3.3.1 Primary chimeric design It is currently not known what part of SERINC5 is required for its antiviral activity against HIV-1. To explore the potential motif, we took advantage of the non-antiviral SERINC1 and swapped three large regions encompassing 3, 4, and 3 transmembrane domains from SERINC5 to create gain of function chimeras and 4, 4, and 3 transmembrane domains from

SERINC1 to create loss of function chimeras (Figure 24). These six chimeras were synthesized and then cloned into pQCXIP to overexpress them in the subsequent experiments.

Figure 24: SERINC1/SERINC5 chimera designs SERINC5 transmembrane domains (TM) are displayed in green while SERIN1 TMs are in orange. Intracellular domains are shown in blue and extracellular domains in red. The domain structure was constructed with data from TMHMM domain prediction server. The first three chimeras (C1-3) consists of a base of SERINC5 with the first 3, middle 4, and final 3 TMs replaced with the corresponding 4, 4, and 3 TMs of SERINC1, respectively. These first three indicate loss-of-function mutants as SERINC1 does not have antiviral activity and is replacing parts of the antiviral SERINC5. The gain-of- function mutants (C4-6) are the opposite of the C1-3 and utilize a non-antiviral SERINC1 base.

3.3.2 SERINC5 downregulates HIV-1 in a dose-dependent manner Before finding the region of importance, we had to establish a baseline for whether SERINC1 and SERINC5 exhibit an antiviral effect in a dose-dependent manner. For this experiment, we co-transfected 5, 10, 25, 50, 100, and 200ng of FLAG-tagged SERINC1 or SERINC5 into HEK 293T cells along with 500ng G2A HIV-1, infected TZM-bl cells with the viral supernatant, and measured the infectivity via luciferase assay (Figure 25). G2A HIV-1 is a mutated variant of HIV-1 where the second glycine of the Nef sequence is mutated to arginine thus creating a myristoylation-defective and therefore, Nef-defective HIV-1255. Since Nef downregulates HIV-1, we used this Nef-defective mutant to ensure antiviral activity measurements. In the end, SERINC5 was found to downregulate HIV-1 infectivity in a dose dependent manner with increasing doses contributing to a lower infectivity while SERINC1 consistently did not have an antiviral effect even at the high 200ng dose. In addition, SERINC5 was only detectable at higher dosages (specifically 100ng and 200ng) while SERINC1 was visible at dosages as low as 50ng in the anti-FLAG immunoblot.

Figure 25: Titration of SERINC5 and SERINC1 activity against HIV-1. (A) Varying dosages of SERINC5 or SERINC 1 (S1/S5) were transfected into HEK 293T cells along with 500ng of G2A HIV-1. These graphics represent relative infectivity and fold inhibition data after measurement of infected TZM-bl reporter cells and resulting dosage curves. (B) Immunoblots for gp120 and p24 for HIV-1, FLAG (for SERINC5 and SERINC1), and tubulin were used for controls. Experiment was replicated twice (n=2).

3.3.3 Central region of SERINC5 is necessary for antiviral activity After the initial six S1/S5 chimeras described above were generated, we set out to determine the dosages needed for optimal and comparable expression to SERINC5. We co- transfected 50, 100, and 200ng of FLAG tagged- SERINC1, SERINC5, or C1-C6 into HEK 293T cells along with 500ng G2A HIV-1, infected TZM-bl cells were then used to measure the infectivity via luciferase assay (Figure 26). As can be seen in the luciferase data summary graphics (Figure 26A), All clones except for C2 and SERINC1 displayed a dose-dependent

increase in inhibition against HIV-1. In addition, the majority of the chimeras except for C3 and C5 were detected on the immunoblot by the 100ng dosage as seen in the anti-FLAG immunoblot. Surprisingly, at the 200ng dosage, previously visible chimeras such as C2 were not detected while a signal for C3 appeared.

Figure 26: Determination of optimal S1/S5 chimera dosage. Varying dosages of SERINC1, SERINC5, or their chimeras were transfected into HEK 293T cells along with 500ng of G2A HIV-1. (A) These graphics represent relative infectivity and fold inhibition data after measurement of infected TZM-bl reporter cells. (B) Immunoblots for gp120 and p24 for HIV-1, and tubulin were used for controls. Anti-FLAG immunoblot was used in conjunction with the infectivity data to find the optimal dosage. Experiment was replicated three times (n=3).

Due to the seeming instability of the C2 chimera at 200ng based on its non-patternistic detection and decrease in fold inhibition, 100ng was chosen as the optimal dose. In addition, flow cytometry was used rather than western blot to detect FLAG as immunoblotting was deemed to lack adequate sensitivity to detect SERINC1, 5, and the chimeras. In Figure 27, 100ng of SERINC1, SERINC5, or C1-C6, and 25ng SERINC1 or SERINC5 were transfected into HEK 293T cells along with 500ng G2A HIV-1, infected TZM-bl cells were then used to measure the infectivity via luciferase assay. As can be seen in the luciferase data summary graphics (Figure 27A), the greatest decrease in fold inhibition was in C2. C1 showed a marked decrease in fold inhibition along with the subsequent greatest increase in fold inhibition in its corresponding gain of function chimera 4. C2 however, had over a 100-fold decrease in inhibition compared to SERINC5. C3 showed no significant change thus eliminating the importance of the final third of SERINC5 in the function of anti-HIV-1 activity. Lastly, the gain of function chimeras C5 and C6 corresponding to their loss of function chimeras, C2 and C3, respectively, were not deemed to be significantly different from each other and as such, the central 143-311 amino acids of SERINC5 was concluded to contain a zone or motif required for its antiviral activity against HIV-1. In addition, flow cytometry analysis of FLAG (Figure 27B, D) proved that the chimeras were without a doubt being expressed.


Figure 27: 100ng optimal dosage transfection for S1/S5 chimeras. 100ng of the S1/S5 chimeras were transfected alongside G2A HIV-1 into HEK 293T cells. Infectivity and fold inhibition normalized via RT assay are shown in (A). Anti-FLAG flow cytometry and its corresponding summary graphic gauged the expression of SERINC1/5 and the chimeras (B, D). Anti-gp120, p24, and tubulin immunoblots were used for controls (C). Experiment was replicated twice (n=2).

3.3.4 A second set of chimeras indicate that Amino acids 176-311 are required for SERINC5 antiviral activity With the knowledge that the central region of SERINC5 is integral in its antiviral activity, we set out to construct more focused chimeras to narrow down the region. To do this, we utilized the existing loss-of-function FLAG-tagged C2 with its SERINC5 central region swapped with SERINC1 and used a unique restriction site on FLAG-tagged SERINC5 to insert parts of SERINC1 into SERINC5. The resulting two loss-of-function chimeras, C2C1 and


C2C5, replace 97 nucleotides and 394 nucleotides, respectively, compared to the 501 nucleotides replaced for the original C2 (Figure 28).

Figure 28: C2 Chimera Designs. SERINC5 transmembrane domains (TM) are displayed in green while SERINC1 TMs are in orange. Intracellular domains are shown in blue and extracellular domains in red. The domain structure was constructed with data from TMHMM domain prediction server. Both chimeras are loss-of-function mutants and contain FLAG tags on their C-termini. The red flags indicate restriction sites. C2C1 replaces the first 32 amino acids from the C2 region of the SERINC5 with SERINC1 while C2C5 replaces the last 131 amino acids from the C2 region of SERINC5 with SERINC1.

These chimeras were inferred to express to a similar degree to C2 and therefore HEK 293T cells were transfected with a 100ng dose of the chimeras, 25ng of SERINC1, or a dosage curve of SERINC5 from 5-100ng. 500ng of G2A HIV-1 was also transfected alongside these constructs and the viral supernatant was used to infect TZM-bl cells which were then used to measure the infectivity via luciferase assay. (Figure 29). As can be seen in the fold-inhibition summary graphic (Figure 29A), both the original C2 and all three replicates of C2C5 had the largest decrease in fold inhibition compared to SERINC5. C2C1 on the other hand had the highest fold inhibition thus eliminating that region’s importance. Additionally, using flow


cytometry (Figure 29B, D), all constructs could be seen to express to a significant degree. In the end, the region of anti-HIV-1 activity was narrowed down to amino acids 176-311.

Figure 29: C2 chimeras reveal region of antiviral activity in SERINC5. 100ng of both C2 chimeras were transfected three times into HEK 293T cells along with 500ng G2A HIV-1. RT- normalized infectivity and fold inhibition was compared with corresponding values of SERINC1 and SERINC5 (A). Anti-FLAG flow cytometry and its corresponding summary graphic gauged the expression of SERINC1/5 and the chimeras (B, D). Anti-gp120, p24, and tubulin immunoblots were used for controls (C). Experiment was replicated twice (n=2).


Chapter 4: Discussion Multiple co-immunoprecipitation experiments with SERINC5 and ubiquitin showed without a doubt, that SERINC5 is ubiquitinated (see Figure 16). Further investigation into the nature of this ubiquitination and its purpose, however, opened many more questions. Using proteasomal and lysosomal inhibitors did not increase the retention nor increase the amount of ubiquitinated SERINC5 indicating that SERINC5 is not degraded due to ubiquitination (see Figure 17). Contrary to that conclusion, we found that SERINC5 is likely to be polyubiquitinated with K63 linkages through the detection of ubiquitination in the presence of K63-mutant ubiquitin. Furthermore, due to the absence of ubiquitination in the presence of the ubiquitin KO mutant, which is not able to form linkages, SERINC5 is not likely to be monoubiquitated thus reopening the possibility of degradation via ubiquitination (see Figure 18). Polyubiquitination with K63 linkages however, is not necessarily an absolute sentence for degradation and may serve another purpose. For example, K63 ubiquitin linkages have been shown to be associated with intracellular trafficking and involved in the activation of the NFκB256,257. As such, activation of NFκB in the case of an HIV-1 infection, may increase the amount of SERINC5 targeted to the plasma membrane thus countering HIV-1 viral infectivity from that producer cell. Lastly, we presented data suggesting that Nef may not increase the degradation nor the ubiquitination of SERINC5 (see Figure 19). This corroborates the evidence put forth by the Göttlinger and Pizzato groups indicating that SERINC5 is sequestered in late endosomes due to the presence of Nef231,232. Interestingly, the omission of MG132 protease inhibitor increased the amount of detected SERINC5, likely indicating a potential toxic effect due to the addition of the inhibitor. Going forward, an alternate type of polyubiquitination could be determined by utilizing more mutants targeting K6, K11, K27, K39, K33, M1, and the N- terminus of ubiquitin239. Different ubiquitin enzymes can be knocked out or inhibited to test for the effect on SERINC5 especially regarding its downregulation of HIV-1 infectivity. Finally, Ubiquitin sites may also be predicted on SERINC5 using software such as UbPred, mutating these sites, and preforming a co-immunoprecipitation with ubiquitin258. Results from this prediction software have indicated a potential ubiquitination site at the 343rd residue of SERINC1 and the 282nd, 373rd, and 383rd residues of SERINC5. As only SERINC5 has this lysine at the 282nd position which happens to be within the C2 region which we determined to


have the highest antiviral activity against HIV-1, ubiquitination at this position could potentially play a role in the antiviral activity of SERINC5. With regards to the SERINC5-CD4 chimera internalization assay, inconsistencies in the trends with IC1 appearing to interact with Nef more strongly in one repeat than another where IC5 was the clear leader led to the investigation of the viability of our experimental setup (see Figure 22). In the end, we found that the chimeras were expressing to an adequate degree therefore leading to the conclusion that the assay was not viable (see Figure 23). Some of the main problems with the setup were that Nef was never proven to interact directly with the intracellular domain of SERINC5 and the intracellular domains that were conjugated were predicted using the TMHMM protein domain prediction software which is only an approximation of the domain organization. In addition, replacing the Nef binding site of CD4 with intracellular domains of SERINC5 may change the overall protein structure especially during folding thereby invalidating any structural and organizational predictions made previously. Furthermore, this assay was also operated under the unproved assumption that if Nef were to bind the CD4-conjugated intracellular domain of SERINC5, that CD4 would be internalized. All in all, there were too many variables that were unaccounted for and moving forward, a bioluminescent resonance energy transfer (BRET) experiment using these chimeras could be used to determine if Nef does indeed interact directly with one of the SERINC5 intracellular domains through the detection of the proximity of these proteins. In addition, the di-leucine containing motif of Nef at residues 164 and 165 required for CD4 downregulation could also be mutated or deleted to determine if Nef directly interacts with SERINC5 in a similar manner as it internalizes CD4259. Lastly, SERINC1-SERINC5 gain-of-function and loss-of-function chimeras in conjunction with a luciferase assay using TZM-bl reporter cells were used to initially determine that the area most responsible for the antiviral activity of SERINC5 was the central region encompassing amino acids 144-311. This was clearly indicated by the 164-fold loss of antiviral activity with C2 followed closely by C1 with a 134-fold inhibition loss (see Figure 27). The gain of function mutants (C4-C6) only saw modest gains in fold inhibition, but due to the similarities in the degree to which they differed, these results were not acted upon. Using this information, we created two new chimeras, C2C1 and C2C5, that focused on smaller subsets of this central region, and with the same assay, determined that the region required for antiviral activity

encompasses amino acids 176-311. This was indicated by a significant and consistent loss of fold inhibition in C2C5 versus C2C1 which had a similar level to the original C2 (see Figure 29). This study represents a major step in finding a specific antiviral motif for SERINC5. On the other hand, unpublished data by A. Chande, was presented at Cold Spring Harbor Retroviruses conference in May of 2016, indicating that the antiviral activity of SERINC5 was most likely shared by the whole protein and is slightly more concentrated at the ninth transmembrane domain. This data is directly contrary to what is presented in this work however, where the C3 chimera which lacks the SERINC5 TM9 domain is able to downregulate HIV-1 infectivity normally. Using non-antiviral SERINC2 versus SERINC1 to create chimeras with SERINC5 could also explain the difference as SERINC2 may be similar to SERINC5 in terms of the C2 region, but not the TM9 region where SERINC1 is the opposite. Furthermore, there are still many unanswered questions with regards to SERINC5 and its status as a host restriction factor. SERINC5 is not upregulated by interferon and has been shown to not have been involved in an evolutionary arms race with Nef231,238. As such, it is not unthinkable that this bulky protein is simply in the right place at the right time and is not specifically targeting HIV-1. SERINC proteins may also be required for efficient budding of certain HIV-1 strains as they have been found to facilitate the formations of phospholipids for subsequent lipid raft domains. In addition, upon aligning SERINC1 and SERINC5 with the clustal omega server and focusing on amino acids 176-311, one can see two internal and external domains, and transmembrane domains 5, 6, and 7260. Comparing residues from SERINC5 and SERINC1 indicates that SERINC5 contains 88 unique residues in this region. Moving forward, chimeras focusing on additional sections of the later end of the C2 region will be synthesized to further narrow down the region of antiviral activity and the amount of potential point mutations that can be tested. Another approach which could be taken is to mutate sites of interest such as the predicted SERINC5 ubiquitination site, lysine 282 which is the only unique ubiquitination site out of the three to be within the C2 region. If SERINC5 is indeed a traditional host restriction factor, and is trafficked using K63 ubiquitin linkages in concordance with NFκB signaling and therefore HIV-1 infection, mutating K282 may indicate that this residue is required for HIV-1 downregulation. Besides this speculation, all the experiments completed for this project utilize overexpression promoters and may not be perfectly indicative of what happens in nature. As such a more natural expression

vector along with transfection into peripheral blood mononuclear cells (PBMCs) rather than HEK 293T cells could provide more applicable evidence. Taken together our study demonstrates that SERINC5 is ubiquitinated and that this ubiquitination is not involved in its degradation, and the region encompassing amino acids 176- 311 is required for SERINC5 antiviral activity against HIV-1. With the high mutation rate of HIV-1, it is still as important as ever to study host restriction factors. These endogenous defenses have and will continue to illuminate different countermeasures against HIV-1 that could be utilized by ARVs, such as is the case with Enfuvirtide which mimics a part of gp41 to prevent proper viral fusion, or could point out HIV-1 countermeasures against host restriction which could be interfered with72. This study provides the basis for further experimentation to narrow down both the biological role of ubiquitination regarding SERINC5 and the domain or moiety responsible for its downregulation of HIV-1 Infectivity.

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