Impact of natural HIV-1 alleles and polymorphisms on SERINC3/5 downregulation

by Steven W. Jin

B.Sc., Simon Fraser University, 2016

Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science

in the Master of Science Program Faculty of Health Sciences

© Steven W. Jin 2019 SIMON FRASER UNIVERSITY Spring 2019

Copyright in this work rests with the author. Please ensure that any reproduction or re-use is done in accordance with the relevant national copyright legislation. Approval

Name: Steven W. Jin Degree: Master of Science Title: Impact of natural HIV-1 Nef alleles and polymorphisms on SERINC3/5 downregulation Examining Committee: Chair: Kanna Hayashi Assistant Professor Mark Brockman Senior Supervisor Associate Professor Masahiro Niikura Supervisor Associate Professor Ralph Pantophlet Supervisor Associate Professor Lisa Craig Examiner Professor Department of Molecular Biology and Biochemistry

Date Defended/Approved: April 25, 2019

ii Ethics Statement

iii Abstract

HIV-1 Nef is a multifunctional accessory required for efficient viral pathogenesis. It was recently identified that the serine incorporators (SERINC) 3 and 5 are host restriction factors that decrease the infectivity of HIV-1 when incorporated into newly formed virions. However, Nef counteracts these effects by downregulating SERINC from the cell surface. Currently, there lacks a comprehensive study investigating the impact of primary Nef alleles on SERINC downregulation, as most studies to date utilize lab- adapted or reference HIV strains. In this thesis, I characterized and compared SERINC downregulation from >400 Nef alleles isolated from patients with distinct clinical outcomes and subtypes. I found that primary Nef alleles displayed a dynamic range of SERINC downregulation abilities, thus allowing naturally-occurring polymorphisms that modulate this activity to be identified. In addition, I found that Nef alleles isolated from patients with better clinical outcomes had a poorer ability to counteract SERINC, suggesting that variation in this activity may contribute to differences in HIV-1 pathogenesis.

Keywords: HIV/AIDS; Nef; SERINC3; SERINC5; viral diversity; viral infectivity

iv Dedication

To my mom, who has given up so much of her life so I can have mine, for that, I am forever in your debt.

v Acknowledgements

The completion of a Master’s degree program is as much an intellectual journey as it is a personal one; in this regard, there is an army of individuals who have shaped both aspects of my journey.

Foremost, I would like to extend my sincerest thanks to my senior supervisor Dr. Mark Brockman, as well as Dr. Zabrina Brumme, who have given me the platform to train and grow as a young scientist; my successes and accomplishments would not have been possible without your continuous support and generosity. Lastly, to every single lab member who I have met since joining the lab, you have all made this journey an unforgettable experience and I’m grateful to be able to call some of you my lifelong friends.

vi Table of Contents

Approval ...... ii Ethics Statement ...... iii Abstract ...... iv Dedication ...... v Acknowledgements ...... vi Table of Contents ...... vii List of Tables ...... ix List of Figures...... x List of Acronyms ...... xi

Chapter 1. Role of HIV-1 accessory in viral pathogenesis ...... 1 1.1. Introduction to HIV-1 ...... 1 1.2. HIV-1 life cycle and pathogenesis ...... 1 1.3. HIV-1 accessory proteins...... 2 1.3.1. Virulence infectivity factor (Vif) ...... 3 1.3.2. Virulence protein R () ...... 3 1.3.3. U (Vpu) ...... 4 1.3.4. Negative factor (Nef) ...... 4 1.4. Sequence and function variation in primary Nef alleles ...... 7 1.5. Conclusions...... 9 1.6. Thesis Objectives ...... 10 1.7. References ...... 11

Chapter 2. HIV-1 Nef alleles isolated from elite controllers are impaired for SERINC5 downregulation activity ...... 21 2.1. Abstract ...... 21 2.2. Introduction ...... 22 2.3. Materials and Methods ...... 23 2.3.1. Study Participants ...... 23 2.3.2. Reagents...... 23 2.3.3. Generation of HIV-1 nef expression and proviral constructs ...... 23 2.3.4. Generation of SERINC5 knockout Jurkat LTR-GFP reporter cells ...... 24 2.3.5. SERINC5 downregulation assays ...... 24 2.3.6. CD4 and HLA class I downregulation assays ...... 25 2.3.7. Infectivity and replication capacity ...... 25 2.3.8. Western blot ...... 26 2.3.9. Statistical analysis ...... 27 2.4. Results ...... 27 2.4.1. Primary HIV-1 Nef alleles display variable abilities to internalize SERINC5 . 27 2.4.2. Natural Nef polymorphisms are associated with SERINC5 downregulation function………...... 30

vii 2.4.3. Nef-mediated SERINC5 downregulation correlates more strongly with CD4 internalization compared to HLA internalization ...... 32 2.4.4. Viral adaptation to host cytotoxic T lymphocytes contributes to variability in Nef-mediated SERINC5 downregulation activity ...... 34 2.4.5. CTL escape mutations K94E and H116N selectively impair Nef-mediated SERINC5 downregulation function ...... 34 2.5. Discussion ...... 38 2.6. References ...... 40

Chapter 3. Differential ability of HIV-1 Nef alleles across viral subtypes to downregulate SERINC3 and SERINC5 ...... 44 3.1. Abstract ...... 44 3.2. Introduction ...... 45 3.3. Materials and Methods ...... 46 3.3.1. Study Participants ...... 46 3.3.2. Plasmids ...... 46 3.3.3. Site-directed mutagenesis ...... 47 3.3.4. SERINC, CD4 and HLA-I downregulation...... 47 3.3.5. Generation of a SERINC3 knockout HEK293T cell line ...... 47 3.3.6. Infectivity Assay ...... 48 3.3.7. Proximity Ligase Assay (PLA) ...... 48 3.3.8. Western blot ...... 49 3.3.9. Statistical analysis ...... 49 3.4. Results ...... 49 3.4.1. Dynamic range of Nef-mediated SERINC downregulation activity among primary isolates ...... 49 3.4.2. Nef-mediated antagonism of SERINC3 and SERINC5 enhances viral infectivity ...... 52 3.4.3. Polymorphisms associated with modulation of SERINC ...... 55 3.4.4. Mutations at residues 8 and 11 of Nef selectively impair SERINC3 downregulation ...... 57 3.4.5. Nef codons 8 and 11 are critical for co-localization with SERINC3 ...... 60 3.5. Discussion ...... 61 3.6. References ...... 63

Chapter 4. Thesis Summary ...... 67 4.1. References ...... 70

Appendix A. Extended Tables ...... 72

viii List of Tables

Table 2.1. Nef polymorphisms associated with SERINC5 downregulation(N≥5, p<0.05, q<0.35) ...... 31 Table 3.1. Nef polymorphisms associated with differential SERINC5 modulation in HIV-1 subtypes A, B, C and D ...... 56 Table 3.2. Nef polymorphisms associated with differential SERINC3 modulation in HIV-1 subtypes A, B, C and D ...... 57 Table A.3.1. Extended list of Nef polymorphisms associated with differential SERINC5 downregulation in HIV-1 subtypes A, B, C and D (p<0.05)...... 72 Table A.3.2. Extended list of Nef polymorphisms associated with differential SERINC5 downregulation in HIV-1 subtypes A, B, C and D (p<0.05)...... 75

ix List of Figures

Figure 1.1 HIV-1 ...... 3 Figure 1.2. Model of SERINC antagonism by HIV-1 Nef ...... 7 Figure 1.3. HIV-1 Nef sequence, motifs and structure ...... 9 Figure 2.1. Method used to measure downregulation of SERINC5 by primary Nef alleles ...... 28 Figure 2.2. Characterization of SERINC5 downregulation and antagonism activity in HIV-1 Nef isolates from controllers and progressors...... 29 Figure 2.3. Validation of natural polymorphisms in Nef that modulate SERINC5 downregulation ...... 32 Figure 2.4. Associations between Nef-mediated downregulation functions and protein stability ...... 33 Figure 2.5. Impact of K94E and H116N Nef mutants on infectivity and viral replication ...... 36 Figure 2.6. Impact of K94E and H116N Nef mutants on downregulation activities ...... 38 Figure 3.1. Nef-mediated downregulation of SERINC3 and SERINC5 assay ...... 50 Figure 3.2. Nef-mediated SERINC3 and SERINC5 downregulation by primary Nef alleles representing globally relevant subtypes ...... 51 Figure 3.3. Validation of HEK293T SERINC3 knockout cell line ...... 53 Figure 3.4. Downregulation of SERINC3 and SERINC5 correlates with viral infectivity ...... 55 Figure 3.5. Polymorphisms at Nef codons 8 and 11 selectively impair SERINC3 downregulation ...... 59 Figure 3.6. Characterization of Nef-SERINC3 co-localization using proximity ligase assay ...... 61

x List of Acronyms

ADCC Antibody-dependent cellular cytotoxicity AIDS Acquired immunodeficiency syndrome AP Adaptor protein BST2 Bone marrow stromal antigen 2 cART Combination antiretroviral therapy CCR5 C-C chemokine receptor type 5 CD4 Cluster of differentiation 4 CP Chronic progressors CRISPR Clustered regularly interspaced short palindromic repeat CTL Cytotoxic CD8+ T-lymphocytes CXCR4 C-X-C chemokine receptor type 4 EC Elite controllers ELISA -linked immunosorbent spot GFP Green fluorescent protein HIV-1 Human immunodeficiency virus type I HLA-I Human leukocyte antigen class I kDa Kilodalton KO Knockout LTR Long-terminal repeat PACS Phosphofurin acidic cluster sorting protein PBMC Peripheral blood mononuclear cells PIC Pre-integration complex pVL Plasma viral load MFI Median fluorescence intensity Nef Negative factor NK Natural killer SERINC Serine incorporator TGN Trans-Golgi network Vif Virulence infectivity factor Vpr Virulence protein R Vpu Viral protein U WT Wildtype

xi Chapter 1.

Role of HIV-1 accessory proteins in viral pathogenesis

1.1. Introduction to HIV-1

Human immunodeficiency virus type 1 (HIV-1) is the causative agent of acquired immunodeficiency syndrome (AIDS), a global pandemic that has resulted in over 39 million deaths since its discovery in 1981 [1]. Of the 36.9 million people currently living with HIV, an estimated 75,000 are in Canada [2, 3]. With approximately 26.6 million cases, sub-Saharan Africa is the most affected region in the world and has the highest rate of new HIV infections yearly [3]. Although there are currently no effective vaccines or cures for HIV, treatment with combination antiretroviral therapy (cART) can control the virus to undetectable levels in the blood and significantly reduce the risk of transmission [4]. However, cART is costly and is not easily accessible in all parts of the world, with only ~59% of HIV infected individuals receiving treatment [1]. Furthermore, HIV/AIDS is still very much an active and prevalent pandemic. In 2017, there were approximately 1.8 million cases of new infections and almost 1 million cases of AIDS-related death [3]. Therefore, the development of new drugs to treat HIV, effective vaccines to prevent infection and strategies to cure individuals living with HIV are needed to circumvent the pandemic.

1.2. HIV-1 life cycle and pathogenesis

HIV-1 is a ~10 kb plus-sense single-stranded (ss) RNA virus that primarily infects CD4+ T-cells. Initial infection is established by the interaction between the (gp160) on an HIV virion with the CD4 receptor and CCR5 or CXCR4 co- receptor on the human target cell [5-8]. Engagement of the viral glycoprotein with the host cell receptors leads to pore formation and fusion between the virus and the target cell membrane, allowing the viral contents to be released into the cytoplasm [9]. One of the first steps immediately after viral uncoating is the process of converting the ssRNA HIV genome into double-stranded (ds) DNA, mediated by the packaged reverse

1 transcriptase enzyme [10]. The newly converted dsDNA HIV genome is then transported into the nucleus and permanently integrated into the host , mediated by the viral enzyme [11]. From here, HIV can either manifest as a latently infected cell or continue its lytic life cycle by synthesizing new viral transcripts and proteins where they will assemble at the plasma membrane to form and bud off as progeny virions that can propagate new rounds of infection [12].

Because HIV-1 targets primarily CD4+ T-cells (the helper cells required for priming and modulating the adaptive immune system), depletion of these cells to less than 200 cells/mm3 leads to the final clinical stage of HIV infection known as AIDS [13, 14]. During the onset of AIDS, the body’s immune defense is compromised and is prone to increased risk of mortality by typically manageable opportunistic infections and diseases [15]. The rate of onset to AIDS from the initial infection varies, where clinical, immunological and host genetic properties have been described to influence the rate of disease progression [16-21]. However, less is known about the influence of the biological properties of viral factors on HIV pathogenesis in patients, as laboratory reference strains are typically used to characterize the mechanisms of viral proteins.

1.3. HIV-1 accessory proteins

The HIV-1 genome encodes for 9 including two structural proteins (Gag and Envelope) to produce and form the physical structure of an HIV virion, three enzymatic proteins (Polymerase, Integrase and Protease) to carry out the necessary steps in the viral replication life cycle, two regulatory proteins (Tat, Rev) to modulate viral expression and lastly four accessory proteins (Vif, Vpr, Vpu, and Nef) to enhance viral infectivity and pathogenicity of the virus (Figure 1.1) [22]. While accessory proteins are dispensable for viral replication in vitro (hence the term “accessory”), they play an ancillary role in overcoming major barriers that the virus is confronted with in its human host and considerably enhance the pathogenicity and fitness of HIV in vivo [23].

2 Figure 1.1 HIV-1 genome Genetic map of the HIV-1 HXB2 reference genome. The four accessory proteins Vif, Vpr, Vpu and Nef are highlighted in blue. Figure adapted from Los Alamos National Laboratory database (https://www.hiv.lanl.gov/content/sequence/HIV/MAP/landmark.html)

1.3.1. Virulence infectivity factor (Vif)

HIV-1 Virulence infectivity factor (Vif) is a 23 kDa, 192 amino acid accessory protein expressed in relatively high amounts in the late stages of the viral life cycle [24]. Vif is required for efficient viral replication, where Vif-defective viruses produced in certain cell lines display lower infectivity compared to wildtype viruses [25]. The mechanism for this phenotype was later determined to be mediated by Vif’s ability to counteract the host restriction factor APOBEC3 cytidine deaminases in the virus producer cell [26]. In the absence of Vif, APOBEC3 proteins are packaged into progeny virions and when released into the target cell, APOBEC3 induces cytidine to uracil hypermutations in the minus-sense ssDNA HIV genome during the reverse transcription step, resulting in guanidine to adenine hypermutations in the respective plus-sense strand [27]. These APOBEC3-mediated hypermutations lead to degradation of the reverse transcribed DNA and/or introduce lethal mutations rendering the virus defective [28]. However, Vif antagonizes the effects of APOBEC3 by targeting it for degradation in the proteasome via ubiquitinylation [29, 30]. Therefore, Vif plays an important role in pathogenesis by overcoming the innate antiretroviral immune defense exerted by APOBEC3 restriction factors during the HIV life cycle.

1.3.2. Virulence protein R (Vpr)

HIV-1 Virulence protein R (Vpr) is a 14 kDa, 96 amino acid late expression accessory protein that possesses multiple functions that are pertinent in mediating HIV infection [31, 32]. One of its most notable functions is promoting the import of the pre- integration complex (PIC) into the nucleus by facilitating the interaction between importins and nucleoporins with the PIC [33]. Vpr is also known to interact with a

3 plethora of other host factors to modulate the transcription of the viral genome, induce apoptosis, disrupt cell-cycle control, suppress immune activation and improve the fidelity of the reverse transcription process [34-38]. Hence, Vpr plays a biologically relevant role in creating a conducive cell environment for the establishment of an optimal HIV infection.

1.3.3. Viral protein U (Vpu)

HIV-1 Viral protein U (Vpu) is a 16 kDa, 81 amino acid membrane-associated accessory protein expressed late in the viral life cycle [39, 40]. The two main functions of Vpu are targeting newly synthesized CD4 molecules in the endoplasmic reticulum (ER) for proteasomal degradation and promoting the release of progeny virions from the producer cell by counteracting the effects of the tetherin/BST2 restriction factor [41-43]. Vpu-mediated degradation of CD4 decreases the expression of CD4 on the cell surface, which may provide a few biological advantages to HIV. Downregulation of CD4 prevents superinfection, where an already infected cell is infected again with a second strain of HIV, which could lead to premature cell death [44]. Newly synthesized CD4 may also trap the envelope glycoprotein precursor gp160 in the ER, while cell surface CD4 can interact with the assembled viral , hindering the release of nascent virions [45-47]. Therefore, Vpu-mediated degradation of CD4 in an infected cell can alleviate unwanted interactions between CD4 and Envelope glycoproteins from different virions or those that are newly synthesized. On the other hand, tetherin/BST-2 is a host restriction protein induced by interferon-alpha cytokines and functions to trap the release of at the final stage of the HIV life cycle [48]. Vpu counteracts the antiretroviral effects of tetherin and enhances virion release by internalizing tetherin from the cell surface and targeting it for degradation via an endosome-lysosome pathway [49]. Together, these two Vpu activities function together to offset the negative consequences inflicted by the expression and presence of CD4 and tetherin.

1.3.4. Negative factor (Nef)

HIV-1 “Negative factor” (Nef) is a 27-35 kDa, 206 amino acid myristoylated accessory protein crucial for the maintenance of high viral loads and progression to AIDS [50-52]. It is expressed abundantly and early in the HIV life cycle [53]. Early reports demonstrated that Nef had a suppressive effect on HIV replication, hence the name

4 “Negative factor”, however, it has been since shown that Nef has a positive effect on enhancing pathogenesis [54, 55]. The strongest clinical evidence for Nef’s critical role in pathogenesis is demonstrated in a cohort where eight blood recipients were infected with the same Nef-defective HIV-1 strain from a single HIV infected blood donor [52]. All eight blood recipient patients displayed delayed disease progression and eventually became either long-term non-progressors or elite controllers [50, 56, 57].

Nef has been described to perform multiple functions in vitro despite lacking enzymatic activity [58]. Instead, Nef acts as an adapter protein to interact with and manipulate a myriad of host proteins to exert its functions [58]. For example, one of Nef’s most conserved and characterized function is its ability to downregulate CD4 molecules from the surface of infected cells [59, 60]. Phenotypically, both Nef and Vpu reduce the expression cell surface CD4 but their mechanisms differ. While Vpu targets newly synthesized CD4 molecules for degradation, Nef traffics mature CD4 molecules away from the cell surface by interacting with the host adapter protein 2 (AP-2) complexes to mediate clathrin-dependent internalization of CD4 for lysosomal degradation [59, 61, 62]. Therefore, during both the early and late stages of the HIV life cycle, CD4 is continuously removed by two separate viral proteins, highlighting the importance of this activity and potential consequences of retaining CD4 expression on the surface of infected cells [63]. In addition to preventing superinfection and promoting virion release, downregulation of CD4 by Nef has also been recently described to allow infected cells to escape from antibody-dependent cellular cytotoxicity (ADCC) because conformational changes induced by the CD4-envelope interaction exposes vulnerable gp160 epitopes to neutralizing antibodies that can be targeted for ADCC [64-66].

Another well-characterized immune evasion function mediated by Nef is the downregulation of human leukocyte antigen class I (HLA-I) molecules, particularly HLA- A and HLA-B [67]. In an infected cell, HLA-I molecules present viral peptide antigens to cytotoxic CD8+ T-lymphocytes (CTL) to signal for elimination. However, Nef protects infected cells from immune surveillance by CTLs by downregulating HLA-I molecules from the cell surface [68]. Furthermore, specificity for HLA-A and HLA-B but not HLA-C or HLA-E protects infected cells from cell lysis and death by natural killer (NK) cells. NK cells target deviant cells devoid of HLA-I expression on the cell surface but the maintenance of HLA-C and HLA-E expression in an HIV infected cell acts as inhibitory ligands for NK cells [69, 70]. While Nef downregulates cell surface CD4 molecules by

5 promoting the formation of clathrin-coated vesicles mediated by AP-2, it has been proposed that Nef forms a complex with AP-1 and newly synthesized HLA-I molecules in the trans-Golgi network (TGN), preventing its translocation to the plasma membrane [71]. Nef can also promote internalization of surface HLA-I in a clathrin-independent mechanism through its interaction with Phosphofurin acidic cluster sorting protein 2 (PACS-2) [72-74]. Ultimately, both mechanisms traffic HLA-I molecules to the endosome/lysosome degradation pathway, reducing the expression of HLA-I on the surface available for antigen presentation.

One of Nef’s most enigmatic and least understood activity was its ability to enhance the infectivity of progeny virions [75-77]. It was recently elucidated that this phenotype was attributed to Nef’s ability to antagonize the host serine incorporators 3 and 5 (SERINC3/5) that restrict retroviral infectivity when incorporated into newly formed virions by inhibiting the formation of fusion pores with target cells (with SERINC5 being more potent) (Figure 1.2A) [78, 79]. The SERINC restriction factors belong to a family of homologous multipass transmembrane proteins (SERINC1-5), although only SERINC3 and SERINC5 contain antiviral activity. The natural function of the human SERINC proteins have only been described in one prior study; SERINC proteins facilitate the synthesis of sphingolipids and phosphatidylserine by enhancing the incorporation of the amino acid serine into membranes [80]. However, this biological property of SERINC3 and SERINC5 remain independent from the mechanisms involved in the restriction of HIV-1 infectivity [81]. There is currently limited evidence to suggest that SERINC may in part reduce HIV fusion pore formation by interacting with Envelope, promoting or facilitating conformational changes in the glycoprotein rendering it unable to perform its membrane fusion functions [82]. To counteract the antiviral effects of the SERINC3 and SERINC5 restriction factors, Nef promotes internalization of SERINC from the cell surface and traffics it for lysosomal degradation, preventing its incorporation into budding virions (Figure 1.2B) [83]. Although the precise mechanism for Nef-mediated downregulation of SERINC3/5 remains incompletely characterized, this activity utilizes similar trafficking pathways needed for the downregulation of CD4, where a dileucine motif in Nef is required to promote the formation of clathrin-coated vesicular endocytic complexes with AP-2 [84].

6

Figure 1.2. Model of SERINC antagonism by HIV-1 Nef (A) In the absence of an antagonist (Nef), the SERINC3 and SERINC5 host restrictions factors are incorporated into budding virions, which subsequently reduces the formation of fusion pores between the virion and target cell, inhibiting cytoplasmic release of viral contents. (B) Nef antagonizes the antiviral effects of SERINC by internalizing the host restriction factors from the cell surface, preventing its incorporation into newly formed virions. Figure adapted from Database Center for Life Science (http://first.lifesciencedb.jp/archives/11809).

1.4. Sequence and function variation in primary Nef alleles

Nef is one of the most polymorphic HIV-1 proteins in part because Nef is highly immunogenic and frequently targeted by CTLs during primary infection and the high evolutionary rate during HIV replication allows the virus to rapidly adapt to evade host immune pressures [85-87]. HLA-I molecules of an infected cell presents virus-derived peptide epitopes to CTL, where peptide-HLA binding is determined by the HLA allele and the amino acid sequence of the peptide. This allows CTL to recognize a broad range of epitopes derived from foreign pathogens [86]. As a result, HLA-I restricted immune pressure can drive the selection of predictable CTL escape mutations or polymorphisms in HIV proteins based on the HLA-I alleles of the patient [88-90], where polymorphism is defined as amino acid variation from the subtype consensus sequence. Therefore, the predictable nature of HIV adaptation to evade HLA-I mediated immune response has allowed researchers to identify and classify HLA-associated polymorphisms using large datasets of linked host HLA and virus sequences [90]. There is evidence to suggest that a major proportion of within-host HIV-1 evolution during the first year of infection can be attributed to CTL escape [91]. However, in some cases, HLA-I driven CTL escape mutations can also carry a fitness cost to the virus, especially if the mutation occurs in a sequence-conserved region of the virus [92]. However, given

7 the adaptable nature of the HIV virus, compensatory mutations can arise at secondary sites to regain viral fitness [93].

Another contributing factor to the extensive genetic diversity of HIV is the occurrence of HIV subtypes and recombination events. HIV-1 group major (M), the predominant circulating strain responsible for the global pandemic consists of 9 subtypes determined based on the phylogenetic distance from one another [94-96]. Within a subtype, there can be up to 15-20% in genetic variation, whereas between subtypes, there can be up to 25-35% in genetic variation [97]. There are also circulating recombinant forms (CRFs) that arises when a patient is infected by two distinct HIV subtype strains, further expanding the ever-growing genetic complexity and diversity of HIV-1 [95].

It is therefore plausible that the extensive sequence variation in Nef could also result in variation in Nef’s different functions. Indeed, previous studies from our group and others have demonstrated a dynamic range of activity in patient-derived Nef alleles for most functions including modulation of CD4 and HLA-I molecules [64, 98-105]. Results from these studies support the notion that Nef could be a correlate of HIV pathogenesis wherein patients exhibiting slower disease progression typically harboured Nefs with poorer activity for most functions despite being genetically intact. While studies with laboratory-adapted or reference Nef strains can inform key residues that are absolutely required for function, these residues are almost 100% conserved in patient- derived sequences [106]. That is because these residues encode canonical motifs in Nef that are required for optimal localization, structure formation and/or its ability to interact with host proteins (Figure 1.3). For example, a G2A mutation in Nef’s myrsitoylation motif renders the protein defective for most of its functions such as downregulation of CD4, HLA-I, and SERINC molecules because it is unable to localize to the plasma membrane [107]. Alanine substitutions of the dileucine motif (LL164/165AA) of Nef impairs its ability to interact with the host adaptor protein complexes (i.e. AP-1 and AP-2) that facilitate clathrin-mediated endocytosis of CD4 and SERINC [59, 108, 109] and a M20A mutation selectively impairs Nef’s ability to downregulate HLA-I molecules [110]. However, these mutations described are never found in circulating HIV strains and so our understanding of the mutational landscape of Nef polymorphisms and its functional implication are limited [106]. Currently, the impact of naturally-occurring Nef alleles and

8 polymorphisms on SERINC downregulation and its role in pathogenesis remains the least understood given its recent discovery.

Figure 1.3. HIV-1 Nef amino acid sequence, motifs and structure (A) Amino acid sequence of HIV-1 Nef (NL4-3 strain). Canonical motifs in Nef that are highly conserved in circulating sequences and required for critical activities modulated by Nef. Figure adapted from Roeth et al., Microbiology and Molecular Biology Reviews (2006). (B) Structure of Nef which contains a N-terminal arm domain (codons 1-57), C-terminal folded core (codons 58- 149 and 181-206) and a flexible C-terminal loop (150-180). Mutations highlighted G2A, D123A, and LL165AA disrupt Nef myristoylation, dimerization and interaction with host adaptor proteins, respectively.

1.5. Conclusions

In summary, while the four HIV-1 accessory proteins may be dispensable in certain in vitro conditions for viral replication, in combination, they exude a plethora of activities that are crucial for promoting the virulence and pathogenesis of HIV in its natural host. In the absence of these accessory proteins, the human immune defense system set in place should, in theory, be able to eliminate or at the very least, control the

9 virus, preventing or slowing down transmissibility and progression to AIDS. Therefore, a detailed understanding of the interplay between the virus and host could better inform interactions that could either be intervened, harnessed or exploited in novel therapeutic strategies to combat HIV/AIDS and other similar viral pathogens.

1.6. Thesis Objectives

The main objective of this thesis was to investigate the impact of primary Nef alleles on the downregulation and antagonism of the SERINC3/5 restriction factors. The overall hypothesis is that Nef-mediated SERINC downregulation varies widely at the population-level and that Nef alleles isolated from patients with slower disease progression will display poorer ability to counteract SERINC. Furthermore, by characterizing the functional variability of a large panel of patient-derived Nef sequences, I expect to be able to use statistical methods to identify naturally-occurring polymorphisms in Nef that modulate its ability to downregulate SERINC proteins. Chapter 2 describes a research study that examined the SERINC5 downregulation activities of Nef clones isolated from rare elite controllers compared to those of Nef clones isolated from chronic progressors. I found that controller-derived Nef clones displayed significantly poorer ability to counteract SERINC5 compared to progressor- derived Nef clones. Furthermore, I identified two CTL escape mutations in Nef that impaired its ability to downregulate SERINC5, while other Nef-mediated downregulation functions were retained. In Chapter 3, I characterized the SERINC3 and SERINC5 downregulation functions of Nef clones isolated from patients infected with either HIV-1 subtype A, B, C or D. I observed differential subtype hierarchy for these SERINC downregulation activities. Whereas subtype B-derived Nef clones displayed the best overall ability to downregulate SERINC5, they displayed the worst overall ability to downregulate SERINC3 when compared to the other subtypes. Furthermore, I identified polymorphisms at Nef residues 8 and 11 that were critical for SERINC3 downregulation but remained fully functional for downregulation of CD4, HLA-I, and SERINC5. My results indicate that these residues in the N-terminal domain of Nef contribute to its ability to co-localize with SERINC3. Finally, Chapter 4 provides a brief conclusion on the thesis and provides insight into the advantages of studying patient-derived sequences in complementation with lab-adapted Nef strains.

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20 Chapter 2.

HIV-1 Nef alleles isolated from elite controllers are impaired for SERINC5 downregulation activity

2.1. Abstract

HIV-1 Nef enhances virion infectivity by counteracting host restriction factor SERINC5; however, the impact of natural Nef polymorphisms on this function is largely unknown. I characterized SERINC5 downregulation activity of 91 primary HIV-1 subtype B nef alleles, including isolates from 45 elite controllers and 46 chronic progressors. Controller-derived Nef clones displayed a lower ability to downregulate SERINC5 (median 80% [IQR 38-95%] activity) compared to progressor-derived clones (96% [IQR 75-100%]) (p=0.0005). I identified 18 Nef polymorphisms associated with differential function, including two CTL escape mutations that contributed to lower SERINC5 downregulation: K94E, driven by HLA-B*08; and H116N, driven by the protective allele HLA-B*57. HIV-1 strains encoding Nef K94E and/or H116N displayed lower infectivity and replication capacity in the presence of SERINC5. These results demonstrate that natural polymorphisms in HIV-1 Nef can impair its ability to internalize SERINC5, indicating that variation in this recently described function contributes to differences in viral pathogenesis.

21 2.2. Introduction

Nef is 27-32 kDa myristoylated HIV-1 accessory protein that is crucial for viral pathogenesis [1-3]. It displays no enzymatic activity, rather it acts as an adapter protein to modulate diverse cellular events related to vesicular transport, signal transduction and actin cytoskeletal remodeling [4-7]. Nef’s well-characterized abilities to internalize CD4 and HLA class I from the infected cell surface allow HIV-1 to evade antibody-dependent cellular cytotoxicity (ADCC) and cytotoxic T lymphocytes (CTL) [8-13]. Other important Nef functions include stimulation of viral replication and enhancement of virion infectivity [14-19]. Collectively, these Nef activities increase HIV-1 persistence, but their individual contributions to disease progression remain largely undefined [20].

Nef-mediated enhancement of virion infectivity was recently shown to be due to its ability to counteract members of the serine incorporator (SERINC) family of host restriction factors, of which SERINC5 is the most potent [21, 22]. SERINC5 incorporates into the membrane of progeny virions and inhibits their fusion with target cells [23]. Nef prevents this by internalizing SERINC5 from the infected cell surface and trafficking it to lysosomes via an endosomal route that is similar to that used by Nef to downregulate CD4 [24]. Several canonical Nef mutations have been reported to impair its ability to antagonize SERINC5, including G2A, D123A and LL165AA, which block myristoylation, dimerization and interaction with AP-2 trafficking complexes, respectively [25]; however, while nef exhibits extensive genetic diversity [26, 27], these mutations are rare in circulating HIV-1 Nef strains (all >99% conserved, HIV Sequence Database; www.hiv.lanl.gov). Studies to examine the impact of naturally occurring HIV-1 Nef polymorphisms on its ability to counteract SERINC5 have not been conducted. In a prior report, I demonstrated that Nef clones isolated from HIV-1 elite controllers, who spontaneously suppress plasma viremia without therapy [28], displayed functional impairments in vitro despite the absence of obvious genetic defects or deletions [29]. Rather, reduced function was linked to natural variation in primary nef sequences, including mutations selected by the protective HLA allele B*57 (HLA allele associated with lower viral loads and enriched in HIV controllers), indicating that viral adaptation to host immune selection pressure contributed to attenuation in at least some cases.

22 2.3. Materials and Methods

2.3.1. Study Participants

Clinical characteristics of participants have been described previously [29]. Briefly, the elite controller cohort (N=45) displayed a median plasma viral load (pVL) of 2 RNA copies/mL (interquartile range [IQR] 0.2 – 14) and a median CD4 count of 811 cells per mm3 (IQR 612 – 1022). The chronic progressor cohort (N=46) displayed a median pVL of 80,500 RNA copies per mL (IQR 25,121 – 221,250) and a median CD4 count of 293 cells per mm3 (IQR 73 – 440). All participants were recruited from the Boston area, provided written informed consent, and were not receiving antiretroviral therapy at the time of blood sample collection. This study was approved by the Research Ethics Boards at the Massachusetts General Hospital (Boston, MA USA) and Simon Fraser University (Burnaby, BC Canada).

2.3.2. Reagents

The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 NL4-3 Infectious Molecular Clone (pNL4-3) (Cat# 114), from Dr. Malcolm Martin [40]; Jurkat LTR-GFP CCR5+ Cells (JLTRG-R5) (Cat #11586), from Dr. Olaf Kutsch [41, 42]; and TZM-bl cells (Cat#8129) from Dr. John C. Kappes and Dr. Xiaoyun Wu [43-48].

2.3.3. Generation of HIV-1 nef expression and proviral constructs

Primary HIV-1 subtype-B nef alleles were amplified from plasma viral RNA and a single phylogenetically representative nef clone was isolated from each participant in a previous study [29]. Genbank accession numbers are JX171199-JX171243 (for elite controllers) and JX440926-JX440971 (for chronic progressors). Nef alleles were transferred into pSELECT-GFPzeo (InvivoGen), which features a composite hEF1-HTLV promoter driving nef and an independent CMV promoter driving expression of GFP. To do this, I modified the multiple cloning site in pSELECT-GFPzeo to incorporate Asc I and Sac II sites. Each nef gene was amplified by PCR using degenerate primers incorporating these restriction sites (Fwd: 5’-AGAGCACCGG CGCGCCTCCA CATACCTASA AGAATMAGAC ARG-3’, HXB2 nt 8746-8772 underlined, Asc I site in

23 bold; Rev: 5’-GCCTCCGCGG ATCGATCAGG CCACRCCTCC CTGGAAASKC CC-3’, HXB2 nt 9474-9449 underlined, SacII site bolded). The same strategy was used to clone nef from HIV-1 subtype B reference strains SF2 and NL4.3, which served as the positive controls. Point mutations were introduced into nef sequences using overlap extension PCR, including G2A substitutions in both reference clones that served as negative controls. All Nef clones and mutations were validated by Sanger sequencing.

Primary nef sequences and mutants were introduced into the NL4.3 reference strain backbone as described previously [29]. Wild-type NL4.3 served as a positive control, while strains encoding G2A or ∆Nef (premature stop codons at positions 31 & 33) were used as negative controls for infectivity and replication assays.

Nef polymorphisms are reported using HXB2 numbering convention [49]. Sequences were pairwise-aligned to the reference strain HXB2 (GenBank accession number K03055) and insertions with respect to HXB2 were removed using an in-house alignment algorithm based on the HyPhy platform [50].

2.3.4. Generation of SERINC5 knockout Jurkat LTR-GFP reporter cells

Jurkat T cell lines express high endogenous SERINC5 [21]. CRISPR/Cas9 methods were used to disrupt the SERINC5 gene in Jurkat LTR-GFP R5 cells. To do this, cells were co-transfected with px330-based plasmids [51,52] encoding target sequences described in Rosa et al, 2016 along with pMAX-GFP for use as a transfection control. Following transfection, GFP+ cells were isolated as single cells into R20+ media (RPMI-1640 supplemented with 2 mM L-glutamine, 1000 U/ml Penicillin, 1 mg/ml Streptomycin and 20% vol/vol FBS, all from Sigma-Aldrich) and expanded in 96-well flat bottom plates. Stable SERINC5 knockout (KO) clones were validated by Western blot using a rabbit polyclonal anti-SERINC5 antiserum (Abcam).

2.3.5. SERINC5 downregulation assays

To assess Nef-mediated internalization of SERINC5 from the cell surface, 1 × 106 CEM-A*02 T cells were co-transfected with 1 µg of pSELECT-GFPzeo encoding nef and 5 µg of pSELECT-SERINC5-internal HA tag (iHA)-∆GFP (sub-cloned from pBJ5- SERINC5(iHA) ([22]) by electroporation in 150 µL OPTI-mem medium (Thermo Fisher

24 Scientific) using a BioRad GenePulser MXCellTM instrument (square wave protocol: 250 V, 2000 µF, infinite Ω, 25 millisecond single pulse). Cultures were recovered for 20 hours with 350 µl of R10+ medium (RPMI-1640 supplemented with 2 mM L-glutamine, 1000

U/ml Penicillin and 1 mg/ml Streptomycin, all from Sigma-Aldrich) at 37ºC plus 5% CO2. Following this, 2.5 × 105 cells were stained with 0.5 µg of Alexa Fluor® 647 anti-HA.11 (BioLegend) and analyzed by flow cytometry for GFP expression (a marker for transfected cells) and cell surface SERINC5 expression (HA tag stain) using a Millipore Guava 8HT instrument. A minimum of 25,000 cells were assessed in each case. The median fluorescence intensity (MFI) values of SERINC5 for each Nef clone were normalized to the positive (pSELECT-nefWT-GFPzeo) and negative (pSELECT-∆nef) controls using the formula: (MFI∆Nef – MFICLONE)/(MFI∆Nef – MFIWT) × 100, such that Nef function less than or greater than wild type Nef is represented by values of <100% or >100%, respectively. The activity of each primary Nef clone was normalized to Nef (SF2 strain) to be consistent with our previous studies. Since point mutations were introduced into Nef (NL4.3 strain), that strain was used to normalize results from those studies. The SERINC5 antagonism activity of each Nef clone is reported as the mean ± S.D. based on at least three independent transfection experiments.

2.3.6. CD4 and HLA class I downregulation assays

Selected primary Nef clones and mutant were evaluated for their ability to internalize CD4 and HLA-A*02 (as a representative class I HLA molecule) as described previously [29]. Briefly, 2.5 × 105 transfected CEM-A*02+ CD4 T cells from the same pool of transfected cells described above were stained with anti-CD4-APC and anti-HLA- A*02-PE (BD Biosciences). The MFI of surface CD4 and HLA-A*02 expression in the GFP+ cell subsets were determined by flow cytometry and normalized using the same formula as described for SERINC5.

2.3.7. Infectivity and replication capacity

Virus stocks were generated by co-transfecting 8 × 105 HEK293T cells seeded in one well of a 6-well plate with 2.5 μg of pNL4.3 and either 30 ng of pSELECT- SERINC5(iHA)-ΔGFP or empty pSELECT-ΔGFP vector using DNAfectin 2100 (Applied Biological Materials). Culture supernatants were harvested 48 hours post-transfection and stored at -80°C prior to use. To assess SERINC5 surface expression on virus-

25 producing cells, the transfected cells were collected using Trypsin/EDTA (Sigma-Aldrich) and then prepared for analysis by flow cytometry. Briefly, cells were stained with 0.5 μg of Alexa Fluor® 647 anti-HA.11 (BioLegend), washed, treated with Fix/Perm solution (BD Biosciences), and then stained with PE anti-Gag/p24 (KC57; Beckman Coulter). A minimum of 10,000 cells were assessed in each case. Virus particles in the supernatant were quantified by p24 ELISA (XpressBio) and infectivity was determined by exposing 1× 104 TZM-bl reporter cells to a standardized amount of each virus (1 or 5 ng p24) on a 96-well flat bottom plate. Luminescence activity of TZM-bl cells was measured 48 hours later using the Steady-Glo. Luciferase Assay (Promega) and a Tecan Infinite M200 PRO plate reader. Relative infectivity was calculated for each viral strain using the following formula: (absolute light units (ALU) of virus generated in HEK293T/SERINC5+)/(ALU of virus generated in HEK293T/empty vector). To determine the infectivity of viruses produced in primary PBMC isolated from HIV-uninfected donors, VSV-g pseudotyped (0.2 μg) viruses (produced in HEK293T cells using transfection conditions as described above) were used to infect 5 × 105 activated PBMC (stimulated using 5 μg/ml PHA for 72 hours) in 96-well U bottom plates by spinoculation (800 g for 1 hour at room temperature). PBMC were incubated with the virus inoculum at 37°C for 8 hours and then removed, washed once with PBS and resuspended in R10+ media supplemented with 100 U/mL human IL-2. Newly produced viruses were harvested, quantified using p24 ELISA and then infectivity was measured using TZM-bl cells as described above. Infectivity was assessed in at least two independent experiments for each viral strain. Replication capacity was determined using viruses produced in the absence of SERINC5 by infecting 1 × 106 Jurkat LTR-GFP CCR5+ and Jurkat SERINC5 KO LTR- GFP CCR5+ cells at a multiplicity of infection (MOI) of 0.003. The proportion of GFP+ cells in culture was measured on days 2 to 8 and results are shown as the fold-increase in %GFP+ cells based on day 2 values. Replication was assessed in at least two independent experiments for each mutant viral strain.

2.3.8. Western blot

To assess steady-state protein expression of primary Nef alleles and mutants, 5 × 106 CEM-A*02 CD4 T cells were transfected with 10 µg of pSELECT-nef-GFPzeo alone via the electroporation settings as described above. After 24 hours, cells were pelleted, lysed and prepared as described previously [29]. Nef was labeled using a

26 polyclonal rabbit serum (cat. # ARP444; NIBSC Center for AIDS Reagents) (1:2,000) followed by staining with donkey anti-rabbit HRP-conjugated secondary antibody (GE Healthcare) (1:30,000). To validate CRISPR/Cas9-mediated knockout of SERINC5 in the Jurkat LTR-GFP cells, 2 × 106 parental or KO cells were pelleted and lysed. SERINC5 protein expression was detected using rabbit polyclonal anti-SERINC5 antibody (Abcam ab204400) at a 1:300 dilution, followed by staining with donkey anti- rabbit HRP-conjugated secondary antibody (GE Healthcare) (1:30,000). Proteins were detected using Clarity Western ECL substrate (Bio-Rad) and visualized on an ImageQuant LAS 4000 imager (GE healthcare).

2.3.9. Statistical analysis

All statistical analyses were performed using Prism v.7 (Graphpad). Results of two-tailed tests were considered significant if the p-value was less than 0.05. The nonparametric Mann-Whitney U test was used to compare differences in median Nef function between EC and CP cohorts and to identify Nef polymorphisms associated with differential in vitro activity. Multiple comparisons were addressed using q-values, the p- value analogue of the false discovery rate (FDR). The FDR is the expected proportion of false positives among results deemed significant at a given p-value threshold (e.g. at a q ≤ 0.2, I expect 20% of identified associations to be false positives). Following normalization of data positive and negative controls, I used the unpaired t-test to determine if the observed function of each Nef mutant was significantly different from that of the WT Nef.

2.4. Results

2.4.1. Primary HIV-1 Nef alleles display variable abilities to internalize SERINC5

To assess whether SERINC5 antagonism differs among circulating HIV-1 strains, I characterized the ability of 91 primary subtype B nef alleles (isolated from 45 elite controllers and 46 chronic progressors during untreated infection) to downregulate SERINC5 using a transfection-based assay (Figure 2.1). The function of each Nef clone was normalized to that of a control subtype B Nef isolate (SF2 strain), such that activities

27 better or worse than Nef (SF2) are reported as >100% or <100%, respectively. Empty vector (∆Nef) and the Nef G2A mutant were included as negative controls.

ΔNef G2A Nef WT Nef Primary Nef A 104

103

5

C N

I 102

R

E S 101

MFI: 54 MFI: 61 MFI: 17 MFI: 29 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 GFP (Nef)

B C (MFI – MFI ) ∆Nef CLONE x 100 (MFI∆Nef – MFIWT)

∆Nef WT Nef Patient Nef

Figure 2.1. Method used to measure downregulation of SERINC5 by primary Nef alleles (A) SERINC5 expression on the cell surface was assessed by flow cytometry following transient expression of Nef and a SERINC5 variant encoding an internal HA epitope tag (SERINC-iHA). Results are shown for two negative controls, empty vector (∆Nef) and Nef G2A mutant, a positive control, WT Nef (SF2 strain), and one representative primary Nef allele. Median fluorescence intensity (MFI) for SERINC5 (y-axis) in the transfected cell population (GFP+, x-axis) are indicated. In repeated experiments, the MFI values obtained using empty vector (∆Nef) and Nef G2A mutant were not discernable. (B) SERINC5 MFI values for each primary Nef allele were normalized to those of WT Nef (set to 100%) and ∆Nef (set to 0%) using the indicated formula. (C) Normalized SERINC5 downregulation activity (mean ± S.D.) is shown for controls and the representative primary Nef allele, based on three independent experiments.

I observed that controller-derived Nef clones displayed lower SERINC5 downregulation activity (median 80% [IQR 38-95%]) compared to progressor-derived clones (96% [IQR 75-100%]) (p=0.0005) (Figure 2.2A). To confirm this observation, I engineered the HIV-1 subtype B reference strain NL4.3 to encode each of 24 primary nef alleles that were selected to display a range of SERINC5 downregulation phenotypes, plus control nef sequences, and produced virions in the absence or presence of SERINC5. As expected, relative viral infectivity, calculated as the ratio of infectivity for each strain produced in the presence versus absence of SERINC5, varied

28 widely (Figure 2.2B). I observed a strong association between SERINC5 downregulation function and relative infectivity (Spearman R=0.62, p=0.0004; Figure 2.2C), which is consistent with internalization of SERINC5 being a key driver of Nef’s ability to counteract this host restriction factor. These data also indicated that ~80% normalized SERINC5 downregulation function was necessary to enhance viral infectivity in our assays, suggesting that Nef must maintain relatively high SERINC5 internalization activity to be effective – and conversely, that relatively modest impairment of this function may be biologically relevant. While additional studies are necessary to assess the activity of each primary Nef allele against a wider range of SERINC5 expression levels, I noted that more than half of controller-derived Nef clones displayed a normalized SERINC5 downregulation activity below 80%, compared to only one-quarter of progressor-derived clones (p=0.02). Together, these results demonstrate that the ability of Nef to internalize SERINC5 varies among primary isolates, and that this function is particularly impaired in Nef clones isolated from spontaneous HIV-1 elite controllers.

Figure 2.2. Characterization of SERINC5 downregulation and antagonism activity in HIV-1 Nef isolates from controllers and progressors (A) The ability of Nef clones isolated from 45 Elite Controllers (EC; red) and 46 Chronic Progressors (CP; blue) to downregulate SERINC5 is shown (Mann-Whitney U-test; p<0.001). Function was assessed by flow cytometry following transient expression of a CEM-derived T cell line with Nef and SERINC5(iHA) as described in Figure 2.1. For each Nef clone, downregulation function was normalized to Nef (SF2 strain) such that better or worse function than Nef (SF2) is reported as >100% or <100%, respectively. Results are reported as the median normalized function for each clone based on triplicate data obtained from at least three independent experiments. Bars represent the median and interquartile ranges. (B) Relative infectivity of NL4.3- derived viral strains encoding 24 primary Nef alleles (14 EC, red; 10 CP, blue) and four controls (G2A, ΔNef, WT NL4.3 Nef and WT SF2 Nef, black) was assessed by quantifying the infectivity of each strain generated in HEK-293T cells in the presence of SERINC5 divided by that of each strain generated in the absence of SERINC5. All viruses were tested at least twice in independent experiments. Mean results based on triplicate data from one representative experiment are shown. (C) Correlation between Nef-mediated SERINC5 downregulation (x-axis) and relative viral infectivity (y-axis) is shown for NL4.3-derived strains described in panel B (Spearman R=0.62; p=0.0004).

29 2.4.2. Natural Nef polymorphisms are associated with SERINC5 downregulation function

Using the linked genotype-phenotype dataset, I identified 18 naturally occurring Nef polymorphisms (located at 14 codons) that were associated with differential SERINC5 downregulation activity (all p<0.05) (Table 2.1). The most statistically significant correlation was observed at codon 51, where clones encoding threonine (N=43) displayed higher activity (median 95.3%) compared to clones that did not (N=45, typically asparagine) (81%) (p=0.004). I validated eight polymorphisms by introducing the mutation into a subtype B Nef isolate (NL4.3 strain), each of which resulted in the expected change in function (all p<0.05) (Figure 2.3A). Specifically, downregulation function was increased modestly for N51T, I114V and S163C mutants, which displayed ~5% higher activity compared to control Nef (NL4.3); whereas downregulation was impaired for C55S, K94E, H116N, V148L and S163R mutants, which displayed 5 to 50% lower activities compared to Nef (NL4.3). Codon 51 has been associated with viral infectivity previously [30], while codon 163 is located adjacent to the dileucine motif that is critical for SERINC5 antagonism and has been shown to affect Nef binding to clathrin adapter protein complexes [31]. While steady-state expression of these Nef mutants appeared to be variable (Figure 2.3), SERINC5 downregulation activity did not correlate with our ability to detect Nef by Western blot, indicating that these changes in Nef function were unlikely to be due to differences in protein stability.

30 Table 2.1. Nef polymorphisms associated with SERINC5 downregulation(N≥5, p<0.05, q<0.35) Number of Median Nef Activity Individuals Amino With Without With Without Codona Impactb p-value q-value Acid (AA) AA AA AA AA 11 A 100.5 86.5 6 75 + 14 0.005 0.22 19 K 100.7 85.6 9 82 + 15 0.011 0.25 28 D 85.1 93.4 47 44 - 8 0.047 0.34 43 * V 39.4 88.4 5 85 - 49 0.025 0.33 51 T 95.3 80 43 45 + 15 0.004 0.22 51 N 80 96 42 46 - 16 0.005 0.22 55 C 90.8 73.7 82 9 + 17 0.03 0.33 65 E 84.9 98.3 84 7 - 13 0.01 0.25 94 * E 50.3 88.6 5 86 - 38 0.015 0.28 94 K 88.4 55.2 83 8 + 33 0.047 0.34 114 V 89.8 74.9 70 21 + 15 0.037 0.34 116 H 95.3 80.6 59 32 + 15 0.012 0.25 116 * N 80.9 95.3 31 60 - 14 0.02 0.3 148 V 89.8 66.8 78 13 + 23 0.041 0.34 163 C 97.2 83.6 27 64 + 14 0.028 0.33 163 R 39.4 87.6 7 84 - 48 0.033 0.33 170 L 81 91.1 55 36 -10 0.049 0.34 182 Q 97.9 85.1 10 81 + 13 0.021 0.3 a HXB2-aligned residues; Nef polymorphisms associated with evasion from CD8+ T cells are indicated by * [27] b Median Nef activity with AA – median Nef activity without AA, rounded to nearest whole number.

31

Figure 2.3. Validation of natural polymorphisms in Nef that modulate SERINC5 downregulation (A) Eight Nef polymorphisms that were significantly associated with SERINC5 modulation were confirmed by site-directed mutagenesis in Nef (NL4.3 strain). Green bars represent mutants that were anticipated to improve SERINC5 downregulation function relative to WT Nef (100%) and red bars represent mutants that were anticipated to impair this function. Results are reported as the mean function for each Nef mutant, based on at least three independent experiments, plus standard deviations. Statistically significant differences compared to WT Nef are indicated by asterisks, * (p <0.05) ** (p<0.01), or *** (p<0.001) (unpaired T-test). (B) Steady-state protein expression for all Nef mutants was determined by Western blot (upper panel), compared to cellular β-actin controls (lower blot). Detection of each Nef mutant, quantified as the % expression relative to WT Nef, is shown.

2.4.3. Nef-mediated SERINC5 downregulation correlates more strongly with CD4 internalization compared to HLA internalization

The mechanisms of Nef-mediated SERINC5 downregulation and CD4 downregulation share numerous features, including reliance on Nef’s dileucine motif (LL164/165) [21, 22, 24] and trafficking of the internalized receptors to lysosomes through early (Rab5+), late (Rab7+) and recycling (Rab11+) endosomal compartments [24]. To explore potential associations between Nef functions, I compared the SERINC5 downregulation activity of our 91 primary clones to previously obtained CD4 and HLA downregulation results as well as protein stability data for these isolates (reported in [29]) (Figure 2.4). Notably, SERINC5 downregulation activity correlated more strongly with the ability of Nef clones to internalize CD4 (Spearman R=0.55, p<0.0001) compared

32 to HLA (R=0.31, p=0.0025). In addition, polymorphisms at S163 that are adjacent to the dileucine motif were similarly associated with differential ability of Nef to downregulate CD4 [29]. I observed no correlation between steady-state Nef expression levels detected by Western blot and downregulation of SERINC5, CD4, or HLA, indicating that associations between these functions were not due simply to differences in protein stability. These results suggest that similar polymorphisms in primary nef alleles contribute to variation in SERINC5 and CD4 downregulation functions, while effects on HLA downregulation activity are more independent.

Figure 2.4. Associations between Nef-mediated downregulation functions and protein stability (A, B) For each primary Nef allele isolated from an Elite Controller (EC, red) or Chronic Progressor (CP, blue), normalized downregulation activity for SERINC5 (x-axis) was compared to that for CD4 (A) and HLA class I (B). A stronger correlation was observed between downregulation of SERINC5 and CD4 compared to HLA, suggesting a greater shared impact of Nef polymorphisms on these two functions. (C, D, E) The ability of each Nef clone to downregulate CD4 (C), HLA class I (D), or SERINC5 (E) was compared to their steady-state protein expression as detected by Western blot. No significant associations were observed between Nef detection and any of these functions, suggesting that differences in Nef downregulation activity were not driven primarily by variation in protein expression or stability. Correlations were assessed using the Spearman test. Data for Nef-mediated CD4 and HLA downregulation and Western blot detection were reported previously [29].

33 2.4.4. Viral adaptation to host cytotoxic T lymphocytes contributes to variability in Nef-mediated SERINC5 downregulation activity

Notably, three Nef polymorphisms that were associated with a substantial reduction in SERINC5 downregulation function, I43V (-49% activity), K94E (-38%) and H116N (-14%) (Table 2.1), are HIV-1 escape mutations selected in CD8+ T cell epitopes restricted by HLA-C*03, B*08 and B*57, respectively [32, 33]. This indicates that immune pressure on Nef may attenuate its ability to antagonize SERINC5. Notably, H116N is often selected early following infection in individuals expressing the protective B*57 allele [32] and it is the predominant escape variant observed for this epitope. Thus, it is tempting to speculate that reduced Nef-mediated SERINC5 downregulation contributes to lower viremia in these cases. In contrast, K94E is selected more slowly in individuals expressing the non-protective B*08 allele [32] and E is not the primary escape variant observed for this epitope (rather K92R). This suggests that HIV-1 can adapt to B*08 without enduring the detrimental impact of this polymorphism on SERINC5 downregulation. Additional studies will be necessary to explore these links between host immune pressure and Nef function.

2.4.5. CTL escape mutations K94E and H116N selectively impair Nef- mediated SERINC5 downregulation function

To directly assess the impact of K94E and H116N on viral phenotypes, I generated HIV-1 NL4.3-derived strains encoding these mutations in the absence or presence of SERINC5. Consistent with our downregulation results, I observed lower SERINC5 surface expression on HEK-293T cells producing wild type NL4.3 compared to cells producing viruses that lacked Nef (∆Nef) or those that encoded Nef mutations that were anticipated to be detrimental to this function (G2A, K94E, H116N, or K94E/H116N) (Figure 2.5A). Moreover, while virion infectivity was comparable for all NL4.3-derived strains generated in the absence of SERINC5 (Figure 2.5B, white bars), substantial differences in infectivity were seen among viruses generated in the presence of SERINC5. Specifically, ∆Nef virus was 41-fold less infectious compared to wild type NL4.3, while G2A, K94E, H116N and double-mutation viruses were 11-, 4-, 3- and 5.5- fold less infectious than NL4.3, respectively (Figure 2.5B, black bars) (all p<0.05). Infectivity of the ∆Nef mutant was modestly impaired in the absence of SERINC5 (1.7- fold lower, compared to NL4.3), which may be due to SERINC3 expression by HEK-

34 293T cells. Notably, NL4.3-derived viruses encoding Nef K94E, H116N or the double- mutation also displayed reduced infectivity when they were harvested following infection of primary PBMC (Figure 2.5C), indicating that these polymorphisms altered viral phenotypes in the presence of endogenous levels of SERINC5. The results of multi- cycle viral replication capacity assays using Jurkat-derived GFP reporter T cells, which express high endogenous levels of SERINC5 [21], were entirely consistent with results obtained for Nef-mediated SERINC5 downregulation and viral infectivity enhancement by each mutant (Figure 2.5D). Specifically, substantial delays in cell-to-cell spread were observed for viruses encoding Nef K94E, H116N or the double-mutation. To confirm that differences in replication were mediated by SERINC5, I generated a SERINC5 knockout (KO) clone of the Jurkat-GFP reporter T cell line (Figure 2.5G). I observed no significant differences in replication between NL4.3-derived viruses encoding wild type Nef versus Nef K94E, H116N or the double-mutation in cells lacking SERINC5 (Figure 2.5E). Unexpectedly, the ∆Nef virus displayed enhanced replication capacity compared to viruses encoding Nef in this SERINC5 KO cell line, which is consistent with some early reports suggesting that Nef inhibits viral replication in certain immortalized cell lines [34, 35]. While I cannot explain this observation, I confirmed that this strain indeed lacked Nef (Figure 2.5H). Finally, I performed replication assays in primary PBMC by infecting PHA-stimulated cells with equivalent amounts of each virus (5ng p24) and measured p24 in culture supernatants at days 3 and 6. Consistent with our prior observations, viruses encoding Nef K94E, H116N or the double-mutation displayed lower replication in primary cells compared to wild type NL4.3 (Figure 2.5F). Together, these results demonstrate that K94E and H116N impair viral infectivity and replication capacity in the presence of SERINC5.

35

Figure 2.5. Impact of K94E and H116N Nef mutants on infectivity and viral replication (A) Representative flow cytometry plots display the expression of surface SERINC5 and intracellular p24 in HEK-293T cells co-transfected with pSERINC5-iHA and pNL4.3 encoding WT Nef or mutants. MFI values for SERINC5 on cells in the p24+ gate are shown. (B) Infectivity values (y-axis) are shown for NL4.3-derived viruses produced in the absence (white) or presence (black) of SERINC5-iHA. Results reflect luminescence values (absolute light units (ALU); log10) obtained following incubation of each virus with TZM-bl reporter cells, normalized to p24. Unpaired t-tests were used to compare each Nef mutant to the appropriate WT NL4.3 control (i.e. absence or presence of SERINC5). *** = p<0.001. (C) Infectivity values (y-axis) are shown for NL4.3 derived viruses following passaging in healthy donor PBMC. Results reflect luminescence

36 values (ALU, log10) obtained using TZM-bl reporter cells, normalized to p24. Unpaired t-tests were used to compare each Nef mutant to WT NL4.3 control. *** = p<0.001. (D and E) Replication capacity of NL4.3-derived viruses was examined using Jurkat LTR-GFP R5 cells (D), which express high endogenous levels of SERINC5, or Jurkat-LTR-GFP R5 cells in which SERINC5 was knocked out using CRISPR/Cas9 methods (E), respectively. In each case, cells were inoculated at MOI= 0.0003 and viral spread was monitored by flow cytometry. The mean (±S.D.) fold-increase in %GFP+ cells (from day 2) is reported for each Nef mutant virus based on triplicate infections. All viruses were tested at least three times in independent experiments. (F) Replication capacity of NL4.3-derived viruses was assessed in healthy donor PBMC stimulated using PHA. Cells were spinoculated with each VSV-g pseudotyped virus (5 ng p24), supernatant was harvested at days 3 and 6, and viral replication was detected using p24 ELISA. (G) CRISPR/Cas9 methods were used to disrupt the SERINC5 gene in Jurkat LTR-GFP reporter cells. Clonal knockout (KO) cell lines were then isolated. Loss of SERINC5 expression was confirmed by Western blot using a rabbit polyclonal anti-SERINC5 antiserum. (H) Western blot analysis was used to confirm the presence or absence of Nef in NL4.3-derived viruses following infection of Jurkat LTR-GFP SERINC5 KO cells. Cells were harvested on day 9 of the replication assay shown in Figure 2.5F

To assess if the impairment of SERINC5 downregulation function by K94E and H116N was selective, I next tested the ability of Nef mutants encoding these substitutions to internalize CD4 and HLA class I (Figure 2.6, left side). Compared to wild type Nef (NL4.3 strain), K94E reduced HLA downregulation activity modestly (to 87%) (p<0.05) but had no effect on CD4 internalization. Conversely, H116N reduced CD4 downregulation activity modestly (to 89%) (p<0.05) but had no effect on HLA internalization. All three Nef-mediated downregulation functions were impaired in the mutant encoding both K94E and H116N, indicating that the impact of these polymorphisms can be additive. In particular, SERINC5 downregulation activity was reduced markedly in the Nef double-mutant (to 19%) (p<0.001), while more moderate effects were seen for downregulation of CD4 (69%) and HLA (87%) (both p<0.05). To explore this further, I identified an EC-derived Nef clone (EC48) encoding E94 and N116 that displayed poor SERINC5 downregulation activity (32%) but retained moderate CD4 and HLA downregulation functions (78% and 92%, respectively). To confirm that E94 and N116 were determinants of impaired SERINC5 internalization by this primary Nef clone, I reverted these polymorphisms individually or simultaneously to the consensus residue (Figure 2.6, right side). Consistent with results for NL4.3-derived Nef, reversion of E94K or N116H partially rescued SERINC5 downregulation activity (to 69% and 59%, respectively) (both p<0.05); while reversion of both polymorphisms enhanced this activity further (to 89%) (p<0.001). In contrast to results obtained using NL4.3-derived Nef, the E94K and double reversion mutant displayed lower HLA downregulation activity compared to the primary Nef clone (56% and 51%, respectively) (p<0.01), suggesting

37 that unidentified compensatory polymorphisms in the EC48 nef sequence contributed to maintain this function. Together, these results demonstrate that K94E and H116N reduce Nef’s ability to downregulate SERINC5 to a greater extent compared to CD4 or HLA class I, and further indicate that their impact on all three Nef functions may be modulated by other sequence variation present in primary nef alleles.

Figure 2.6. Impact of K94E and H116N Nef mutants on downregulation activities The ability of Nef (NL4.3) site-directed mutants encoding K94E and/or H116N or primary Nef EC48 reversion mutants encoding E94K and/or N116H to downregulate SERINC5, CD4 and HLA is shown. Bar graphs represent the mean downregulation function based on at least 3 independent experiments, plus standard deviation. Results were normalized to WT NL4.3 (for NL4.3 mutants) or WT SF2 (for EC48 mutants). The unpaired t-test was used to compare each mutant with it representative parental control, either WT Nef (NL4.3) or Nef EC48. Significant differences are indicated by asterisks: * = p<0.05, ** = p<0.01 and *** = p<0.001.

2.5. Discussion

I have demonstrated that primary HIV-1 subtype B nef alleles exhibit substantial variability in SERINC5 downregulation activity. Nef clones isolated from HIV-1 elite controllers displayed lower in vitro function compared to those from progressors, suggesting that impaired SERINC5 antagonism may contribute to lower plasma viremia and slower disease progression in this rare group of individuals. Consistent with the observation that even limited amounts of SERINC5 incorporation into viral particles reduces infectivity [36], I found that Nef-mediated infectivity enhancement required ~80% normalized SERINC5 downregulation activity in our assays, a threshold that was not met by 51% of controller-derived clones.

While a prior study examined the ability of diverse HIV and SIV Nef isolates to antagonize SERINC5 [37], it assessed relatively few (~15) HIV-1 group M Nef clones. Our analysis of 91 primary HIV-1 subtype B Nef clones thus provides greater power to

38 explore functional variation among patient-derived sequences. To date, it has been difficult to separate Nef’s multiple in vitro functions genetically. Here, I identified 18 polymorphisms that were associated with differential SERINC5 downregulation activity. I validated eight of these polymorphisms using site-directed mutagenesis, including two well-characterized CD8+ T cell escape mutations, K94E and H116N. While mechanisms responsible for impaired SERINC5 internalization by primary nef alleles or site-directed mutants have not been defined in this study, it is intriguing that K94E and H116N had only modest effects on Nef’s ability to downregulate CD4 or HLA class I, suggesting that K94 and H116 lie within domains of Nef that are particularly important to antagonize SERINC5 rather than to internalize CD4 or HLA.

Our study illustrates tremendous diversity in SERINC5 downregulation activity among primary HIV-1 nef isolates; however, the clinical implications of this observation are unclear. In particular, the importance of Nef-mediated SERINC5 antagonism for HIV- 1 pathogenesis remains unproven, a fact that is complicated by Nef’s multiple functions and the still recent discovery of SERINC5 as a host restriction factor. In addition, Nef’s ability to counteract SERINC5 is highly dependent on the expression levels of both proteins [36, 38], which may not be recapitulated fully by in vitro assays. Furthermore, the HIV-1 Envelope contributes to viral sensitivity to SERINC5 [39], but its relative impact compared to Nef has not been elucidated and detailed analyses of functional interactions between primary nef and env alleles have not been conducted. Our results using primary PBMC indicate that Nef polymorphisms can impair viral infectivity and replication in the context of endogenous SERINC5. These observations are consistent with natural variation in Nef-mediated SERINC5 antagonism being a novel contributor to clinical outcomes; however, additional research will be necessary to address this issue. This study also highlights the constraints placed on HIV-1 Nef by the interplay between adaptive host immunity and intrinsic host restriction mechanisms that may result in viral attenuation during the course of natural infection.

39 2.6. References

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43 Chapter 3.

Differential ability of HIV-1 Nef alleles across viral subtypes to downregulate SERINC3 and SERINC5

3.1. Abstract

SERINC3 and SERINC5 are recently identified restriction factors that inhibit retroviral infectivity when incorporated into newly formed virions but are counteracted by HIV-1 Nef. Since subtype B reference Nef strains are commonly used, our understanding of Nef sequence and polymorphism diversity at the population level and its impact on SERINC antagonism function is limited. Here, I characterized SERINC3 and SERINC5 downregulation in 339 primary HIV-1 Nef alleles representing globally relevant subtypes A, B, C and D. There was a distinct subtype hierarchy for Nef- mediated SERINC5 downregulation (B > D > C/A) and SERINC3 downregulation activity (A/C/D > B) (both p<0.001). With linked genotype-phenotype analyses, I identified that the absence of S8, I11 or V11 was associated with impaired SERINC3 downregulation activity and was confirmed by point mutations S8R and I11G in NL4-3 Nef (50% and 0% relative activity, respectively) with negligible impact on CD4, HLA-I and SERINC5 downregulation activities. In addition, using proximity ligase assay, I showed that S8R and I11G reduced Nef’s ability to co-localize with SERINC3. Altogether, our results demonstrate that Nef’s ability to counteract SERINC3/5 differs among primary isolates and variation in this activity could contribute to observed differences in pathogenesis between HIV-1 subtypes. Lastly, I identified two residues in Nef’s N-terminal domain that are crucial for downregulation of SERINC3, but not SERINC5, confirming that these activities are genetically separable.

44 3.2. Introduction

The encodes several cellular restriction factors that act as blockades against the HIV-1 life cycle and other viral pathogens [1-5]. However, HIV-1 has adapted to evade these innate antiviral activities of restriction factors by either evasion or counteraction mechanisms. The most recent restriction factor to be identified belong to the family of serine incorporator (SERINC) multipass transmembrane proteins, particularly SERINC3 and SERINC5 (with the latter more potent), but are antagonized by HIV-1 Nef [6, 7]. Incorporation of SERINC3/5 in newly formed virions interferes with the formation and establishment of fusion pores with the uninfected target cell, resulting in a restriction of viral infectivity phenotype [8].

Nef is a highly polymorphic ~206 amino acid (27-35 kDa) multifunctional accessory protein required for efficient viral pathogenesis in vivo [9-11]. Despite the exceptionally polymorphic, immunogenic and adaptable nature of Nef, it also contains functional motifs that are highly or almost exclusively conserved in circulating sequences [12-14]. One of the most essential is the myristoylation motif in the N-terminal arm (residues 1-7) that allows Nef to attach to the plasma membrane [15] and enable it to carry out some of its critical functions such as co-localization with and ultimately downregulation of CD4, HLA class I and SERINC cell surface proteins [6, 7, 16-18]. Another indispensable motif is the dileucine residues at positions 164/165 of Nef, which is essential for the formation of clathrin-coated pits through its interactions with the adaptor-protein complexes, promoting the internalization and downregulation of its target proteins [21, 22]. Downregulation of CD4 allows infected cells to evade antibody- dependent cellular cytotoxicity (ADCC) responses while downregulation of HLA-I avoids recognition by CD8+ cytotoxic lymphocytes [19, 20]. Likewise, Nef counteracts the antiviral effects of the SERINC proteins by downregulating them from the cell surface to prevent its incorporation into progeny virions. While mutations at these motifs, namely G2A and LL164/165AA, do in fact attenuate Nef function, they are not selective against SERINC antagonism and are rarely found in natural isolates [12, 23].

Since most studies to date emphasize on SERINC5 antagonism using laboratory reference subtype B Nef strains (i.e. NL4-3, SF2 or LAI), they do not capture the global diversity of Nef sequence and function of primary alleles within and across different major HIV-1 subtypes, as well as their ability to antagonize the less potent SERINC3.

45 Furthermore, the lack of characterization of the impact of primary Nef alleles on SERINC antagonism limits our insight of naturally-occurring polymorphisms that could disrupt this activity aside from the canonical mutations. Given that Nef displays extensive genetic and functional diversity within and between subtypes [24-26], I would also expect there to be a dynamic range of SERINC downregulation activity in a large cohort of globally representative Nef alleles. In addition, there is growing evidence that HIV subtype is a determinant of disease progression [27, 28]. For example, subtype A and C infection is associated with lower mortality rates compared to subtype D infection when controlled for other variables [29-31]. Thus, differences in the biological properties of HIV-1 subtypes, particularly viral protein function, could potentially contribute to differential disease progression, transmissibility and pathogenesis. Therefore, in this study, I aimed to characterize Nef-mediated downregulation and antagonism of SERINC3 and SERINC5 by >300 primary alleles across the four major HIV-1 subtypes (A, B, C and D).

3.3. Materials and Methods

3.3.1. Study Participants

Nef sequences and clones from chronically infected, treatment naïve individuals were characterized previously [24]. Briefly, the study cohort consists of Nef clones derived from 360 patients infected with HIV-1 of different subtypes. However, for this study, Nef clones that displayed poor steady-state detection by western blot were excluded from analysis, resulting in a total of 339 Nef clones (92 subtype A. 91 subtype B, 71 subtype C and 85 subtype D) for characterization. Nef sequences in this study are available as Genbank accession numbers KC906733 – KC907077.

3.3.2. Plasmids

Nef clones used in this study were previously described [24]. Briefly, patient- derived HIV-1 nef amplicons were amplified from plasma with nested RT-PCR and cloned into pSELECT-GFPzeo expression plasmid (Invivogen) which features a hEF1- HTLV promoter and an independent CMV promoter driving expression of GFP. Expression of SERINC5 with an internal HA tag (iHA) was sub-cloned from pBJ5- SERINC5(iHA) [7] into the pSELECT-∆GFP plasmid. A SERINC3(iHA) construct was synthesized as a gBlock Gene Fragment (Integrated DNA Technologies; Accession

46 Number NM_006811) with an internal HA tag inserted between amino acids 311 and 312, a Kozak sequence (GCCGCCACC) upstream of the start codon and flanking restriction enzyme cut sites BamHI and SacII at the 5’ and 3’ ends, respectively and subsequently cloned into the pSELECT-∆GFP expression vector. The HIV-1 NL4-3 Infectious Molecular Clone (pNL4-3) was obtained from the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH (Cat #114).

3.3.3. Site-directed mutagenesis

Point mutations in reference and primary Nef clones were introduced using overlap PCR extension methods as previously described [39].

3.3.4. SERINC, CD4 and HLA-I downregulation

To assess Nef-mediated downregulation of SERINC3 or SERINC5 from the cell surface, 1 × 106 CEM-A*02 CD4 T cells were co-transfected with 1 µg of pSELECT- GFPzeo encoding nef and 5 µg of pSELECT-SERINC3(iHA)-∆GFP or pSELECT- SERINC5(iHA)-∆GFP by electroporation in 150 µL OPTI-mem medium (Thermo Fisher Scientific) using a BioRad GenePulser MXCellTM instrument (square wave protocol: 250 V, 2000 µF, infinite Ω, 25 millisecond single pulse). Cultures were recovered for 20-22 hours with 350 µl of R10+ medium (RPMI-1640 supplemented with 2 mM L-glutamine, 1000 U/ml Penicillin and 1 mg/ml Streptomycin, all from Sigma-Aldrich) at 37ºC plus 5%

5 CO2. Following this, 2.5 × 10 cells were stained with 0.5 µg of Alexa Fluor® 647 anti- HA.11 (BioLegend) and analyzed by flow cytometry for GFP expression (a marker for transfected cells) and cell surface SERINC expression (HA tag stain) using a Millipore Guava 8HT instrument. The median fluorescence intensity (MFI) values of SERINC3/5 for each Nef clone were normalized to the positive (pSELECT-nefWT (NL4-3)-GFPzeo) and negative (pSELECT-∆nef-GFPzeo) controls using the formula: (MFI∆Nef –

MFICLONE)/(MFI∆Nef – MFIWT) × 100, such that Nef function less than or greater than wild type Nef is represented by values of <100% or >100%, respectively.

3.3.5. Generation of a SERINC3 knockout HEK293T cell line

To rule out the influence of high endogenous levels of SERINC3 in our virus production system, I generated a HEK293T SERINC3 knockout (KO) cell line using

47 CRISPR/Cas9 technology. Parental HEK293T cells were transfected with pX330-based plasmids [40, 41] encoding target sequences described in Rosa et al., 2015 using DNAfectin 2100 (Applied Biological Materials) and serially diluted into 96-wells to allow for clonal expansion. Knockout of endogenous SERINC3 in HEK293T clones were confirmed using Western blot with rabbit polyclonal anti-SERINC3.

3.3.6. Infectivity Assay

To assess the effect of SERINC antagonism by Nef on infectivity, 8 × 105 HEK293T SERINC3 KO cells were seeded on 6 well plates and transfected with 2 µg pNL4-3 ∆Nef, 30 ng pSELECT-SERINC3 or 5 (iHA)- ∆GFP and 10 ng pSELECT-nef- GFP using DNAfectin 2100 (Applied Biological Materials). Viruses from the culture supernatants were harvested 48-hours post-transfection, spun down to separate the supernatant from residual cells and stored in cryovials at -80°C until use. The concentration of HIV-1 viruses in the supernatant was quantified using p24 ELISA (XpressBio) and a standardized amount viruses were used to infect 1 x 104 TZM-bl reporter cells on a 96-well flat bottom plate for 48 hours in triplicate. Infectivity reading was measured using Steady-Glo® Luciferase (Promega) on a Tecan Infinite M200 PRO plate reader.

3.3.7. Proximity Ligase Assay (PLA)

PLA was conducted using Duolink® flowPLA Detection Kit - Far Red Mouse/Rabbit kit (Millipore-Sigma). Briefly, 1 x 106 CEM A*02 T cells were transfected with 1 µg of pSELECT-nef-GFP and 5 µg pSELECT-SERINC(iHA)-∆GFP as described above. After 20 hours, cells were treated with BD Cytofix-Cytoperm TM solution (BD Biosciences), and co-stained with rabbit polyclonal anti-Nef (cat. # ARP444; NIBSC Center for AIDS Reagents) and mouse anti-HA.11 (Biolegend, clone 16B12) overnight at 4°C. Secondary incubation anti-rabbit PLUS and anti-mouse MINUS probes, ligation, amplification and wash steps were completed in solution as directed by the manufacturer for flow cytometry applications. Cells were analyzed on GUAVA flow cytometer (Millipore). PLA signal from Nef and SERINC co-localization was normalized to WT NL4- 3 Nef (100%) and empty vector (0%).

48 3.3.8. Western blot

Steady-state levels of Nef expression was assessed by transfecting 2.5 x 106 CEM-A*02 CD4 T cells with 10 µg of pSELECT-nef-GFPzeo via electroporation settings as described above. After 24 hours, cells were pelleted, lysed and prepared as described previously. Nef was labeled using a polyclonal rabbit serum (cat. # ARP444; NIBSC Center for AIDS Reagents) (1:2000) followed by staining with HRP Donkey anti- rabbit IgG antibody (BioLegend) (1:5000). To validate CRISPR/Cas9-mediated knockout of SERINC3 in HEK293T cells, 2 x 106 parental or SERINC3 KO HEK293T cells were pelleted and lysed. Endogenous SERINC3 expression was detected using rabbit polyclonal anti-SERINC3 antibody (Abcam ab65218) (1:1000 dilution) followed by staining with HRP Donkey anti-rabbit IgG antibody (BioLegend). Proteins were detected using Clarity Western ECL substrate (Bio-rad) and visualized on ImageQuant LAS 4000 imager (GE healthcare).

3.3.9. Statistical analysis

All statistical analyses were performed using Prism v.7 (Graphad). Results of two-tailed tests were considered significant if the p-value was less than 0.05. The nonparametric Mann-Whitney U test was used to compare differences in median Nef function between subtypes and to identify Nef polymorphisms associated with differential in vitro activity. Multiple comparisons were addressed using q-values, the p-value analogue of the false discovery rate (FDR). The FDR is the expected proportion of false positives among results deemed significant at a given p-value threshold (e.g. at a q≤0.1, I expect 10% of identified associations to be false positives).

3.4. Results

3.4.1. Dynamic range of Nef-mediated SERINC downregulation activity among primary isolates

The 339 patient-derived HIV-1 Nef clones in our study and NL4-3 Nef (black line) distinctively cluster into their respective subtype (A, B, C or D) shown in the phylogenetic tree (Figure 3.2A). Each Nef clone was characterized for their ability to downregulate the SERINC3 and SERINC5 restriction factors from the cell surface. Briefly, the assay

49 entails measuring SERINC expression on the cell surface with flow cytometry following co-transfection of CEM CD4 T-cells with Nef and SERINC with an internal HA tag (iHA). Consistent with the original study [7], SF2 Nef can downregulate SERINC5 efficiently but not SERINC3, while NL4-3 Nef is able to downregulate both (Figure 3.1).

Figure 3.1. Nef-mediated downregulation of SERINC3 and SERINC5 assay Representative flow cytometry plots of CEM CD4-T cells stained with anti-HA-Alexa Fluor®647 following co-transfection of Nef with either SERINC3(iHA) [top panel] or SERINC5(iHA) [bottom panel]. Median fluorescence intensity (MFI) is shown in the gate representing transfected cells. Empty vector and G2A Nef mutant are unable to downregulate SERINC3 or SERINC5, while WT NL4-3 Nef downregulates both.

To compare the relative activities of primary Nef alleles for SERINC3/5 downregulation, NL4-3 Nef was used as our reference strain for the remainder of this study whereby the activity of NL4-3 Nef was normalized to 100% function and 0% function for vector control. I observed a dynamic range of SERINC downregulation activity among our cohort of 339 patient-derived Nef clones. The median activity for SERINC3 downregulation was 78 [interquartile range (IQR) 48-97] % relative to NL4-3 Nef and 94 [IQR 77-102] % for SERINC5 downregulation activity for the same clones (Figure 3.2B; p<0.0001). These results suggest that SERINC5 downregulation activity is more conserved than SERINC3 downregulation in circulating Nef strains. When the SERINC downregulation activities were stratified into their respective subtypes, I observed distinct hierarchical rankings for SERINC3 and SERINC5 downregulation. There were no statistically significant differences between subtypes A, C and D for

50 SERINC3 downregulation, albeit subtype D displayed the highest median activity with 88 [IQR 64-97] %, followed by subtype A (83 [IQR 50-100] %) and subtype C (82 [IQR 50- 96] %) (Figure 3.2C). Most interestingly, primary subtype B Nef clones displayed significantly the poorest ability to downregulate SERINC3 with a median activity of 55 [28-92] % (all p<0.05). In contrast, for SERINC5 downregulation, subtype B Nef clones displayed the highest activity (102 [IQR 96-110] %), followed by subtype D (98 [IQR 74- 104] %), subtype C (89 [IQR 74-99] %) and lastly subtype A (83 [IQR 67-95] %) (all p<0.001) (Figure 3.2D). Indeed, our results do demonstrate that SERINC downregulation activity is highly variable among primary Nef alleles intra- and inter- subtype, with subtype B Nef clones displaying the best SERINC5 downregulation abilities but the worst for SERINC3 downregulation.

Figure 3.2. Nef-mediated SERINC3 and SERINC5 downregulation by primary Nef alleles representing globally relevant subtypes

51 (A) Maximum likelihood phylogenetic tree of 339 patient-derived Nef clones in the study. Subtype A is represented in red, subtype B in orange, subtype C in green and subtype D in blue. Laboratory-adapted subtype B NL4-3 Nef strain (black) was included in the phylogenetic tree as a reference. (B) Downregulation activity of SERINC3 (left) and SERINC5 (right) of the 339 Nef clones relative to NL4-3 Nef (100%). The bars represent the median and interquartile range. SERINC downregulation was assessed by flow cytometry following co-transfection of CEM CD4 T-cell line with Nef and SERINC(iHA) (Figure 3.1). Mann-Whitney U-test statistical analysis was performed. (C) Relative SERINC3 downregulation activity of the 339 Nef clones stratified into their respective subtypes. Asterisks * = p<0.05, ** = p≤0.01, *** = p≤0.001 and **** = ≤0.0001 as determined by Mann-Whitney U-test. (D) Relative SERINC5 downregulation activity of the same Nef clones stratified into their respective subtypes.

3.4.2. Nef-mediated antagonism of SERINC3 and SERINC5 enhances viral infectivity

Next, I wanted to investigate the correlation of SERINC downregulation in our transfection system (Figure 3.2) with the infectivity of progeny virions produced in the presence of different nef alleles and SERINC. Typically, HEK293T cells have been the HIV-1 producer cell line of choice because of the ease of handling. However, for recent SERINC5 antagonism studies using this cell line, SERINC5 expression needed to be supplemented through transient expression because the endogenous protein is expressed in low amounts [32, 33]. In contrast, HEK293T cells endogenously express relatively high levels of the SERINC3 protein compared to CD4 T-cell lines [7], and supplementing the producer cell with SERINC3 plasmids may result in progeny virions incorporating an exceedingly overwhelming amount of SERINC, diminishing the Nef effect on infectivity enhancement [34]. To rule out this confounding variable in our experimental system so that I can study the effects of Nef-mediated antagonism of a standardized amount in trans SERINC expression, I used CRISPR/Cas9 technology to knockout (KO) endogenous SERINC3 in HEK293T cells. The HEK293T SERINC3 KO cell line was validated by Western blot (Figure 3.3A; inset). A further step of validation was performed by comparing the infectivity fold difference for viruses produced in parental versus SERINC3 KO HEK293T cells. For WT NL4-3 HIV-1 viruses produced in parental HEK293T cells, viruses were roughly 3-fold more infectious than ∆Nef HIV-1 viruses, consistent with a modest Nef-mediated infectivity enhancement in HEK293T cells as reported in previous studies [6, 35]. On the other hand, when the same two viruses were produced in the HEK293T SERINC3 KO cells, the modest 3-fold difference between WT and ∆Nef viruses was reduced to only a modest 1.1-fold, suggesting that residual infectivity enhancement typically observed in HEK293T cells can be attributed to

52 high expression of SERINC3. In the absence of a SERINC antagonist, transfection of a standardized amount (30 ng) of pSERINC3(iHA) or pSERINC5(iHA) in the producer cell inhibited virion infectivity by 8-fold and 55-fold, respectively (Figure 3.3A). As expected, SERINC5 was a more potent restriction factor of virion infectivity in our assay. Through validation of our HEK293T SERINC3 KO cell line and confirming that our internally HA- tagged SERINC plasmids retained antiviral activity, I utilized this system to confirm the SERINC antagonism activities of our primary Nef clones.

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Figure 3.3. Validation of HEK293T SERINC3 knockout cell line Inset: Western blot validation of CRISPR/Cas9-mediated knockout of SERINC3 in a HEK293T clone using rabbit polyclonal anti-SERINC3. Cellular β-actin was stained and imaged as a loading control. Bar graph: Infectivity (reported as absolute light units [ALU] luciferase reading from TZM- bl reporter cells following infection with a standardized amount of p24 HIV-1) of NL4-3 WT or ∆Nef HIV-1 viruses produced in different conditions. HIV-1 producer cells were either parental HEK293T cells or SERINC3 KO HEK293T cells (co-transfected with empty vector or SERINC plasmids).

I generated NL4-3 HIV-1 virions with different Nef alleles and SERINC expressed in trans. In our first virus production condition, only SERINC3 was transiently expressed in the HEK293T producer cell. As anticipated, the infectivity of viruses produced with these conditions correlated more strongly with SERINC3 downregulation activity (r=0.62, p<0.0001) compared to SERINC5 downregulation (r=0.31, p=0.004) (Figure 3.4A). Conversely, when viruses obtained from the HEK293T producer cell contained only exogenous SERINC5, the viral infectivity of these viruses correlated more strongly with SERINC5 downregulation (r=0.63, p<0.0001) compared to SERINC3 downregulation (r=0.32, p=0.003) (Figure 3.4B). Therefore, our results show that our Nef-mediated SERINC downregulation data is a predictive indicator for Nef-mediated SERINC antagonism activity in an in vitro infection system. Lastly, when both SERINC3 and SERINC5 were transiently expressed in equal amounts (15 ng of each plasmid) in the producer cell, viral infectivity correlated with SERINC5 downregulation (r=0.50,

53 p<0.0001) but not SERINC3 downregulation (r=0.15, p=0.13) (Figure 3.4C), indicating the ability to downregulate SERINC5 when both SERINCs are present is a more predictive measure of viral infectivity, which is not surprising since it is the more potent restriction factor.

54 Figure 3.4. Downregulation of SERINC3 and SERINC5 correlates with viral infectivity A) Correlation of relative SERINC3 downregulation (left) and SERINC5 downregulation (right) activity (x-axis) with infectivity measurements (y-axis; ALU per ng p24; log10) of NL4-3 ∆Nef viruses produced in SERINC3 KO HEK293T cells with Nef (~20 primary Nef clones per subtype) and SERINC3 expressed in trans. (C) Correlation of relative SERINC5 downregulation (left) and SERINC5 downregulation (right) activity (x-axis) with infectivity measurements (y-axis; ALU per ng p24; log10) of NL4-3 ∆Nef viruses produced in SERINC3 KO HEK293T cells with Nef (~20 primary Nef clones per subtype) and SERINC5 expressed in trans. (C) Correlation of relative SERINC3 downregulation (left) and SERINC5 downregulation (right) activity (x-axis) with infectivity measurements (y-axis; ALU per ng p24; log10) of NL4-3 ∆Nef viruses produced in SERINC3 KO HEK293T cells with Nef (~20 primary Nef clones per subtype) and equal amounts of SERINC3 and SERINC5 expressed in trans.

3.4.3. Polymorphisms associated with modulation of SERINC

With a large dataset of linked sequence and functional data (Figure 3.2), I performed statistical analyses to determine whether there are polymorphisms at specific Nef residues that are significantly associated with differential SERINC downregulation activity that could potentially influence some of the dynamic range of function I observed. In this analysis, I identified subtype-specific polymorphisms in Nef at distinct residues modulating SERINC5 downregulation (Table 3.1; complete table in Table A.3.1). Some naturally occurring polymorphisms (i.e. K94E in subtype B Nef) were also identified independently in a previous analysis of SERINC5 downregulation in a cohort of 91 subtype B Nef clones from chronically infected elite controllers and chronic progressors (Chapter 2; Table 2.1). More interestingly, for the SERINC3 downregulation sequence- function analysis (Table 3.2; complete table in Table A.3.2), there was an extremely powerful statistical association at Nef codons 8 and 11, identified independently in all four subtypes, where the absence of S8, I11 or V11 at these positions were associated with highly impaired activity. I, therefore, chose to study these two residues (positions 8 and 11) in further detail.

55 Table 3.1. Nef polymorphisms associated with differential SERINC5 modulation in HIV-1 subtypes A, B, C and D

Relative Nef Number of Impact Amino activity (%) subjects p – q – Subtype Codon Acid value value With Without N N AA AA (With) (Without) A 60 E 45.6 85.3 4 88 -40 0.008 0.44 A 60 A 85.3 51.4 87 5 +34 0.030 0.44 A 157 N 76.2 88.9 32 60 -13 0.035 0.44 A 157 T 88.9 76.2 60 32 +13 0.035 0.44 A 180 T 76.5 90 54 38 -14 0.005 0.44 A 180 V 90 76.5 38 54 +14 0.005 0.44 B 61 Q 109.2 89.9 86 5 +19 0.009 0.2 B 61 Y 82.3 108.3 3 88 -26 0.010 0.2 B 94 K 110.1 96 77 14 +14 0.000 0.02 B 94 E 82.3 109.2 7 84 -27 0.006 0.2 B 120 F 84.4 108.9 6 85 -25 0.012 0.2 B 120 Y 108.9 84.4 85 6 +25 0.012 0.2 C 32 A 87.3 100.5 64 7 -13 0.005 0.27 C 32 T 101.2 88.3 3 68 +13 0.018 0.36 C 88 G 70.3 91.4 15 56 -21 0.008 0.27 C 88 S 91.4 70.3 56 15 +21 0.008 0.27 C 98 D 82.1 95 34 37 -13 0.006 0.27 C 98 E 95 82.1 37 34 +13 0.006 0.27 D 23 A 55.7 99.7 7 78 -44 0.020 0.76 D 23 T 99.7 82.7 76 9 +17 0.031 0.76 D 114 V 100.5 64.2 73 12 +36 0.003 0.69 D 114 I 76 100.5 10 75 -25 0.020 0.76 D 176 E 47.5 99.7 3 82 -52 0.025 0.76

56 Table 3.2. Nef polymorphisms associated with differential SERINC3 modulation in HIV-1 subtypes A, B, C and D

Relative Nef Number of Impact Amino activity (%) subjects p – q – Subtype Codon Acid value value With Without N N AA AA (With) (Without) A 11 V 95.9 36.6 72 16 +59.3 1.2E-06 0.00 A 8 S 95.7 39.8 73 17 +55.9 4.1E-06 0.00 B 11 V 93.7 43.0 30 47 +50.7 9.4E-07 0.00 B 8 S 92.1 50.7 37 46 +41.4 5.8E-06 0.00 C 11 V 90.6 48.2 52 14 +42.4 1.1E-03 0.13 C 8 R 46.8 87.7 5 63 -40.9 1.6E-02 0.23 D 8 S 93.9 67.2 69 13 +26.7 1.6E-04 0.04 D 8 R 70 92.5 10 72 -22.5 6.5E-03 0.20

3.4.4. Mutations at residues 8 and 11 of Nef selectively impair SERINC3 downregulation

To validate the association identified in Table 3.2, I introduced point mutations S8R and I11G in NL4-3 Nef (since these were the most common polymorphisms occurring at these two sites) and characterized it for CD4, HLA-I and SERINC downregulation. Either mutation had no or negligible impact on CD4, HLA-I or SERINC5 downregulation, but was severely impaired for SERINC3 downregulation (50% activity for S8R and 0% activity for I11G) (Figure 3.5A). While these results certainly suggest that mutations at codons 8 and 11 in Nef selectively impair SERINC3 downregulation, NL4-3 sequence and function is not always representative of primary strains [36]. To confirm that mutations at these residues also impact primary Nef alleles similarly, a representative functional Nef clone from each subtype was chosen for validation. S8R or I/V11G mutations in a subtype A, C and D patient-derived Nef clone all impaired SERINC3 downregulation, although the extent of impairment varied (Figure 3.5B). Since most primary subtype B Nef clones encode the R8 and G11 polymorphisms and are non- functional for this activity, I picked a representative subtype B Nef clone with relatively poor SERINC3 downregulation activity and attempted to rescue function by reverting the amino acids at codons 8 and 11 to the functional residues. Individual reversions R8S and G11V in the patient-derived subtype B Nef clone did not rescue function, however, a double reversion (R8S+G11V) was able to rescue function from 45 to 79% (Figure

57 3.5C). Additionally, amino acid probability plots composed from >3000 sequences obtained from the Los Alamos database show that serine and valine are highly conserved at positions 8 and 11, respectively, for subtypes A, C and D but not subtype B, where those sites are highly polymorphic and variable (Figure 3.5D). The amino acid probability plot is consistent with the sequences I obtained from our dataset of 339 primary Nef clones for all four subtypes, and thus explains why SERINC3 downregulation activity is conserved in subtypes A, C and D but poorly conserved for subtype B. Lastly, I also wanted to confirm that the S8R and I11G mutations in proviral NL4-3 impairs infectivity enhancement when viruses are produced in HEK293T +pSERINC3 cells. ∆Nef HIV-1 viruses were 4-fold less infectious than WT HIV-1, whereas S8R and I11G HIV-1 viruses were 3.3-fold and 4.4-fold less infectious, respectively (Figure 3.5E).

58

Figure 3.5. Polymorphisms at Nef codons 8 and 11 selectively impair SERINC3 downregulation (A) Relative CD4 (blue), HLA-I (green), SERINC5 (purple) and SERINC3 (black) downregulation activity (y-axis) of NL4-3 Nef mutants S8R and I11G normalized to WT Nef (100%). Downregulation activity was assessed by flow cytometry following transfection of a CEM T-cell line as described in the Methods. Statistically significant differences compared to WT or parental Nef are indicated by asterisks, * (p<0.05) ** (p<0.01) or *** (p<0.001) (unpaired t-test). (B) Relative SERINC3 downregulation activity (y-axis) of a single representative patient-derived Nef clone (Parental) from subtype A (red), subtype C (green) and subtype D (blue) and their respective mutants at positions 8 and 11. (C) Relative SERINC3 downregulation activity (y-axis) of a representative patient-derived subtype B Nef clone and its respective reversion mutations at positions 8 and 11. (D) Amino acid probability plot of Nef codons 8 and 11 composed from Nef sequences (N>3000) downloaded from the Los Alamos database for subtypes A, B, C and D. (E) Relative infectivity (y-axis) of viruses with S8R and I11G mutations in the proviral NL4-3 plasmid when produced in HEK293T cells and exogenous SERINC3. Unpaired t-test was used to determine statistically significant differences from the infectivity of WT HIV-1 virus.

59 3.4.5. Nef codons 8 and 11 are critical for co-localization with SERINC3

Since point mutations at positions 8 and 11 in Nef selectively impaired downregulation and antagonism of SERINC3 but had a negligible impact on downregulation of CD4, HLA-I or SERINC5 molecules (Figure 3.5A), it is plausible that these two residues are critical sites for co-localizing with SERINC3. To test this, I employed proximity ligase assay (PLA) technology to quantify Nef-SERINC3(iHA) co- localization in vitro by flow cytometry. In PLA, the proximity of two primary target antibodies (i.e. anti-Nef and anti-HA) produces a fluorescent signal when the two proteins of interest are less than 40 nm apart in distance [37], thus providing greater sensitivity than analogous assays such as bimolecular fluorescence complementation without the need for genetic modification. There was a strong 5.5-fold induction of PLA signal with WT Nef and SERINC3 in comparison to the absence of Nef (Figure 3.6A). While there was a noticeable Nef-SERINC3 PLA signal for S8R and I11G Nef, they were significantly weaker (59% and 28% normalized PLA signal, respectively) relative to WT Nef (Figure 3.6B). The PLA results mirror the SERINC3 downregulation data observed for these mutations (Figure 3.5A), suggesting that impaired activity in S8R and I11G Nef could be largely due to a lack of direct contact and co-localization with SERINC3. Lastly, to rule out that our PLA results were not confounded by weaker antibody recognition or protein expression of S8R and I11G Nef, I performed a Western blot stained with the same Nef antibody to assess the steady-state expression of these mutants. Both S8R and I11G Nef were readily detected by Western blot (Figure 3.6C), confirming that impaired Nef-SERINC3 co-localization and downregulation activity is not influenced by weaker protein expression.

60 Figure 3.6. Characterization of Nef-SERINC3 co-localization using proximity ligase assay (A) Flow cytometry histogram plots of proximity ligase assay (PLA) signal from Nef and SERINC3(iHA). PLA was performed on CEM T-cells following co-transfection of Nef and SERINC3(iHA) plasmids, where cells were fixed and intracellularly stained with anti-Nef and anti- HA antibodies, followed by a secondary antibody stain containing PLA probes that were detected and measured by flow cytometry. (B) Normalized Nef and SERINC3 co-localization relative to WT Nef as measured by PLA. (C) Steady-state protein expression for all Nef mutants determined by Western blot using the same anti-Nef antibody used in the PLA experiment (Figure 3.6A and 3.6B), compared to cellular β-actin controls (lower blot).

3.5. Discussion

To date, this study represents the most comprehensive characterization of SERINC3 and SERINC5 downregulation and antagonism activity by primary HIV-1 Nef alleles (N=339) across the major circulating subtypes A, B, C and D. I demonstrated substantial and significant differences in SERINC downregulation abilities between subtypes. Primary Nef clones from subtype A and C displayed the poorest ability to downregulate SERINC5 compared to those from subtype D, suggesting that a relatively attenuated ability to counteract SERINC5 may contribute to decreased pathogenesis in subtype A or C infection, as observed in regions where these subtypes co-circulate [29]. In an analysis where both SERINC3 and SERINC5 downregulation abilities were taken into consideration, subtype D Nef clones in our study were the most versatile and efficient at counteracting both restriction factors. Over 51% of subtype D clones had above median activity for downregulation of both SERINC3 and SERINC5, while only 35% of subtype C and 26% of subtype A Nef clones fell into this category (data not shown). Conversely, 41% of subtype A clones had below median activity for downregulation of both SERINC3 and SERINC5, whereas only 35% of subtype C and 26% subtype D clones fell into this category. Although SERINC3 downregulation activity did not correlate significantly with the infectivity of viruses produced in HEK293T+pSERINC3/5 cells (Figure 3.4; left plot), statistical significance was achieved when subtype B samples were excluded from the analysis (r=0.37, p=0.002; data not shown). Therefore, the subtype ranking D > C > A for Nef-mediated SERINC downregulation activity in our study supports previous reports that showed subtype A may be the least virulent strain among the three subtypes.

Another significant finding from this study is that most subtype B Nef clones were unable to counteract SERINC3, while this function was conserved in most subtype A, C

61 and D Nef clones. This key difference was explained by two amino acid differences at codons 8 and 11 of Nef, where S8 and V11 were required for SERINC3 co-localization and downregulation. While I showed that impairments in SERINC3 downregulation was due in part to decreased co-localization with Nef, further experiments are needed to elucidate the precise mechanism. One possibility is that codons 8 and 11 of Nef are direct interaction sites with SERINC3 and this could be validated with fluorescence resonance energy transfer (FRET) experiments complemented with microscopy. In addition, because codons 8 and 11 are in the N-terminal arm of Nef, which is important for membrane localization, another possibility is that polymorphisms at these codons alter the localization of Nef to different regions of the lipid membrane, away from regions where SERINC3 proteins may typically be found.

Interestingly, the consensus residues in subtype B were R8 and G11, rendering most alleles from this subtype attenuated for this function (Figure 3.2C). The reason for this phenomenon was not explored in this study, however, a few speculations can be made. Since S8R and I11G did not confer an increase in activity for CD4, HLA-I or SERINC5 downregulation, the mechanism is likely not explained by functional compensatory measures. Since SERINC3 downregulation activity is preserved in subtypes A, C and D that mainly circulates in sub-Saharan Africa suggests that there may be some type of evolutionary host pressure on Nef to retain this function in those populations. For example, there could be variation in single nucleotide polymorphisms (SNPs) or copy-number variation in the SERINC3 gene potentially resulting in a different expression profile in patients of African ancestry. If the gene expression profile is indeed different in those from African ancestry compared to those from European ethnicities, it could also partly explain why subtype B HIV-1 may not be prevalent in African regions, due to the inability of Nef to efficiently antagonize the effects of SERINC3 in the African populations, while this activity could be dispensable in regions populated with individuals of European ancestry. There is currently some evidence that endogenous SERINC gene expression in primary PBMC varies widely between donors [38], and therefore warrants future studies to further investigate the potential impact of variation in SERINC3 expression in patients on viral genetics and function.

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64 23. Trautz, B., V. Pierini, R. Wombacher, B. Stolp, A.J. Chase, M. Pizzato, and O.T. Fackler, The Antagonism of HIV-1 Nef to SERINC5 Particle Infectivity Restriction Involves the Counteraction of Virion-Associated Pools of the Restriction Factor. J Virol, 2016. 90(23): p. 10915-10927.

24. Mann, J.K., H. Byakwaga, X.T. Kuang, A.Q. Le, C.J. Brumme, P. Mwimanzi, S. Omarjee, E. Martin, G.Q. Lee, B. Baraki, R. Danroth, R. McCloskey, C. Muzoora, D.R. Bangsberg, P.W. Hunt, P.J. Goulder, B.D. Walker, P.R. Harrigan, J.N. Martin, T. Ndung'u, M.A. Brockman, and Z.L. Brumme, Ability of HIV-1 Nef to downregulate CD4 and HLA class I differs among viral subtypes. Retrovirology, 2013. 10: p. 100.

25. Jubier-Maurin, V., S. Saragosti, J.L. Perret, E. Mpoudi, E. Esu-Williams, C. Mulanga, F. Liegeois, M. Ekwalanga, E. Delaporte, and M. Peeters, Genetic characterization of the nef gene from human immunodeficiency virus type 1 group M strains representing genetic subtypes A, B, C, E, F, G, and H. AIDS Res Hum Retroviruses, 1999. 15(1): p. 23-32.

26. Omondi, F.H., S. Chandrarathna, S. Mujib, C.J. Brumme, S.W. Jin, H. Sudderuddin, R. Miller, A. Rahimi, O. Laeyendecker, P. Bonner, F.Y. Yue, E. Benko, C.M. Kovacs, M.A. Brockman, M. Ostrowski, and Z.L. Brumme, HIV subtype and Nef-mediated immune evasion function correlate with viral reservoir size in early-treated individuals. J Virol, 2019.

27. Kuritzkes, D.R., HIV-1 subtype as a determinant of disease progression. J Infect Dis, 2008. 197(5): p. 638-9.

28. Kanki, P.J., D.J. Hamel, J.L. Sankale, C. Hsieh, I. Thior, F. Barin, S.A. Woodcock, A. Gueye-Ndiaye, E. Zhang, M. Montano, T. Siby, R. Marlink, N.D. I, M.E. Essex, and M.B. S, Human immunodeficiency virus type 1 subtypes differ in disease progression. J Infect Dis, 1999. 179(1): p. 68-73.

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30. Kiwanuka, N., O. Laeyendecker, M. Robb, G. Kigozi, M. Arroyo, F. McCutchan, L.A. Eller, M. Eller, F. Makumbi, D. Birx, F. Wabwire-Mangen, D. Serwadda, N.K. Sewankambo, T.C. Quinn, M. Wawer, and R. Gray, Effect of human immunodeficiency virus Type 1 (HIV-1) subtype on disease progression in persons from Rakai, Uganda, with incident HIV-1 infection. J Infect Dis, 2008. 197(5): p. 707-13.

31. McPhee, E., M.K. Grabowski, R.H. Gray, A. Ndyanabo, J. Ssekasanvu, G. Kigozi, F. Makumbi, D. Serwadda, T.C. Quinn, and O. Laeyendecker, Short Communication: The Interaction of HIV Set Point Viral Load and Subtype on Disease Progression. AIDS Res Hum Retroviruses, 2019. 35(1): p. 49-51.

32. Kmiec, D., B. Akbil, S. Ananth, D. Hotter, K.M.J. Sparrer, C.M. Sturzel, B. Trautz, A. Ayouba, M. Peeters, Z. Yao, I. Stagljar, V. Passos, T. Zillinger, C. Goffinet, D.

65 Sauter, O.T. Fackler, and F. Kirchhoff, SIVcol Nef counteracts SERINC5 by promoting its proteasomal degradation but does not efficiently enhance HIV-1 replication in human CD4+ T cells and lymphoid tissue. PLoS Pathog, 2018. 14(8): p. e1007269.

33. Heigele, A., D. Kmiec, K. Regensburger, S. Langer, L. Peiffer, C.M. Sturzel, D. Sauter, M. Peeters, M. Pizzato, G.H. Learn, B.H. Hahn, and F. Kirchhoff, The Potency of Nef-Mediated SERINC5 Antagonism Correlates with the Prevalence of Primate Lentiviruses in the Wild. Cell Host Microbe, 2016. 20(3): p. 381-391.

34. Zhang, X., T. Zhou, J. Yang, Y. Lin, J. Shi, X. Zhang, D.A. Frabutt, X. Zeng, S. Li, P.J. Venta, and Y.H. Zheng, Identification of SERINC5-001 as the Predominant Spliced Isoform for HIV-1 Restriction. J Virol, 2017. 91(10).

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66 Chapter 4.

Thesis Summary

In October 2015, two breakthrough studies published in Nature elucidated that the long-sought mechanism for HIV-1 Nef’s ability to enhance virion infectivity was dependent on antagonizing the newly identified host restriction factors SERINC3 and SERINC5 [1, 2]. The SERINC proteins are incorporated into the lipid envelope of progeny virions and decrease HIV-1 infectivity by impairing fusion with target cells. Notably, this effect is counteracted by Nef, which internalizes SERINC3/5 from the cell surface, thus preventing its incorporation into virion particles [1, 2]. Given its recent discovery, little is known about the variability of this Nef function at the population-level or the types of naturally-occurring Nef polymorphisms that can modulate this activity, as most studies utilize laboratory-adapted viral strains such as NL4-3 [3-7]. Furthermore, the impact of Nef sequence variation on SERINC downregulation function has only been described for a small number of Nef mutations located in highly conserved residues that are almost never observed in natural isolates [4, 8]. My thesis addressed this knowledge gap by characterizing Nef-mediated SERINC downregulation in over 400 primary Nef alleles isolated from patients with distinct infection characteristics to better understand the role of this Nef function in HIV pathogenesis.

In this thesis, I have demonstrated that Nef-mediated SERINC downregulation activity varies widely among patient-derived alleles. In my first study, I compared the activity of Nef clones isolated from elite controllers (rare HIV infected individuals that spontaneously suppress their viral loads to undetectable levels in the absence of treatment) and chronic progressors. In this analysis, I observed that controller-derived Nef clones displayed significantly poorer ability to downregulate SERINC5 compared to progressor-derived Nef clones, suggesting that impairment for this function may contribute to the maintenance of low viral loads in some cases of natural HIV control. From this study, I also identified two CTL escape mutations in Nef, namely K94E and H116N [9], shown to impair SERINC5 downregulation activity, suggests that there are HLA-I restricted immune responses that can drive the virus to become less fit at the cost of escaping from CTL-mediated cell death. Notably, Nef H116N is selected in individuals who possess the protective HLA-B*57 allele, one of the well-established host genetic

67 determinants of slower disease progression in HIV infected individuals [9-11]. Therefore, certain host immune responses that selectively drive viral adaptation into an attenuate strain could be exploited in CTL-based vaccine or functional cure strategies.

In my second study, I characterized SERINC3 and SERINC5 downregulation activity in Nef clones isolated from a large panel of patients infected with one of the four major circulating group M HIV-1 subtypes (A, B, C or D). Since HIV subtype is also a potential determinant of disease progression [12, 13], I hypothesized that there may be differential Nef-mediated SERINC downregulation abilities between subtypes. Indeed, I observed a distinct subtype hierarchy, where subtype B Nef clones displayed the highest overall activity for SERINC5 downregulation, followed by subtype D, subtype C and lastly subtype A Nef clones. This result is consistent with prior reports demonstrating that clinical and disease outcomes in subtype A infection are typically more favorable compared to subtype D [12]. Therefore, functional differences in SERINC5 downregulation could contribute to the observed differences in pathogenesis between HIV-1 subtypes. Using the same clones, I observed that most subtype B Nef clones were unable to downregulate SERINC3, while this activity was maintained in most subtype A, C and D Nef clones. Linked sequence-function statistical analyses identified

S8 and I11 or V11 in Nef as key residues for modulating SERINC3, where mutations at these residues selectively impaired co-localization and downregulation of SERINC3, but not SERINC5, providing the first evidence that these activities are genetically separable.

In conclusion, this dissertation contains the most comprehensive study of SERINC downregulation activity using primary Nef alleles, providing greater insight into the sequence and functional properties of Nef strains that are derived from the current HIV/AIDS pandemic. Results from this thesis provide supporting evidence that HIV control or slower disease progression is associated with poorer ability to downregulate and counteract the SERINC restriction factors, and targeting this virus-host interaction could be exploited in novel therapeutic strategies. Lastly, by characterizing patient- derived Nef sequences, naturally-occurring polymorphisms that selectively impair SERINC3 or SERINC5 downregulation were identified and validated experimentally. Among the polymorphisms identified, some were known CTL escape mutations, while others may be novel crucial interaction residues between Nef and SERINC. All in all, this thesis highlights the importance and advantages of integrating patient-derived samples

68 alongside reference strains in research studies as it provides greater insight into the properties of circulating HIV-1 strains that we aim to combat.

69 4.1. References

1. Usami, Y., Y. Wu, and H.G. Gottlinger, SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef. Nature, 2015. 526(7572): p. 218-23.

2. Rosa, A., A. Chande, S. Ziglio, V. De Sanctis, R. Bertorelli, S.L. Goh, S.M. McCauley, A. Nowosielska, S.E. Antonarakis, J. Luban, F.A. Santoni, and M. Pizzato, HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation. Nature, 2015. 526(7572): p. 212-7.

3. Kmiec, D., B. Akbil, S. Ananth, D. Hotter, K.M.J. Sparrer, C.M. Sturzel, B. Trautz, A. Ayouba, M. Peeters, Z. Yao, I. Stagljar, V. Passos, T. Zillinger, C. Goffinet, D. Sauter, O.T. Fackler, and F. Kirchhoff, SIVcol Nef counteracts SERINC5 by promoting its proteasomal degradation but does not efficiently enhance HIV-1 replication in human CD4+ T cells and lymphoid tissue. PLoS Pathog, 2018. 14(8): p. e1007269.

4. Trautz, B., V. Pierini, R. Wombacher, B. Stolp, A.J. Chase, M. Pizzato, and O.T. Fackler, The Antagonism of HIV-1 Nef to SERINC5 Particle Infectivity Restriction Involves the Counteraction of Virion-Associated Pools of the Restriction Factor. J Virol, 2016. 90(23): p. 10915-10927.

5. Trautz, B., H. Wiedemann, C. Luchtenborg, V. Pierini, J. Kranich, B. Glass, H.G. Krausslich, T. Brocker, M. Pizzato, A. Ruggieri, B. Brugger, and O.T. Fackler, The host-cell restriction factor SERINC5 restricts HIV-1 infectivity without altering the lipid composition and organization of viral particles. J Biol Chem, 2017. 292(33): p. 13702-13713.

6. Heigele, A., D. Kmiec, K. Regensburger, S. Langer, L. Peiffer, C.M. Sturzel, D. Sauter, M. Peeters, M. Pizzato, G.H. Learn, B.H. Hahn, and F. Kirchhoff, The Potency of Nef-Mediated SERINC5 Antagonism Correlates with the Prevalence of Primate Lentiviruses in the Wild. Cell Host Microbe, 2016. 20(3): p. 381-391.

7. Zhang, X., T. Zhou, J. Yang, Y. Lin, J. Shi, X. Zhang, D.A. Frabutt, X. Zeng, S. Li, P.J. Venta, and Y.H. Zheng, Identification of SERINC5-001 as the Predominant Spliced Isoform for HIV-1 Restriction. J Virol, 2017. 91(10).

8. O'Neill, E., L.S. Kuo, J.F. Krisko, D.R. Tomchick, J.V. Garcia, and J.L. Foster, Dynamic evolution of the human immunodeficiency virus type 1 pathogenic factor, Nef. J Virol, 2006. 80(3): p. 1311-20.

9. Brumme, Z.L., M. John, J.M. Carlson, C.J. Brumme, D. Chan, M.A. Brockman, L.C. Swenson, I. Tao, S. Szeto, P. Rosato, J. Sela, C.M. Kadie, N. Frahm, C. Brander, D.W. Haas, S.A. Riddler, R. Haubrich, B.D. Walker, P.R. Harrigan, D. Heckerman, and S. Mallal, HLA-associated immune escape pathways in HIV-1 subtype B Gag, Pol and Nef proteins. PLoS One, 2009. 4(8): p. e6687.

10. Altfeld, M., M.M. Addo, E.S. Rosenberg, F.M. Hecht, P.K. Lee, M. Vogel, X.G. Yu, R. Draenert, M.N. Johnston, D. Strick, T.M. Allen, M.E. Feeney, J.O. Kahn, R.P. Sekaly, J.A. Levy, J.K. Rockstroh, P.J. Goulder, and B.D. Walker, Influence

70 of HLA-B57 on clinical presentation and viral control during acute HIV-1 infection. AIDS, 2003. 17(18): p. 2581-91.

11. Navis, M., I. Schellens, D. van Baarle, J. Borghans, P. van Swieten, F. Miedema, N. Kootstra, and H. Schuitemaker, Viral replication capacity as a correlate of HLA B57/B5801-associated nonprogressive HIV-1 infection. J Immunol, 2007. 179(5): p. 3133-43.

12. Baeten, J.M., B. Chohan, L. Lavreys, V. Chohan, R.S. McClelland, L. Certain, K. Mandaliya, W. Jaoko, and J. Overbaugh, HIV-1 subtype D infection is associated with faster disease progression than subtype A in spite of similar plasma HIV-1 loads. J Infect Dis, 2007. 195(8): p. 1177-80.

13. Kuritzkes, D.R., HIV-1 subtype as a determinant of disease progression. J Infect Dis, 2008. 197(5): p. 638-9.

71 Appendix A.

Extended Tables

Table A.3.1. Extended list of Nef polymorphisms associated with differential SERINC5 downregulation in HIV-1 subtypes A, B, C and D (p<0.05)

Relative Nef Number of Impact Amino activity (%) subjects p – q – Subtype Codon Acid value value With Without N N AA AA (With) (Without) A 180 T 76.5 90.0 54 38 -14 0.01 0.44 A 180 V 90.0 76.5 38 54 +14 0.01 0.44 A 60 E 45.6 85.3 4 88 -40 0.01 0.44 A 168 V 49.8 85.3 4 88 -36 0.01 0.44 A 20 L 54.0 86.1 8 84 -32 0.02 0.44 A 15 K 57.5 85.3 5 87 -28 0.02 0.44 A 149 E 74.5 88.9 16 76 -14 0.02 0.44 A 55 N 57.3 85.3 6 86 -28 0.03 0.44 A 194 V 51.4 85.3 5 87 -34 0.03 0.44 A 60 A 85.3 51.4 87 5 +34 0.03 0.44 A 157 N 76.2 88.9 32 60 -13 0.04 0.44 A 157 T 88.9 76.2 60 32 +13 0.04 0.44 A 184 R 71.6 88.5 15 77 -17 0.04 0.44 A 24 A 97.7 83.2 7 82 +15 0.04 0.44 A 15 E 86.1 67.5 78 14 +19 0.04 0.44 A 202 F 89.4 76.7 57 35 +13 0.04 0.44 A 194 R 89.7 77.0 52 40 +13 0.04 0.44 A 51 T 94.4 80.1 8 84 +14 0.05 0.44 A 155 K 79.5 94.4 72 20 -15 0.05 0.44 B 94 K 110.1 96.0 77 14 +14 0.00 0.02 B 61 Q 109.2 89.9 86 5 +19 0.01 0.20 B 61 Y 82.3 108.3 3 88 -26 0.01 0.20 B 94 E 82.3 109.2 7 84 -27 0.01 0.20 B 120 F 84.4 108.9 6 85 -25 0.01 0.20 B 120 Y 108.9 84.4 85 6 +25 0.01 0.20

72 B 158 K 100.0 109.8 17 74 -10 0.01 0.20 B 169 N 115.2 107.0 10 81 +8 0.01 0.20 B 197 E 108.3 75.8 88 3 +32 0.01 0.20 B 14 S 100.7 109.5 14 77 -9 0.02 0.24 B 157 N 106.1 115.2 74 16 -9 0.02 0.24 B 9 K 99.4 109.5 9 78 -10 0.04 0.27 B 10 A 92.4 108.6 9 68 -16 0.04 0.27 B 14 N 129.7 107.6 3 88 +22 0.04 0.27 B 21 R 110.1 102.0 68 23 +8 0.03 0.27 B 54 D 110.8 102.2 60 31 +9 0.04 0.27 B 63 E 109.5 100.7 63 28 +9 0.03 0.27 B 80 T 108.3 51.6 88 3 +57 0.03 0.27 B 81 F 100.8 109.2 9 82 -8 0.04 0.27 B 168 M 103.8 113.3 56 35 -10 0.04 0.27 B 198 M 115.8 107.3 7 84 +9 0.04 0.27 C 32 A 87.3 100.5 64 7 -13 0.01 0.27 C 98 D 82.1 95.0 34 37 -13 0.01 0.27 C 98 E 95.0 82.1 37 34 +13 0.01 0.27 C 88 G 70.3 91.4 15 56 -21 0.01 0.27 C 88 S 91.4 70.3 56 15 +21 0.01 0.27 C 3 G 93.6 74.8 48 23 +19 0.01 0.27 C 45 S 86.1 96.6 42 29 -11 0.01 0.36 C 32 T 101.2 88.3 3 68 +13 0.02 0.36 C 3 N 61.7 90.5 6 65 -29 0.02 0.36 C 85 V 96.8 86.7 8 63 +10 0.02 0.36 C 184 K 93.3 84.8 37 34 +9 0.04 0.36 C 159 E 55.9 90.5 3 68 -35 0.04 0.36 C 159 G 90.5 55.9 68 3 +35 0.04 0.36 C 76 V 99.6 88.3 3 68 +11 0.04 0.36 C 104 K 85.9 95.8 53 18 -10 0.04 0.36 C 151 S 77.2 91.4 10 61 -14 0.04 0.36 C 85 F 86.7 93.6 59 12 -7 0.04 0.36 C 48 T 90.5 62.7 68 3 +28 0.04 0.36 C 50 P 62.7 90.5 3 68 -28 0.04 0.36 C 142 C 91.0 62.7 64 7 +28 0.04 0.36

73 C 142 P 62.7 91.0 7 64 -28 0.04 0.36 C 39 R 77.7 90.5 8 63 -13 0.05 0.36 C 15 E 74.4 91.4 14 57 -17 0.05 0.36 D 114 V 100.5 64.2 73 12 +36 0.00 0.69 D 57 W 100.5 51.3 81 4 +49 0.01 0.76 D 23 A 55.7 99.7 7 78 -44 0.02 0.76 D 114 I 76.0 100.5 10 75 -25 0.02 0.76 D 176 E 47.5 99.7 3 82 -52 0.03 0.76 D 23 T 99.7 82.7 76 9 +17 0.03 0.76 D 24 D 103.0 95.0 26 59 +8 0.03 0.76 D 104 K 92.3 100.5 15 70 -8 0.04 0.76 D 173 I 104.5 97.4 13 72 +7 0.04 0.76 D 104 P 103.0 97.4 5 80 +6 0.04 0.76

74 Table A.3.2. Extended list of Nef polymorphisms associated with differential SERINC5 downregulation in HIV-1 subtypes A, B, C and D (p<0.05) Relative Nef Number of Impact Amino activity (%) subjects p – q – Subtype Codon Acid value value With Without N N AA AA (With) (Without) A 11 V 95.9 36.6 72 16 +59 1.2E-06 0.00 A 8 S 95.7 39.8 73 17 +56 4.1E-06 0.00 A 10 I 96.8 48.1 65 23 +49 9.2E-06 0.00 A 10 K 35.65 93.75 10 78 -58 5.3E-05 0.00 A 11 A 31.45 91.85 6 82 -60 3.0E-04 0.01 A 9 S 92.85 39.8 80 7 +53 1.9E-03 0.06 A 14 P 91.85 40.75 82 10 +51 1.9E-03 0.06 A 8 R 41.7 92.6 9 81 -51 2.2E-03 0.06 A 3 N 41.75 88.5 8 84 -47 2.6E-03 0.07 A 48 V 49.4 93.1 17 75 -44 3.0E-03 0.07 A 8 N 37.25 88.5 6 84 -51 3.8E-03 0.07 A 180 T 69.65 98.65 54 38 -29 4.1E-03 0.07 A 180 V 98.65 69.65 38 54 +29 4.1E-03 0.07 A 56 T 58.45 93.1 16 75 -35 4.6E-03 0.07 A 56 V 97.2 71 33 58 +26 5.1E-03 0.07 A 178 G 46.25 88.5 10 82 -42 5.3E-03 0.07 A 22 Q 66 94.4 31 61 -28 5.9E-03 0.07 A 12 G 85.9 28.9 85 5 +57 6.0E-03 0.07 A 188 H 98.15 71 22 70 +27 6.3E-03 0.07 A 28 S 28.9 84.25 3 88 -55 9.9E-03 0.11 A 35 R 101.1 78.1 6 86 +23 1.6E-02 0.16 A 182 M 69.9 96.8 43 49 -27 1.7E-02 0.16 A 173 V 34 84.6 7 85 -51 1.7E-02 0.16 A 19 K 39.8 84.6 5 87 -45 2.1E-02 0.19 A 19 R 84.6 39.8 87 5 +45 2.1E-02 0.19 A 184 Q 100.9 77.1 11 81 +24 2.4E-02 0.21 A 23 T 69.9 96.3 47 45 -26 2.5E-02 0.21 A 71 K 100 77.55 14 78 +22 2.8E-02 0.22 A 23 A 96.3 69.9 43 49 +26 3.0E-02 0.22 A 105 R 98.7 76.3 21 71 +22 3.0E-02 0.22 A 9 R 45.6 91.85 5 82 -46 3.1E-02 0.22

75 A 71 R 77.55 100 76 16 -22 3.1E-02 0.22 A 188 R 69.9 96.8 49 43 -27 3.2E-02 0.22 A 22 R 91.85 67.05 48 44 +25 4.0E-02 0.26 A 173 M 84.25 45.9 78 14 +38 4.2E-02 0.27 A 45 N 100.6 78.2 5 87 +22 5.0E-02 0.31 B 11 V 93.7 43 30 47 +51 9.4E-07 0.00 B 8 S 92.1 50.65 37 46 +41 5.8E-06 0.00 B 15 A 69.55 35.8 52 39 +34 2.0E-04 0.01 B 14 P 71.4 39.05 57 34 +32 9.4E-04 0.04 B 28 E 32.3 70.05 31 60 -38 1.1E-03 0.04 B 9 S 69.4 38.5 63 24 +31 1.1E-03 0.04 B 161 N 52.6 95.7 84 7 -43 2.8E-03 0.08 B 8 R 51.45 76.4 28 55 -25 3.8E-03 0.10 B 188 S 100.4 52.6 7 84 +48 4.9E-03 0.11 B 28 V 94 52.25 9 82 +42 5.5E-03 0.11 B 15 T 36.35 58.4 22 69 -22 8.1E-03 0.15 B 148 L 87.45 51 16 75 +36 1.0E-02 0.16 B 182 E 93.55 52.6 6 85 +41 1.1E-02 0.16 B 12 G 57.7 22.9 80 10 +35 1.1E-02 0.16 B 11 S 35.8 68.9 7 70 -33 1.2E-02 0.16 B 21 K 87.45 52.6 12 79 +35 1.9E-02 0.24 B 11 P 2.3 60.45 3 74 -58 2.4E-02 0.29 B 10 I 96.4 54.1 9 68 +42 2.8E-02 0.32 B 148 V 51.9 79.05 73 18 -27 3.2E-02 0.32 B 12 D 10.7 55.6 3 87 -45 3.4E-02 0.32 B 163 T 96.4 52.95 3 88 +43 3.4E-02 0.32 B 24 E 55.6 27.5 83 8 +28 3.5E-02 0.32 B 9 K 26.5 57.7 9 78 -31 3.5E-02 0.32 B 168 V 94.4 52.6 4 87 +42 3.8E-02 0.33 B 11 I 94.4 55.6 8 69 +39 4.2E-02 0.34 B 14 A 30.55 55.6 8 83 -25 4.4E-02 0.34 B 98 D 25.3 55.6 6 85 -30 4.6E-02 0.34 B 178 R 79.6 52.6 21 70 +27 4.6E-02 0.34 B 43 I 52.95 84 78 13 -31 4.8E-02 0.34 C 11 V 90.6 48.2 52 14 +42 1.1E-03 0.13 C 49 A 66.2 94 40 31 -28 5.3E-03 0.23

76 C 40 Y 93.1 67.05 31 40 +26 5.8E-03 0.23 C 49 P 95.9 76.2 11 60 +20 1.1E-02 0.23 C 5 W 87.2 39.45 65 6 +48 1.3E-02 0.23 C 8 R 46.8 87.7 5 63 -41 1.6E-02 0.23 C 9 S 87.7 51.9 63 5 +36 1.9E-02 0.23 C 12 G 87.45 42.7 64 4 +45 2.0E-02 0.23 C 188 S 73.6 95.9 56 15 -22 2.0E-02 0.23 C 151 K 96 74.2 12 59 +22 2.5E-02 0.23 C 40 H 70.2 90.3 38 33 -20 2.5E-02 0.23 C 85 F 78.2 98 59 12 -20 2.8E-02 0.23 C 12 E 35.8 87.2 3 65 -51 2.9E-02 0.23 C 32 A 78.3 98.2 64 7 -20 3.1E-02 0.23 C 85 V 98.15 78.4 8 63 +20 3.2E-02 0.23 C 14 P 85.4 37.45 67 4 +48 3.3E-02 0.23 C 24 A 34.5 85.4 4 67 -51 3.5E-02 0.23 C 11 A 49.6 88.15 4 62 -39 3.7E-02 0.23 C 32 T 98.2 78.6 3 68 +20 3.8E-02 0.23 C 158 E 88.15 67.9 26 45 +20 4.0E-02 0.23 C 161 D 61.9 87.7 12 59 -26 4.2E-02 0.23 C 161 N 87.7 61.9 59 12 +26 4.2E-02 0.23 C 10 K 49.6 88.15 4 62 -39 4.4E-02 0.23 D 8 S 93.9 67.2 69 13 +27 1.6E-04 0.04 D 100 I 103.5 86.4 4 81 +17 2.8E-03 0.20 D 14 P 89.75 37.9 80 5 +52 2.9E-03 0.20 D 64 E 83 95.1 64 21 -12 6.1E-03 0.20 D 8 R 70 92.55 10 72 -23 6.5E-03 0.20 D 14 S 36.7 89.3 4 81 -53 6.8E-03 0.20 D 40 Y 102.6 86.4 4 81 +16 7.0E-03 0.20 D 197 E 65.5 90.7 14 71 -25 8.0E-03 0.20 D 23 T 90.45 66.3 76 9 +24 8.1E-03 0.20 D 9 S 92.3 63.8 75 5 +29 8.7E-03 0.20 D 38 A 52 90.2 6 79 -38 1.1E-02 0.22 D 197 V 96.9 86 11 74 +11 1.3E-02 0.24 D 64 D 95.05 84 20 65 +11 1.7E-02 0.30 D 3 N 50.55 90.2 8 77 -40 2.0E-02 0.33 D 28 A 26.8 89.2 3 82 -62 2.2E-02 0.33

77 D 104 K 71.9 91.5 15 70 -20 2.5E-02 0.36 D 23 A 66.3 89.75 7 78 -23 3.0E-02 0.39 D 24 D 95.3 83.4 26 59 +12 3.5E-02 0.44 D 176 E 63.8 89.2 3 82 -25 3.8E-02 0.45 D 100 L 86.6 100.45 79 6 -14 4.0E-02 0.45

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