The Antiviral Mechanism of SERINC5 on HIV-1 Infection

Austin Blake Featherstone

Dissertation

Submitted to the Faculty of the

Graduate School of Vanderbilt University in

partial fulfillment of the requirements for the

degree of

DOCTOR OF PHILOSOPHY

Microbiology and Immunology

December 12, 2020

Nashville, Tennessee

Approved:

Ivelin Georgiev, Ph.D.

Mark Denison, M.D.

Suman Das, Ph.D.

Todd Graham, Ph.D.

Fernando Villalta, Ph.D.

DEDICATION

I would like to dedicate this section to my family, who has supported me through each step of the way while working towards getting my Ph.D. I would also like to thank my wife, Carrie, who has been alongside me throughout the entire journey. And to my

Grandma and Grandpa Wolf, my Great-Grandmother (Nana) and to my Great Aunt

Mary, who in her dying words, told me to continue to go straight and to shoot for the stars.

ACKNOWLEDGMENTS

The road to the graduate degree is a long and tumultuous road. It takes a village of people to help support a Ph.D. candidate to earn their Ph.D. I first and foremost must thank my research mentor, Chris Aiken, for all of the time and mentorship that he has given me over the past five years while working in his lab. He has taught me to think critically about my data and what the data means. He has also taught me to use the scientific method in my research and to follow the scientific method in order to generate new questions to answer for my thesis project. Lastly, as a fellow Christian, Chris and I have had many discussions about our faith and The Word of God. I greatly appreciate those discussions and will reflect back on them in the future. I also want to the wonderful members of the Aiken lab for all of their support over the past five years. I want to give a special thank you to Jing Zhou, Ph.D., who seemed to always know where everything was at in the lab and was always there if I ever needed anything. And to my “lab mom”, Jiong Shi, M.S., for always being there to talk to and help with anything that I needed. Lastly to my colleagues who left before me, Janani Varadarajan,

Ph.D., Upul Halambage, Ph.D., Fatima Nawaz, Ph.D., Mallori Burse, Ph.D., Jordan

Anderson-Daniels, Ph.D., and Stephanie Lamb, Ph.D.

I also have to thank my thesis committee for all of their time, dedication, helping me to think critically of my research and continually pushing me to be a better scientist.

My thesis committee includes Ivelin Georgiev, Ph.D. (chair), Mark Denison, M.D.,

Suman Das, Ph.D., Todd Graham, Ph.D., and Fernando Villalta, Ph.D. I also need to recognize Meharry Medical College and Dr. Maria Lima, Ph.D for allowing me to be a part of their Molecular Pathogenesis Training Program at Meharry Medical College from

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2017-2019. Without their funding, the research conducted in my thesis project would not have been possible. I would also like to recognize the Initiative for Maximizing Student

Diversity (IMSD) at Vanderbilt University and its leaders, Roger Chalkley, Ph.D., Linda

Chalkley, Ph.D., and Christina Keeton, Ph.D. The IMSD was a wonderful organization to be apart of over the past six years and I want to thank all of them for their hard work in helping me become a better scientist and to meet other scientists from under- represented backgrounds.

Lastly, I would like to thank my core group of friends who I started this journey with over the past six years. We would meet up with on a weekly basis for game nights and for unwinding after long days in the lab. These include Kalen Petersen, Ph.D.,

Kristin Petersen, M.S., Matthew Kent, Ph.D., and Ben Kessler, M.S. I also have to thank my church family at Emmanuel Lutheran Church in Hermitage, TN who became my family away from home. I’ve spent a lot of time over the past six years with this family growing my faith, teaching and outreaching to people in need. I am truly grateful for each and every person who has helped me along this journey and I want to let you know that I love each and every one of you.

TABLE OF CONTENTS

Page

DEDICATION…………………………………………………………………………………... ii

ACKNOWLEDGMENTS ……………………………………………………………………... iii

LIST OF TABLES …………………………………………………………………………….. vii

LIST OF FIGURES ………………………………………………………………………….. viii

LIST OF ABBREVIATIONS………………………………………………………………….. ix

Chapter

1. Background and Research Goals ……………………………………………………...... 1

HIV & AIDS …………………………………………………………………………………. 1 HIV-1 Pathogenesis ……………………………………………………………………….. 2 HIV-1 Structure …………………………………………………………………………….. 5 HIV-1 Fusion ...……………………………………………………………………………... 7 HIV-1 Replication Cycle …………………………………………………………………… 9 Host-Restriction Factors …………………………………………………………………. 13 Goals Of My Thesis Work ..……………………………………………………………… 17

2. SERINC5 Inhibits HIV-1 Infectivity by Altering the Conformation of gp120 on HIV-1 Particles ………………………………………………………………………………………. 19

Introduction ………………………………………………………………………………... 19 Results ……………………………………………………………………………………... 21 SERINC5 reduces the infectivity and the capture of NL4-3 Nef- virus to gp120- specific epitope antibodies …………………………………………………………….. 21 SERINC5 does not affect the antibody-dependent capture of HIV-1 particles bearing a SERINC5-resistant Env protein …………………………………………… 25 SERINC5 does not inhibit capture of HIV-1 particles by CD4 protein ……………. 27

SERINC5 does not prevent HIV-1 Env from undergoing a conformational change upon binding to CD4 …………………………………………………………………… 29 SERINC5’s antiviral activity is correlated with its ability to inhibit virus capture … 31 SERINC5 perturbs the trimer conformation of HIV-1 Env …………………………. 33 Nef prevents the conformational effects of SERINC5 on virion-associated gp120 33 SERINC2 does not inhibit the infectivity and the capture of Nef-deficient HIV-1 .. 36 SERINC5 does not grossly affect Env biosynthesis, processing, or glycosylation 38 Discussion ………………………………………………………………………………... 40

3. Unpublished Results on SERINC5 …………………………………………………….. 44

SERINC5 Retention to the ER Inhibits the Antiviral Activity of SERINC5 on HIV-1 Infectivity ………………………………………………………………………………….. 44

4. Summary and Future Directions ……………………………………………………….. 48

Importance ……………………………………………………………………………….. 58

5. Materials and Methods ………………………………………………………………….. 59

Plasmids ………………………………………………………………………………….. 59 Antibodies and Recombinant Proteins ………………………………………………... 59 Cells and Viruses ………………………………………………………………………... 60 Assay of HIV-1 Infectivity ……………………………………………………………….. 61 Virus Capture Assays …………………………………………………………………… 61 CD4-Virus Capture Assays …………………………………………………………….. 62 Pulse-Chase Analysis of Env Biosynthesis and Processing ……………………….. 64

REFERENCES ……………………………………………………………………………... 66

LIST OF TABLES

Table Page

4-1 Antibodies and Recombinant Proteins Used in Study ………………………………. 63

vii

LIST OF FIGURES

Figure Page

1-1 HIV-1 time course towards the progression of AIDS …………………………………. 4

1-2 Mature HIV-1 structure …………………………………………………………………... 6

1-3 Steps of HIV-1 fusion …………………………………………………………………….. 8

1-4 The HIV-1 replication cycle …………………………………………………………….. 12

1-5 HIV-1 restriction factors ………………………………………………………………… 16

2-1 SERINC5 reduces the infectivity and the capture of NL4-3 Nef- virus to gp120- specific epitope antibodies ………………………………………………………………….. 24 2-2 SERINC5 does not affect the antibody-dependent capture of HIV-1 particles bearing a SERINC5-resistant Env protein ………………………………………………………….. 26 2-3 SERINC5 does not inhibit capture of HIV-1 particles by CD4 protein …………….. 28 2-4 SERINC5 does not prevent HIV-1 Env from undergoing a conformational change upon binding to CD4 ………………………………………………………………………… 30 2-5 SERINC5’s antiviral activity is correlated with its ability to inhibit virus capture …. 32

2-6 SERINC5 perturbs the trimer conformation of HIV-1 Env ………………………….. 34

2-7 Nef prevents the conformational effects of SERINC5 on virion-associated gp120. 35

2-8 SERINC2 does not inhibit the infectivity and the capture of Nef-deficient HIV-1 ... 37

2-9 SERINC5 does not grossly affect Env biosynthesis, processing, or glycosylation. 39

3-1 The Antiviral Activity of SERINC5 is Inhibited When SERINC5 is Retained to the

Endoplasmic Reticulum …………………………………………………………………….. 46

3-2 SERINC5-KDEL is Retained from Being Incorporated into Virus Particles and is

Retained in the Cell …………………………………………………………………………. 47

viii

4-1 The Model for SERINC5 Inhibition of Infectivity …………………………………….. 57

LIST OF ABBREVIATIONS

AIDS acquired immune deficiency syndrome

ALU arbitrary light units

APOBEC3 apolipoprotein B messenger RNA (mRNA)-editing enzyme catalytic

polypeptide-like 3

ART antiretroviral therapy

CA capsid protein, p24

CCR5 C-C chemokine receptor type 5

CD4+ cellular protein marker on the surface of HIV-1 target cells

CXCR4 CX chemokine receptor 4

CypA Cyclophilin A

DMEM Dulbecco’s modified eagle’s medium

ELISA enzyme-linked immunosorbent assay

Env HIV-1 envelope glycoprotein

FBS fetal bovine serum

Gag group specific antigen

GALT gut associated lymphoid tissue gp41 envelope transmembrane glycoprotein

x gp120 envelope surface glycoprotein gp160 full-length, uncleaved envelope glycoprotein

HIV-1 human immunodeficiency virus type 1

IN integrase

IFITM interferon induced transmembrane protein

Kb kilobases

LTR long terminal repeat

MA matrix protein, p17

MLV murine leukemia virus

MxB myxovirus resistance protein 2

NaCl sodium chloride

NC nucleocapsid protein, p7

Nef negative factor

PAGE polyacrylamide gel electrophoresis

PBS phosphate-buffered saline

PEI polyethylenimine

RT reverse transcriptase

SDS sodium dodecyl sulfate

SERINC5 serine incorporator 5

SUN Sad1/UNC-84 domain-containing family of proteins

Tetherin bone marrow stromal cell antigen 2

TRIM5α tripartite-motif-containing 5α

Vif viral infectivity factor

ZAP zinc finger antiviral protein

xii

Chapter 1

Background and Research Goals

Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that reverse transcribes its single stranded RNA into double-stranded DNA. HIV-1 causes persistent infections in people which eventually cause acquired immune deficiency syndrome

(AIDS). There is currently no cure for HIV-1, therefore further research must be conducted on the virus to further understand key steps during HIV-1 replication which can be targeted for developing new therapeutics. In 2015, a new inhibitory cellular factor called SERINC5 was discovered to inhibit the early stages of HIV-1 infection; however the antiviral mechanism for SERINC5 is still not fully understood (1, 2). During my thesis research, I investigated the antiviral mechanism of SERINC5 and identified several epitopes on the HIV-1 envelope that SERINC5 affects. I was able to correlate these effects on HIV-1 Env to its inhibition of HIV-1 infectivity.

HIV & AIDS

HIV-1 is the cause of the worldwide pandemic known as acquired immune deficiency syndrome (AIDS). A novel retrovirus was first discovered to cause the disease known as AIDS in 1983 (3) and was confirmed by another lab in 1984 (4). In

1986, the virus was officially designated as human immunodeficiency virus (HIV). Since

its discovery in 1983, there is still no cure for the virus. As of 2018, 37.9 million people are infected around the world with HIV. As of 2018, there were an estimated 770,000 people who died from AIDS-related illnesses worldwide (5). Antiretroviral therapy (ART) is highly effective at inhibiting the virus. ART is a combination-drug therapy to target

HIV-1 particles during different stages of its life cycle. People who are infected with HIV-

1 can live with undetectable levels of HIV in their bloodstream if they adhere to taking

ART (6). Viral suppression caused by ART leads to partial recovery of the immune system and eliminates the risk for the person infected with HIV-1 to develop AIDS. If

HIV-1 replication is left untreated, it will infect and kill the CD4+ T-cells in the body, which results in an increased risk to secondary infections that can ultimately kill the person infected with HIV. In order to eradicate the HIV-1 pandemic, a better understanding of HIV-1 replication in necessary so new drugs and a vaccine can be developed to protect people from becoming infected by HIV-1.

HIV-1 Pathogenesis

HIV-1 infection starts through the sexual transmission of the virus across mucosal surfaces, by perinatal maternal-infant exposure, and by intravenous inoculation

(7). Once the virus has been transmitted, it replicates in cells in all of the lymphoid tissues that are around the site of exposure. HIV-1 preferentially infects T-cells that express high levels of the primary receptor, CD4, and that express the co-receptors

CXCR4 or CCR5. Trafficking of the virus is influenced through local inflammation or through the virus infecting dendritic cells through the interaction of the viral Env with the dendritic cell receptor, DC-SIGN (8, 9). Upon infection by the virus, T-cells in the gut associated lymphoid tissue (GALT) are significantly depleted. Early replication in these

T-cell subsets is followed by the movement of the virus to the proximal lymphoid organs and then to a systematic infection (10).

Viral pathogenesis and the time it takes to develop a persistent infection depends on each patient. However, disease progression can be categorized into three major phases: acute infection, an asymptomatic phase, and AIDS. Acute infection typically will last from days to weeks if left untreated. There is an initial loss of CD4+ T cells during the acute infection followed by a partial recovery and then a slow decay during the period of clinical latency. The asymptomatic phase can start weeks after the initial infection and can last years until there is a precipitous decline of CD4+ T cells which causes the development of AIDS. AIDS can last the life of the patient until they start antiviral treatment or until the patient dies. HIV-1 replication in the host is depicted in

Figure 1-1.

Progression of AIDS

Figure 1-1: HIV-1 Timecourse Towards the Progression of AIDS. The disease progression towards AIDS is divided into three stages. Stage 1 includes the acute stage of infection which lasts 0-9 weeks. During this time, viremia spikes which strongly reduces the CD4+ lymphocyte count. Stage 2 includes clinical latency, which can occur between 9 weeks post-infection and 8 years. During this stage, the HIV RNA copies per mL plasma slowly increase. The gradual increase of virus particles in the plasma slowly reduces the CD4+ T-cells over time. Stage 3 includes the development of AIDS. The gradual reduction of CD4+ T-cells leads to the development of AIDS, which allows opportunistic infections to occur, which usually leads to the death of the patient. Reproduced with permission from Fauci, A,. et al., 1996 (11), Copyright Massachusetts Medical Society.

HIV Structure

HIV-1 is grouped into the genus Lentiviridae, within the family of Retroviridae

(12). The HIV-1 genome consists of two identical single strands of RNA that are enclosed in a conical capsid that also include the viral enzymes reverse transcriptase

(RT) and integrase (IN) (see Figure 1-2). At the 5’ of the viral genome is gag, which encodes the structural proteins of the outer core membrane, matrix (MA, p17), the capsid protein (CA, p24), the nucleocapsid (NC, p7), and a smaller, nucleic acid- stabilizing protein (p6). Following gag in the viral genome is the viral gene, pol, which encodes several key viral enzymes. These include protease (PR, p10), reverse transcriptase (RT, p51), RNase H (p15), and integrase (IN, p32). Adjacent to the pol gene is the Env gene which encodes for the uncleaved Env protein, gp160. gp160 becomes cleaved by a cellular furin-like protease into the surface transmembrane glycoprotein, gp120, (SU, gp120) and the transmembrane glycoprotein, gp41, (TM, gp41)(13). The mature viral Env spike is a trimer of heterodimers on the surface of the

HIV-1 virion. There are approximately 14 trimers present on the surface of the virus(14,

15). In addition to the Env gene, the HIV-1 viral genome also encodes for several accessory proteins that are necessary for immune evasion. These include the transactivator protein, (tat, p14), RNA splicing regulator, (rev, p19), negative regulating factor, (nef, p27), viral infectivity protein, (vif, p23), virus protein r, (vpr, p15), and virus protein unique, (vpu, p16) (12).

Structure of Mature HIV-1

Figure 1-2: The Structure of the Mature HIV-1 Virus Particle. HIV-1 is a single- stranded, enveloped RNA Retrovirus. The HIV-1 genome consists of two identical single-strands of RNA that are enclosed within a protective fullerene cone structure. Approximately 14 Env trimers span the viral membrane as a trimer of heterodimers. Reproduced with permission from Campbell and Hope, 2015 (16), Copyright Springer Nature BV.

HIV-1 Fusion

HIV-1 fusion starts with binding of the mature virus spike to a susceptible target cell (17). The variable 1/2 (V1/V2) loop on HIV-1 gp120 interacts with the primary receptor, CD4, on T cells (18, 19). Once the V1/V2 loop on gp120 interacts with CD4, this causes a conformational change of the HIV-1 trimer, which allows for the V3 loop of gp120 to interact with the co-receptor, CX chemokine receptor 4 (CXCR4), or C-C chemokine receptor type 5 (CCR5) (20, 21). When gp120 binds to the receptor and the co-receptor, then the amino terminal (N-terminal) portion of gp41 is extended and inserts into the target-cell membrane. This tethers the viral and cell membranes together, allowing for the fusion peptide of each gp41 to bend at the hinge region and bring the two membrane together to fuse. This allows for the amino-terminal helical region (HR-N) and the carboxy-terminal helical region (HR-C) from each gp41 together to form the six-helix bundle (6HB) (22, 23). The formation of the 6HB is the driving force that brings the two membranes together which allows for the fusion pore to form (24).

Once the fusion pore forms, this allows the viral core to enter into the cell and begin trafficking (16). A summary of the process of HIV-1 fusion can be found in Figure 1-3.

Steps of HIV-1 Fusion

CD4 CXCR4/CCR5

Figure 1-3: The Steps of HIV-1 Fusion. HIV-1 Fusion occurs in four steps. Its starts with the attachment of the virus particle to the target cell through the interaction of the V1/V2 loop on HIV-1 gp120 with the primary receptor, CD4. This causes a conformational change in gp120 which allows for the V3 loop to interact with a co- receptor (CXCR4 or CCR5). Once this occurs, the fusion peptide inserts into the target- cell membrane and the refolding of gp41 brings the two membranes together and forms the 6HB. The formation of the 6HB allows for the fusion pore to form and for the HIV-1 core to be able to enter into the target cell and begin trafficking to the nucleus. Reproduced with permission from Rawi, R., et al. PLoS ONE, 2015 (20), Copyright Public Library of Science.

HIV-1 Replication Cycle

Upon entry into the target cell, the virus then undergoes seven processes to replicate: reverse transcription, trafficking to the nucleus, uncoating, entry into the nucleus, integration into the host chromatin, transcription, translation, assembly, viral budding, and maturation. Each of these steps is depicted in Figure 1-4. Once fusion occurs, the viral core begins to traffic towards the nucleus using microtubules and the motor proteins dynein and kinesin (25–29). While the viral core traffics to the nucleus, the virus undergoes reverse transcription. During reverse transcription, the viral single stranded RNA reverse transcribes through help from the viral protein, reverse transcriptase, to become double stranded DNA (dsDNA) (30–32). The dsDNA and the viral proteins inside of the viral core are also known as the pre-integration complex

(PIC). While the HIV-1 viral core traffics to the nucleus and undergoes reverse transcription, the virus core also undergoes a process called uncoating, which allows for the viral DNA (vDNA) to be able to enter into the nucleus and become incorporated into the host genome. There are currently several different models that suggest when uncoating takes place during the early stages of infection (33–39). The first model suggests that the HIV-1 capsid undergoes immediate uncoating when it enters into the cell (33, 34). There are significant issues with this model if the viral core is meant to protect the vRNA/DNA from host sensors or restriction factors. Other studies, including several imaging-based approaches, propose a model in which some of the core disassembles in the cytoplasm during the first hour post-entry, but a majority of the viral core remains associated with the reverse transcription complex (26, 35). A major issue with this model is how the viral core would stay associated if some of the CA monomers

9 disassociate from the viral core? A third model for viral uncoating supports the idea that the viral core remains intact until it docks to the nuclear pore complex, which would allow the virus to protect its replicating genome from cytosolic DNA sensors (40–42).

Issues of timing and discordance with other studies require that this model may need to be further studied. Validation of all three of these models will require further investigation in order to determine which model is correct or in order to postulate a new model that more accurately describes HIV-1 uncoating.

Once the virus undergoes reverse transcription and uncoating, then the viral core docks to the nuclear pore complex in non-dividing cells and the vDNA can enter into the nucleus and incorporate into the host genome (43). After the vDNA has incorporated into the host genome, then the viral protein, Tat, helps transactivate transcription of viral mRNA. The viral mRNA travels from the nucleus to the cytoplasm where it can then be translated by host ribosomes to make new viral proteins (44). Two viral proteins are created through translation: Gag and GagPol. The MA domain of Gag targets Gag to the plasma membrane and promotes incorporation of the viral Env glycoproteins into the budding virions; NC recruits the viral RNA genome into the budding virions and facilitates the assembly process of HIV-1; finally, the p6 domain recruits the endosomal sorting complex required for transport (ESCRT) apparatus, which catalyzes the membrane fission step to complete the budding process (45). Once the virus is released from the host cell, the viral protease cleaves the polyproteins, Gag and Gag-Pol, which then allows the virus to undergo maturation. During maturation, the cleavage of the Gag and Gag-Pol polyproteins allows for the viral proteins to assemble into the mature structure of the HIV-1 virus. The CA core assembles into a fullerene-like cone structure.

The CA monomers form predominantly hexameric rings with seven CA pentamers at the wide end and five CA pentamers at the short end of the fullerene cone (46). When the core assembles into the fullerene-cone structure during maturation, the viral RNA is packaged into the core and forms a stable dimer (47). Once the virus has completed the maturation process, then the virus is ready to undergo another round of infection and this completes the replication cycle for HIV-1.

Figure 1-4: The HIV-1 Replication Cycle: The HIV-1 replication cycle begins with the HIV-1 virus binding to a CD4+ T-cell and interacting with the co-receptor CXCR4 or CCR5. Upon the interaction with the receptor and the co-receptor, the virus undergoes fusion and the viral core is able to enter into the cell. The viral ssRNA then undergoes reverse transcription to become dsDNA (PIC) that can incorporate into the host chromosome. While the virus undergoes reverse transcription, the viral core is trafficked to the nucleus via microtubules and through the motor proteins kinesin and dynein. As the virus travels to the nucleus, the viral core undergoes uncoating which docks to the nuclear pore complex and allows the PIC to enter into the nucleus. Upon import into the nucleus, the PIC incorporates into the host chromosome where it waits until it is transactivated by the viral protein, Tat, and undergoes transcription. The viral mRNA is trafficked to the cytoplasm where the host ribosomes and translate the mRNA and express the viral proteins Gag and GagPol. These proteins help with viral assembly at the cell’s plasma membrane. Once the proteins are assembled at the plasma membrane, the virus can bud and release from the host cell. The viral protease then cleaves the immature viral proteins to allow for viral maturation to occur and this completes the HIV-1 replication cycle. Reproduced with permission from Dismuke, D., 2006 (48).

Host-Restriction Factors

In order for HIV-1 to replicate efficiently, it must bypass inhibition by several cellular antiviral proteins termed restriction factors, which provide an early line of defense for the cell against the virus (49, 50). These restriction factors include apolipoprotein B messenger RNA (mRNA)-editing enzyme catalytic polypeptide-like 3

(APOBEC3) family of proteins, tetherin/bone marrow stromal cell antigen 2

(BST2)/CD317, interferon-induced transmembrane proteins (IFITMs), myxovirus resistance protein 2 (MxB), Sad1/UNC-84 domain-containing family of proteins (SUN), tripartite-motif-containing 5α (TRIM5α), serine incorporator 3 and 5 (SERINC3 and

SERINC5), and the zinc finger antiviral family of proteins (ZAP).

In response to the restriction factors that the cell has evolved against the virus,

HIV-1 has viral proteins that antagonize the cellular restriction factors. The antiviral activity of APOBEC3G (A3G) is antagonized by the viral accessory protein Vif. Vif binds to A3G in the cell and recruits it to the cellular ubiquitin ligase complex to be degraded by the proteasome (50–54). In the absence of Vif, A3G is packaged into assembling

HIV-1 virions at the plasma membrane (55). The resulting infection from the new virus causes A3G to be transferred to the new cell where it associates with RT and deaminates cytidine residues in single-stranded negative-strand cDNA (56–59). The deamination by A3G results in guanosine-to-adenosine hypermutation of the viral plus stranded DNA, which proves to be fatal for the virus.

TRIM5α is another cellular, cytoplasmic restriction factor that inhibits HIV-1.

TRIM5α inhibits HIV-1 infectivity by binding the viral capsid after the virus has entered into the host cell. TRIM5α binding to the viral core prevents the virus from synthesizing

13 a sufficient amount of cDNA to incorporate into the host genome (60, 61). Similarly, another HIV-1 cellular, cytoplasmic restriction factor that recognizes and binds to the

HIV-1 capsid is MxB (62–64). MxB restriction of HIV-1 infection has been correlated to the inhibition of HIV-1 nuclear entry (65).

SUN1/SUN2 are two other cellular restriction factors that inhibit HIV-1 infectivity at the stage of nuclear import of the virus (66, 67). SUN1 and SUN2 are inner nuclear membrane proteins that form the inner part of the linker of nucleoskeleton and cytoskeleton complex. SUN1 and SUN2 form a linker between the nucleoskeleton and the cytoskeleton complex through interactions with the KASH domain of nesprins in the outer nuclear membrane (68–71). SUN1 and SUN2 perform several functions in the cell, including the nuclear morphology, chromosome positioning, and DNA damaging responses (72–74). Overexpression of SUN1 blocks HIV-1 infection in a CypA-CA- dependent manner (66, 67).

ZAPs are cellular, cytoplasmic proteins that can inhibit HIV-1 infectivity through targeting CG-dinucleotide rich regions of the vRNA and binding to the CG-rich regions and removing these regions from the virus (75, 76). HIV-1 and other RNA viruses resist

ZAP inhibition by editing their genome to mimic the low CG content of their hosts (77–

80).

Tetherin is a cellular, membrane protein that inhibits HIV-1 infectivity by preventing the virus from budding from the host cell (81). HIV-1 resists tetherin inhibition through the viral accessory protein, Vpu (81, 82). Vpu colocalizes with Tetherin and removes it from the cell surface so that it cannot antagonize HIV-1 infectivity (83, 84).

IFITMs are cellular restriction factors that are thought to prevent HIV-1 replication by preventing the virus from being able to cleave HIV-1 Env into gp120 and gp41 (85,

86). However, there are several different mechanisms for how IFITMs inhibit HIV-1 infectivity at the moment, so more work needs to be done in order to understand more clearly the antiviral mechanisms of IFITMs.

Human T cells express a 10-12 transmembrane domain protein called SERINC5 that is incorporated into budding HIV-1 particles and inhibits virus-cell fusion. There are five proteins in the human SERINC family, of which, SERINC3 and SERINC5 were identified to inhibit HIV-1 infectivity in 2015 (1, 2). HIV-1 susceptibility to SERINC5 is determined by sequences in the viral Env protein (1, 87). The HIV-1 accessory protein,

Nef, was identified to counteract SERINC5 inhibition, remove SERINC5 from the plasma membrane, and traffic to late endosomes where SERINC5 is degraded (1, 2,

88). However, the mechanism of how SERINC5 inhibits HIV-1 infectivity is still unclear.

SERINC3/5

ZAPs

SUN1/SUN2

Figure 1-5: HIV-1 Restriction Factors: CD4-positive T cells have evolved to express proteins that inhibit HIV-1 at almost every stage of its life cycle. These proteins include IFITMs, which inhibit HIV-1 Env synthesis and cleavage, which therein inhibits viral entry. SERINC3 and SERINC5 inhibit HIV-1 entry through altering the conformation of the HIV-1 Env. Trim5α binds to the viral core when it enters into the cell and prevents sufficient vDNA synthesis to incorporate into the host nucleus. APOBEC3G causes lethal cytidine-to-guanine hypermutations in the virus which prevents successful vDNA synthesis during reverse transcription. MxB/Mx2 inhibits nuclear import of HIV-1 by binding to the viral CA and preventing uncoating. SUN1/SUN2 inhibits HIV-1 nuclear import by binding to the CA/CypA complex. ZAPs bind to CG rich regions of vRNA that are higher than the host’s CG dinucleotide levels and inhibit viral translation. Tetherin inhibits viral budding by preventing the virus from cleaving from the host cell. Reproduced with permission from Doyle, et al. 2015 (89), Copyright Springer Nature BV.

Goals of my Thesis Work Prior to starting my thesis research, there were several key findings that were known about the antiviral mechanism of SERINC5. Previous reports had provided evidence that SERINC5 was Env-dependent (1, 87), that when HIV-1 Nef was expressed, SERINC5 was removed from the plasma membrane of cells and co- localized with Rab7-dependent late endosomes for degradation (1, 2, 88), SERINC5 inhibits HIV-1 fusion (1, 90), and the V3 loop of HIV-1 Env was necessary for

SERINC5’s antiviral activity (87). From these initial reports, I was interested in understanding more about SERINC5’s interaction with the HIV-1 Env for its antiviral activity.

The goals of my thesis work were: 1.) Identify the epitope(s) that SERINC5 targets on HIV-1 Env that prohibit the successful fusion of HIV-1. 2.) Determine whether

SERINC5 has a direct association with HIV-1 Env and link this interaction as a part of

SERINC5’s antiviral mechanism. When I started my thesis project, I used co- immunoprecipitation and western blots to determine whether SERINC5 had a direct association with HIV-1 Env. From my initial western blot data, I was able to identify that both SERINC2 and SERINC5 co-immunoprecipitate with HIV-1 Env. In order to identify whether SERINC5’s antiviral activity was linked due to a direct association with HIV-1

Env, I was going to need to identify an alternative approach in order to answer this question. Others in the field had tried to answer this question through FRET analysis, however, the answer is still unclear whether SERINC5 and HIV-1 Env directly interact with one another, or if they are just in close proximity to one another on the viral membrane. This work is described in chapter 3.

The second part of my thesis project focused on understanding which epitopes

SERINC5 targets on the HIV-1 Env to inhibit HIV-1 infectivity. To answer this question, I used two different in vitro methods. I used an indirect and a direct virus capture assay using Env-specific monoclonal antibodies (mAbs) to identify which epitopes on HIV-1

Env SERINC5 inhibited the capture of the HIV-1 virus to the mAbs. I started by engineering HIV-1 virions with increasing levels of SERINC5 to determine how much

SERINC5 could be co-transfected with the HIV-1 nef-deficient proviral DNA before the amount of virus produced was affected. I then used a panel of HIV-1 Env-specific mAbs and recombinant proteins with the virus capture assays to identify which epitopes

SERINC5 was targeting and link these epitopes with SERINC5’s antiviral activity.

Additionally, I used viral Envs that had previously been reported to be susceptible and resistant to SERINC5 inhibition along with SERINC2 and Nef-positive proviral DNA as controls for my study. My work from this study yielded several key findings, including the identification of 3 epitopes that SERINC5 targeted on HIV-1 Env that appear to be linked to SERINC5’s antiviral activity. From my thesis research, I propose a model where SERINC5 alters the conformation of some, but not all, of the Env molecules on the surface of the HIV-1 virus, and this action is important in the antiviral mechanism for

SERINC5. My work was published in the Journal of Virology and the results from that study are described in Chapter 2 of this dissertation.

Chapter 2

SERINC5 Inhibits HIV-1 Infectivity by Altering the Conformation of gp120 on HIV-1

Particles*

*The results in this chapter were reported in Featherstone, et al. 2020. Journal of Virology, 94(20):

e00594-20 (91).

Introduction

In humans, SERINC (serine incorporator) proteins comprise a family of five transmembrane proteins. Of the five SERINC proteins, only SERINC3 and SERINC5 inhibit HIV-1 infectivity, albeit SERINC5 inhibits HIV-1 infectivity 20-fold greater than

SERINC3 (1, 2). Humans express five alternatively-spiced isoforms of SERINC5 that contain 10 transmembrane domains; however, only the isoform SERINC5-001 effectively inhibits HIV-1 infectivity (92). Both SERINC3 and SERINC5 are incorporated into HIV-1 particles and reduce their infectivity (1, 2, 93), but the viral accessory protein,

Nef, prevents incorporation of SERINC3 and SERINC5 and counteracts its antiviral activity. Expression of Nef relocalizes SERINC5 from the cell membrane to late endosomes/lysosomes where it is degraded (88). SERINC5 is also antagonized by additional retroviral proteins, including the EIAV S2 and MLV GlycoGag proteins (1, 2,

94). Previous work has shown that the antiviral mechanism of SERINC5 depends on sequences in the HIV-1 envelope (Env) protein (1, 2, 87). However, the precise mechanism by which SERINC3 and SERINC5 inhibit Env function is unclear.

HIV-1 Env is a trimer of heterodimers on the HIV-1 virion. It consists of a surface glycoprotein (gp120) and a transmembrane glycoprotein (gp41). During infection of T- cells, the V1/V2 loop on gp120 interacts with the primary cellular receptor, CD4 (18, 19).

Upon binding to CD4, a conformational change in the V1/V2 loop on gp120 occurs and this allows the V3 loop on gp120 to interact with one of two coreceptors present on

CD4+ T-cells; C-X-C chemokine receptor type 4 (CXCR4) or C-C chemokine receptor type 5 (CCR5) (95, 96). As described in Chapter 1, coreceptor binding triggers a conformational change in gp41, resulting in exposure of the fusion peptide insertion into the target cell plasma membrane (97, 98). Subsequently, the gp41 subunits refold into a structure containing a six-helix bundle (22, 23, 99). The formation of the six-helix bundle allows the viral membrane to fuse with the cell membrane, resulting in lipid mixing and formation of a fusion pore (24, 100), which expands, releasing the viral core into the target cell cytoplasm.

While the precise mechanism by which SERINC5 inhibits HIV-1 infectivity was unclear at the start of my research, it was known that the host protein reduces the ability of HIV-1 particles to fuse with target cells (1, 90). The ability of SERINC5 to inhibit HIV-1 infectivity had been shown to depend on the Env glycoprotein. Specifically, Env proteins from many HIV-1 isolates are resistant to SERINC5 (1, 2, 87). Studies with chimeric Env sequences had shown that HIV-1 susceptibility to SERINC5 depends on the V1, V2 and

V3 loop regions in gp120 (87). SERINC5 also appears to restrict expansion of the fusion pore formed between the viral and target cell membranes. The initial findings from

Rosa, et al. provided evidence that SERINC5 restricts expansion of the fusion pore due to a reduction in double-stranded vDNA collected when SERINC5 is expressed on virus

20 particles and from experiments from Sood, et al. where they used FRET on HIV-1 Env molecules during fusion and their results suggest that when SERINC5 is expressed that there is a delay in gp41 refolding into the final six-helix bundle formation (1, 90).

SERINC5 also enhances the susceptibility of HIV-1 to inhibition by some monoclonal antibodies and to a CCR5 antagonist, suggesting that Env conformation is altered (87,

90, 101). However, a previous report provided evidence that SERINC5 does not alter the composition of the viral membrane, which was thought to be a possible mechanism as to how SERINC5 could inhibit HIV-1 infectivity (102).

To directly examine the effects of SERINC5 on HIV-1 Env conformation, I quantified the capture of HIV-1 particles by immobilized Env-specific monoclonal antibodies as probes for Env conformation. SERINC5 inhibited the capture of HIV-1 particles by antibodies recognizing various epitopes on gp120, and this effect was prevented by Nef and was not observed with an Env protein that is resistant to

SERINC5’s antiviral activity. My results demonstrate that SERINC5’s antiviral mechanism is functionally related to its conformational effects on gp120 epitopes and on HIV-1 Env trimer formation and/or stability.

Results

SERINC5 reduces the infectivity and the capture of NL4-3 Nef- virus to

gp120-specific epitope antibodies

Ectopic expression of SERINC5 in HIV-1-producing cells potently reduces HIV-1 infectivity (1, 2, 87, 88, 90, 92–94, 103, 104). To determine whether SERINC5 alters the conformation of HIV-1 Env, I employed an assay that has been previously utilized for

21 quantifying epitope exposure, termed the virus capture assay (105–107). To establish the optimal dose of SERINC5 for these experiments, I generated Nef-defective HIV-1 particles by co-transfection with a range of SERINC5 expression plasmid concentrations and assayed infectivity by single-cycle reporter assays. As previously reported (1, 87,

90, 92, 93, 101, 108), SERINC5 reduced the infectivity of the virus in a dose-dependent manner (Fig. 2-1A). For my mechanistic studies, I selected 2 µg of pBJ5-SERINC5-HA, because this dose strongly inhibited HIV-1 infectivity without reducing virus production

(data not shown). To establish the virus capture assay, I titrated the virus to determine the concentration of HIV-1 particles that provided the best signal-to-noise ratio: i.e., the optimal dose of virus for binding of Env+ vs. Env- particles. For each of the antibodies employed in my study, I observed that the optimal quantity of HIV-1 to use for these assays without saturating the capture of the virus was 3.75 ng of p24 (representative curve shown in Fig. 2-1B). I surveyed a large number of monoclonal antibodies for their utility in the capture assay (Table 4-1); however, only a select few were found to be useful based on an acceptable signal-to-noise ratio. A previous study has shown that for the 2G12 and 2F5 antibodies, the Env-deficient virus can be captured just as strongly as the Env+ virus due to the antibody able to bind to proteins that are post- translationally modified or have a strong affinity for lipids (109). Therefore, I employed an indirect virus capture assay for the 2G12 and 2F5 antibodies, which involved binding of Env-specific antibody to virus followed by pelleting the virus to remove unbound antibody and subsequent capture with an immobilized secondary antibody (Table 4-1).

In these experiments, I determined that the optimal dose of the virus used was also 3.75

22 ng of p24. I also tested 15 additional antibodies that exhibited inadequate signal/noise ratios for use in either direct or indirect capture assay.

Following optimization of the assays, I asked whether SERINC5 alters the ability of HIV-1 particles to be captured by each antibody. From the initial panel tested, I identified three gp120-specific antibodies to which SERINC5 reduced the efficiency of virus capture: 447-52D, which recognizes the V3 loop; 17b, which recognizes a CD4- induced epitope which overlaps the co-receptor binding site; and 2G12, which recognizes an N-linked glycan on gp120 (Fig. 2-1C). SERINC5 reduced the capture of the Env+ virus to these antibodies by 25-50%, with the strongest effect observed with

447-52D. I also observed that HIV-1 capture to two antibodies was unaffected by

SERINC5: b12, which recognizes the CD4-binding site on HIV-1 gp120; and 2F5, which recognizes the membrane proximal external region (MPER) on gp41 (Fig. 2-1D).

My results indicate that SERINC5 reduces the capture of the virus to antibodies recognizing specific epitopes on gp120.

A B b12 0.5 0.4

)

g 0.3

n (

2 0.2 p 0.1 0.0

ng of p24

C 447 52D 17b 2G12 1.0 0.5 1.5

0. 0.4 ) )

)

g g

g 1.0

n (

( 0.6

( 0.3

2 2 0.4 2 0.2

p p p 0.5 0.2 0.1 0.0 0.0 0.0 Env En v Env S5 HA S5 HA S5 HA

b12 2F5

D ns 0.4 0.15 ns

)

0.3 ) g

g 0.10

(

0.2 4 2

2 p p 0.05 0.1

0.0 0.00 En v En v S5 HA S5 HA FIG 2-1 Optimization of the virus capture assay for testing the effects of SERINC5. (A) Titration of SERINC5 levels. These viruses were produced from 293T cells and the viral infectivity was measured by infecting TZM-bl cells. (B) Titration of HIV-1 particles in the virus capture assay using the gp120-specific b12 antibody. (C) Effects of SERINC5 on capture of HIV-1 particles by four gp120-specific monoclonal antibodies. (D) Effects of SERINC5 on capture of HIV-1 particles by b12 and 2F5. Results in this figure are the mean values and standard deviations (error bars) from three independent experiments. Statistical significance was analyzed by unpaired t-test. n.s.: not significant; *: P < 0.05; ***: P < 0.001.

SERINC5 does not affect the antibody-dependent capture of HIV-1 particles

bearing a SERINC5-resistant Env protein

To determine whether the inhibition of capture by SERINC5 is correlated with the inhibition of infectivity, I performed virus capture assays with HIV-1 particles bearing an

HIV-1 Env that is resistant to SERINC5 inhibition, JR-FL (1, 2, 90). In parallel, I employed another HIV-1 Env that is susceptible to SERINC5 inhibition, HXB2 (1, 2, 87,

90, 110). Assays of viral infectivity demonstrated that SERINC5 potently inhibited the

HIV-1(HXB2) virus, whereas the inhibition of HIV-1(JR-FL) was minimal (Fig. 2-2A).

Titration of the viruses in capture assays revealed that, as with HIV-1NL4-3 particles, the optimal quantity was 3.75 ng of p24 (Fig. 2-2B). SERINC5 reduced the capture of HIV-1

HXB2 particles to the gp120-specific antibodies 447-52D, 17b and 2G12, but not b12 or

2F5 (Fig. 2-2C-D). By contrast, SERINC5 did not reduce the capture of HIV-1(JF-FL) particles to any of the antibodies. These results show that SERINC5 does not affect the capture of HIV-1 particles bearing an Env protein that confers resistance to its antiviral activity.

A B b12 b12 300 0.5 0.

0.4 )

) 0.6

g g

i 200

n i

( 0.3

(

c 4

4 0.4

2 2

f 0.2

n p

I 100 0.2 0.1

0 0.0 0.0

ng H B2 Virus ng JR FL Virus

447 52D 17b 2G12 C 1.5 0.15 1.5 ns

ns ns

)

) )

g g

1.0 g 0.10 1.0

(

4 4

2 2

p p p 0.5 0.05 0.5

0.0 0.00 0.0 Env S5 S5 Env S5 S5 Env S5 S5 H B2 JR FL H B2 JR FL H B2 JR FL D b12 2F5 ns ns

0.6 0.3 ns )

) ns g

g 0.4 0.2

(

4 2

2 p p 0.2 0.1

0.0 0.0 Env S5 S5 Env S5 S5 H B2 JR FL H B2 JR FL

FIG 2-2 SERINC5 does not affect the antibody capture of HIV-1 particles bearing the JR-FL Env protein. (A) Effects of SERINC5 on the infectivity of Nef-defective HIV-1 particles bearing the HXB2 vs. JR-FL Env proteins. (B) Titration of Nef-defective HIV-1 particles bearing the HXB2 and JR-FL Env proteins in the virus capture assay using the gp120-specific b12 antibody. (C) Differential effects of SERINC5 on the capture of Nef- defective HIV-1 particles bearing HXB2 vs. JR-FL Env proteins by gp120-specific monoclonal antibodies. (D) SERINC5 does not affect capture of Nef-defective HIV-1 particles bearing HXB2 or JR-FL Env proteins by b12 and 2F5 antibodies. Results in this figure are the mean values and standard deviations (error bars) from three independent experiments. Statistical significance was analyzed by unpaired t-test. n.s.: not significant; *: P < 0.05; **: P < 0.01.

SERINC5 does not inhibit capture of HIV-1 particles by CD4 protein

SERINC5 did not inhibit the capture of the NL4-3, HXB2 or JR-FL virus the b12 monoclonal antibody, which recognizes the gp120 epitope to which CD4 binds.

Therefore, I asked whether SERINC5 affects the binding of gp120 to CD4. To answer this question, I sought to determine a capture assay with immobilized recombinant CD4 protein(111) fused to the hinge and Fc regions of the human IgG1 antibody (CD4-Ig). I observed no significant effect of SERINC5 on capture of any of the three viruses by

CD4-Ig (Fig. 2-3). These observations, together with the b12 capture data, suggested that SERINC5 does not affect HIV-1 binding to CD4. However, one caveat is that CD4 could be binding enough Env that there is not a visible difference in Env binding, even though the conformation of the CD4-binding site is being altered. I will discuss more about this caveat in the Discussion section of Chapter 2.

NL4 3 H B2 JR FL ns 2.5 ns 0.4 0.4 ns

2.0 ) )

) 0.3 0.3

( (

( 1.5

4 4

4 0.2 0.2

2 2

2 1.0

p p p 0.5 0.1 0.1 0.0 0.0 0.0 Env Env Env S5 HA S5 HA S5 HA

FIG 2-3 SERINC5 does not inhibit capture of Nef-defective HIV-1 particles by recombinant CD4 protein. (A) Nef-defective HIV-1 particles bearing HXB2 or JR-FL Env proteins were assayed for capture by plate-bound CD4-Ig protein. Results in this figure are the mean values and standard deviations (error bars) from three independent experiments. Statistical significance was analyzed by unpaired t-test.

SERINC5 does not prevent HIV-1 Env from undergoing a conformational

change upon binding to CD4

I next asked whether SERINC5 affects the CD4-dependent conformational changes in Env. To test this, I employed 17b, a monoclonal antibody that recognizes a gp120 epitope that is induced upon CD4 binding (112–114). I assayed capture of the virus in the presence of various concentrations of added soluble CD4 protein (sCD4). In the absence of sCD4, SERINC5 reduced the capture of HIV-1 particles by 64% (Fig. 2-

4; 0 µg/mL CD4 values). For viruses with and without SERINC5, addition of sCD4 stimulated the capture by 17b, but to a lower maximum level for the SERINC5- containing particles (Fig. 2-4 compare 0.25 µg/mL CD4 values). Analysis of the fold- induction of capture revealed that sCD4 promoted the capture of control particles by

4.3-fold and the capture of SERINC5 particles by 5.3-fold. Thus, although SERINC5 inhibited the capture of HIV-1NL4-3 by 17b, the ability of CD4 to promote capture was not compromised. I conclude that SERINC5 does not alter the ability of Env to undergo the

CD4-dependent conformational change recognized by 17b.

17b Vector SERINC5 HA 3

2 )

(

4 2 p 1

5 5

0 .

. 0

. . 0

0 0

sCD4 ( g mL) FIG 2-4 SERINC5 does not affect CD4-induced exposure of the 17b epitope in gp120 on Nef-defective HIV-1 particles. NL4-3 Nef-deficient virus was co-transfected with/without 2 µg of pBJ5-SERINC5-HA and produced from 293T cells. The viruses were incubated with increasing concentrations of soluble CD4 (sCD4) before being pelleted, resuspended, and added to 17b-coated 96-well plates. Shown are the mean values and standard deviations (error bars) from three independent experiments.

SERINC5’s antiviral activity is correlated with its ability to inhibit virus capture. The observed effects of SERINC5 on antibody capture of HIV-1 suggested that Env conformational effects are related to SERINC5’s antiviral activity. To further test the relationship between the effect of SERINC5 on virus capture and infectivity, I analyzed a panel of viruses produced by co-transfection of the NL4-3.Nef- provirus with increasing quantities of SERINC5 expression plasmid. As expected, the infectivity of the virus decreased with increasing quantities of SERINC5 (Fig. 2-5A). Analysis of the efficiency of capture of these viruses by 447-52D revealed a positive sigmoidal relationship between capture and infectivity (Fig. 2-5B and 2-5C). This suggests that the conformational effect that SERINC5 exerts on HIV-1 Env is quantitatively related to antiviral activity of SERINC5.

A 50 NL4 3 Nef Vector

40 NL4 3 Nef SERINC5

ty i

v 30

t c

f 20

n I 10

g of SERINC5 plasmid B 447 52D 1.0

0. )

g 0.6

(

2 0.4 p

0.2

0.0

C g of SERINC5 plasmid

FIG 2-5 Relationship between SERINC5 inhibition of infectivity and inhibition of gp120-dependent capture. (A) Dose-dependent inhibition of Nef-defective HIV-1 infectivity by SERINC5. (B) Dose-dependent inhibition of capture of Nef-defective HIV-1 particles by SERINC5. (C) X-Y scatter plot depicting the relationship between HIV-1 infectivity and capture efficiency. Shown are the mean values and standard deviations (error bars) from three independent experiments. Statistical significance was analyzed by unpaired t-test. n.s.: not significant; **: P < 0.01; ***: P < 0.001.

SERINC5 perturbs the trimer conformation of HIV-1 Env

In a previous study, Zhang, et al. reported that SERINC5 can disrupt the trimeric structure of Env (104). Disruptions in the trimer conformation of HIV-1 Env can prevent the virus from fusing to the target cell (15, 115). To determine whether SERINC5 affects

HIV-1 Env trimerization, I assayed particle capture by PGT145, a human mAb that has been reported to specifically recognize only HIV-1 Env trimers expressed on cells (116).

I observed that SERINC5 markedly reduced the capture of NL4-3 Nef-deficient virus by

PGT145 (Fig. 2-6). This observation suggests that SERINC5 perturbs the trimeric structure of Env.

Nef prevents the conformational effects of SERINC5 on virion-associated

gp120

SERINC5’s antiviral activity is antagonized by the HIV-1 accessory protein, Nef.

To further probe the relationship between SERINC5’s effects on antibody-dependent capture and inhibition of infectivity, I asked whether Nef can counteract the inhibition of antibody-dependent capture by SERINC5. As previously reported, Nef prevented

SERINC5 from reducing HIV-1 infectivity (Fig. 2-7A) and reduced the levels of

SERINC5 associated with pelleted HIV-1 particles, as detected by immunoblotting (data not shown). By contrast to the Nef- virions, SERINC5 did not inhibit the capture of Nef+ particles by PGT145, 447-52D, 17b, and 2G12 (Fig. 2-7B). I conclude that Nef counteracts the conformational effects of SERINC5 on HIV-1 Env. These results further link the inhibition of capture caused by SERINC5 to its antiviral activity.

0.4 PGT145

0.3

)

n (

0.2

2 p 0.1

0.0 Env S5 HA

FIG 2-6 Effect of SERINC5 on capture of HIV-1 by a monoclonal antibody that recognizes trimeric HIV-1 Env. Nef-defective HIV-1 particles were assayed for capture by monoclonal antibody PGT145. Values are the mean values and standard deviations (error bars) from three independent experiments. Statistical significance was analyzed by unpaired t-test. n.s.: not significant; *: P < 0.05.

ns A 100

ty 0

v i

t 60

c e

f 40

n I 20

B PGT145 447 52D 0.4 ns 1.0 ns

) ) 0.

g 0.3

( (

0.6

4 0.2 4

2 0.4

p p 0.1 0.2

0.0 0.0

17b 2G12 ns ns

0. 1.0 )

) 0. g

g 0.6

( (

0.6

4 0.4 2

2 0.4

p p 0.2 0.2 0.0 0.0

FIG 2-7 Nef prevents the inhibition of Env-dependent HIV-1 capture induced by SERINC5. (A) Nef+ and Nef- HIV-1 particles produced by co-transfection with SERINC5 expression plasmid were assayed for infectivity (A) and capture by monoclonal antibodies (B). Results in this figure are the mean values and standard deviations (error bars) from three independent experiments. Statistical significance was analyzed by unpaired t-test. n.s.: not significant; *: P < 0.05; ***: P < 0.001.

SERINC2 does not inhibit the infectivity and the capture of Nef-deficient

HIV-1

The observed effects of SERINC5 on HIV-1 infectivity and capture suggested that SERINC5’s antiviral activity is linked to its effects on Env. Two of the five human

SERINC proteins exhibit antiviral activity against HIV-1; of these, SERINC5 is the most potent, followed by SERINC3. By contrast, SERINC2 lacks antiviral activity. To further probe the relationship between inhibition of HIV-1 capture and antiviral activity, I asked whether SERINC2 affects HIV-1 capture. Analysis of HIV-1 particles produced by co- transfection of a SERINC2 expression plasmid revealed that, as expected, SERINC2 did not inhibit the infectivity of either HIV-1(HXB2) or HIV-1(JR-FL); rather, it modestly increased the infectivity of both (Fig. 2-8A). Accordingly, the capture of the viruses was not inhibited by SERINC2 (Fig. 2-8B). These results demonstrate that a SERINC protein that lacks antiviral activity does not affect the ability of HIV-1 particles to be captured by

Env-specific monoclonal antibodies.

A 100 400

ty 300

v i

i 60

t c

c 200

e f

f 40

n n

I I 20 100 0 0

447 52D 17b

B 1.5 0.15 ns

ns ns )

) g

g 1.0 0.10

(

2 p 0.5 p 0.05

0.0 0.00 SERINC S 2 S5 S 2 S5 SERINC S 2 S5 S 2 S5 Env Env H B2 JR FL H B2 JR FL

2G12 2F5 ns ns

1.5 ns 0.3 ns ns ns

ns )

)

1.0 g 0.2

(

2 p 0.5 p 0.1

0.0 0.0 SERINC S2 S 5 S 2 S5 SERINC S2 S5 S 2 S5 Env Env H B2 JR FL H B2 JR FL

FIG 2-8 SERINC2 does not inhibit capture of HIV-1 by monoclonal antibodies. Nef- defective HIV-1 particles bearing HXB2 or JR-FL Env proteins were produced by co- transfection with a SERINC2 or SERINC5 expression plasmid and assayed for infectivity (panel A) and capture by monoclonal antibodies (panel B). Results in this figure are the mean values and standard deviations (error bars) from three independent experiments. Statistical significance was analyzed by unpaired t-test. n.s.: not significant; *: P < 0.05; **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.

SERINC5 does not grossly affect Env biosynthesis, processing, or glycosylation

The effect of SERINC5 on capture by 2G12, which recognizes a specific glycan on gp120, suggested the possibility that SERINC5 alters glycosylation and/or egress through the secretory pathway. To examine this, my mentor, Chris Aiken and I, performed pulse-chase analysis of Env biosynthesis in normal 293T cells transfected with Nef-deficient HIV-1 provirus and with or without SERINC5 expression. Cells were radiolabeled for 20 minutes and chased for various times. Immunoprecipitation with

2G12 and with endoglycosidase H (endo H) revealed no obvious difference between the synthesis and processing of gp160 into gp120, nor in the acquisition of resistance to treatment with endoglycosidase H, an enzyme that recognizes high mannose core oligosaccharides added early to glycoproteins and that are further modified to become resistant to digestion by the enzyme (Fig. 2-9A). Immunoblot analysis of HIV-1 particles released from the transfected cells revealed similar quantities of gp120, gp41 and gp160 on the virions (Fig. 2-9B). These results indicate that the expression of SERINC5 does not detectably alter the rate of Env biosynthesis, processing, glycosylation, or degradation in transfected 293T cells.

chase HIV 1 Nef SERINC2 HIV 1 Nef SERINC5 time (h) 0 0.5 1 2 4 0 0.5 1 2 4 EndoH

gp160 gp120

B Anti gp120 (401) Anti gp120 (2G12) Anti HIV 1 (HIV Ig) Phosphorimager 2 anti rab 6 0 2 anti hum 00 2 anti hum 00 Scan

S2 S5 S2 S5 S2 S5 S2 S5 250 250 250 250 150 150 150 gp160 gp160 gp160 150 gp120 gp120 100 100 100 100 75 75 75 75 RTp66 50 50 50 Pr55Gag 50 gp41 37 37 37 37 IN 25 25 25 25 CA 20 20 20 20

15 15 15 MA 15 10 10 10 10 FIG 2-9 SERINC5 does not affect the glycosylation of HIV-1 Env in cell lysates. (A) 293T cell lysates were co-transfected with NL4-3 Nef- and either SERINC2/SERINC5 proviral DNA. Cell lysates were pulse-chase labeled at 0, 0.5, 1, 2, 4 and 8 hours and collected and treated +/- endoglycosidase H. Cell lysates were denatured and run on an SDS-PAGE gel and probed for gp120 with the 2G12 monoclonal antibody. (B) Cell lysates with NL4-3 Nef- and SERINC2/SERINC5 were radiolabeled, collected, normalized by BCA assay, incubated overnight with AG beads bound with HIV-Ig polyclonal antibody, eluted, denatured and run on an SDS-PAGE gel. Gels were fixed and stained with Coomassie blue, dried, and radiolabeled proteins were detected with a phosphoimager.

DISCUSSION

In this study, I sought to further define the mechanism by which SERINC5 reduces HIV-1 infectivity. Previous studies had demonstrated that HIV-1 particles containing SERINC5 are impaired for fusion with target cells, and that sequences in the gp120 protein modulate HIV-1 susceptibility to inhibition by the host protein. However,

SERINC5 can inhibit the infectivity of HIV-1 particles that are pseudotyped with retroviral proteins with little similarity to HIV-1 Env, including Env proteins from murine leukemia viruses (1), suggesting that the mechanism is more complex than sequence- specific targeting. Moreover, SERINC5 does not appear to alter the levels of the Env on the surface of HIV-1 particles. Using immobilized Env-specific monoclonal antibodies to capture native HIV-1 particles, I examined the conformational effects of SERINC5 on selected Env epitopes. I observed that SERINC5 reduced the capture of HIV-1 by antibodies recognizing diverse epitopes on Env, including the third hypervariable domain (recognized by the 447-52D antibody), a conformational epitope that is formed upon binding of CD4 (recognized by the 17b antibody), and an epitope that recognizes

N-linked glycans on gp120 (2G12 antibody). Inhibition of virus capture was connected to the antiviral activity of SERINC5 by the following observations: (1) it was not observed with HIV-1 particles bearing the SERINC5-resistant Env protein from HIV-1JR-FL; (2) it was prevented by expression of Nef during virus production; and (3) it was dependent on SERINC5 expression in a manner that was correlated with dose-dependent inhibition of infectivity. Collectively, these data indicate that SERINC5 reduces HIV-1 infectivity by altering the Env conformation, thus affecting diverse epitopes on Env. My results are consistent with other recent reports that: SERINC5 destabilizes viral Env trimers (104);

SERINC5 alters the conformation of selected Env epitopes on Nef-deficient HIV-1 particles (117).

Conformational effects of SERINC5 on Env may also account for the reported

SERINC5-induced sensitization of HIV-1 particles to neutralizing antibodies (87, 90,

101, 117, 118). It may seem paradoxical that SERINC5 reduces the ability of antibodies to capture virus particles while sensitizing the virus to neutralization. However, I suggest that SERINC5 inactivation of a fraction of Env trimers on the virion surface can reconcile these observations. This would be predicted to: (1) reduce HIV-1 infectivity owing to reduced valency of the Env-receptor interactions; (2) reduce HIV-1 capture by antibodies by providing fewer conformationally intact Env trimers; and (3) enhance sensitivity of the remaining infectious HIV-1 particles to antibody neutralization by rendering the remaining functional Env trimers more critical for cell entry.

In my experiments, SERINC5 did not inhibit the ability of other antibodies (2F5, and b12) to capture HIV-1 particles. Similarly, HIV-1 capture by immobilized CD4 was not affected by SERINC5. I interpret these negative results cautiously. One limitation of the virus capture assay approach is that it might miss some of the effects that are partial or miss effects on epitopes that are harder to access (e.g. MPER with 2F5). This could explain why I did not detect an effect of SERINC5 on HIV-1 capture by the 2F5 mAb, whereas others have (117). In addition, a previous study reported that SERINC5 sensitizes HIV-1 to neutralization by 2F5 (101), suggesting that this gp41 epitope is affected by SERINC5. It is possible that the differing affinity of antibodies to Env could affect the efficiency of the virus capture assay. Incomplete perturbation of Env trimers on the surface of the virus may still permit effective capture of HIV-1 particles by high-

41 affinity antibodies, if a minimal number of Env molecules retain the ability to be bound.

Interestingly, I observed that while SERINC5 did not affect the capture of HIV-1 particles by CD4, 17b binding, which is induced by CD4, was reduced. However, I also observed that addition of CD4 protein effectively stimulated HIV-1 capture by 17b regardless of

SERINC5, though to a lower maximal extent when the particles contained SERINC5. I suggest that SERINC5 inactivates a fraction of Env trimers on HIV-1 particles and that the remaining trimers are competent for CD4 binding and CD4-induced conformational changes. Moreover, in Nef-defective particles produced from T cells, failure to downregulate CD4 from the cell surface could also amplify the antiviral effects of

SERINC5 (104, 117).

How exactly does SERINC5 perturb Env conformation? While my data do not answer this question, the effect of SERINC5 on capture by 2G12 antibody is intriguing.

This neutralizing antibody recognizes a specific, conserved glycan on gp120. In numerous experiments, I have employed 2G12 for detection of gp120 after immunoblotting HIV-1 particles under denaturing conditions, suggesting that the 2G12 epitope is not conformational. I therefore considered the possibility that SERINC5 might affect 2G12 capture of HIV- particles by altering gp120 glycosylation and potentially affecting other epitopes as well. However, my pulse-chase analysis of Env in transfected cells and immunoprecipitation indicates that the 2G12 epitope is formed rapidly upon synthesis and is not affected by SERINC5 when the protein is immunoprecipitated in detergent (RIPA buffer). I therefore suggest that SERINC5 inhibits 2G12 capture of HIV-1 particles by affecting the Env conformation and occluding the antibody access to the glycan epitope. Of course, my observations do not exclude

42 the possibility that SERINC5 affects glycosylation of Env at other sites, thereby altering

Env conformation.

Current models posit that SERINC5, which is incorporated into HIV-1 particles containing either susceptible or resistant Env proteins, acts directly on Env on the surface of HIV-1 particles to affect function. Recent studies indicate that SERINC5 can interact with Env when co-expressed in cells (104), and it seems plausible that the interaction is necessary, if not sufficient, for Env inhibition. Nonetheless, it remains unknown whether SERINC5 irreversibly inactivates Env in a “touch-and-go” manner, or whether the inhibition requires a stable association between SERINC5 and Env on the virion surface. Biochemical studies of the interaction of Env with purified recombinant

SERINC5 (119) will help answer these questions.

Chapter 3

Unpublished Results on SERINC5

SERINC5 Retention to the ER Inhibits the Antiviral Activity of SERINC5 on HIV-1

Infectivity

When I started studying the antiviral mechanism of SERINC5, I was interested in determining whether SERINC5 when necessary to be present on the virus in order to inhibit the infectivity of HIV-1. To test this hypothesis, I engineered an ER-retention signal (KDEL) to the C-terminus of SERINC5-HA, so that when the signal was detected by the receptor, ERD2, COPI-coated vesicles could relocate the protein complex to the

ER instead of being trafficked to the plasma membrane where it could be incorporated into budding HIV-1 virions (120–122). To negate the retention signal, I engineered a point mutation in the KDEL signal and changed it to ADEL in order to confirm that

SERINC5’s antiviral activity was returned to normal.

Once I engineered these small, amino acid sequences to the C-terminus of

SERINC5, I tested the infectivity of NL4-3 Nef- virus with/without SERINC5 and with/without the ER retention signal. After repeating the experiment several times, I was able to determine that at the lower concentrations of SERINC5 co-transfected with the

NL4-3 Nef- virus (0.25 and 0.5 µg) that the KDEL signal inhibited the antiviral activity of

WT SERINC5 virus by about 2-fold (Fig. 3-1). I also observed that incorporation of the

KDEL-containing SERINC5 protein into HIV-1 particles was reduced and that the

44 protein was also reduced in the cell lysate (Fig. 3-1). The results from these experiments suggested that incorporation of SERINC5 into the budding virus particle is necessary for its antiviral effect.

Figure 3-1 The Antiviral Activity of SERINC5 is Inhibited When SERINC5 is Retained to the Endoplasmic Reticulum. (A) SERINC5-HA was engineered with an ER retention signal, KDEL, to determine whether SERINC5’s antiviral activity was associated with its incorporation into the virus particle. The SERINC5-KDEL signal was ablated by creating a point mutation at the lysine residue into an alanine residue, ADEL. Results in this figure are the mean values and standard deviations (error bars) from two independent experiments.

A Concentrated Virus

HIV-1 Nef- SERINC5-HA -KDEL -ADEL

S5-HA ug: 0 0.0625 0.125 0.25 0.0625 0.125 0.25 0.0625 0.125 0.25

α-HA

α-CA

1 2 3 4 5 6 7 8 9 10

B Cell Lysates

HIV-1 Nef- SERINC5-HA -KDEL -ADEL

S5-HA ug: 0 0.0625 0.125 0.25 0.0625 0.125 0.25 0.0625 0.125 0.25

α-HA

α-GAPDH 1 2 3 4 5 6 7 8 9 10

Figure 3-2 SERINC5-KDEL is Retained from Being Incorporated into Virus

Particles and is Retained in the Cell. (A) HIV-1 virus was concentrated and probed for

HA and CA to determine whether less SERINC5 was incorporated into the virus when

SERINC5 was retained to the ER by the ER-retention signal, KDEL. (B) 293T cell lysates were harvested after being transfected with increasing concentrations of

SERINC5-HA, SERINC5-HA-KDEL, or SERINC5-HA-ADEL.

Chapter 4

Summary and Future Directions

The goal of my thesis research was to understand the antiviral mechanism of the host cell integral membrane protein, SERINC5. In Chapter 2, I described how I identified several epitopes on HIV-1 gp120 that SERINC5 affects and showed that these effects are directly correlated with inhibition of HIV-1 infectivity.

The goals of my thesis project were (1) to identify epitopes that SERINC5 targets on HIV-1 gp120 (Chapter 2), and (2) determine whether SERINC5 directly interacts with gp120 in order to inhibit HIV-1 infectivity. The latter aim remains to be resolved. Early on in my thesis research other researchers identified that SERINC5 and SERINC2 were both associated with gp120 using bimolecular fluorescence complementation (BiFC)

(104). This suggests that if SERINC5 directly interacts with HIV-1 Env, that the interaction is not sufficient for its antiviral activity, since the authors also identified

SERINC2 to be in close proximity to HIV-1 Env. The results I described in Chapter 2 suggest that SERINC5 could alter the conformation of gp120 through a direct interaction; however, it is also possible that SERINC5 could interact with gp120 indirectly, through the use of a cellular cofactor. There is currently no data to suggest that a cellular cofactor is helping SERINC5 inhibit HIV-1 infectivity, however, it is still possible that a cofactor does exist and with further research, one may be discovered. If

SERINC5 does directly interact with gp120, it is possible that SERINC5 directly interacts

48 with gp120 during trafficking, since both proteins have to undergo post-translational modifications through the endoplasmic reticulum and the Golgi apparatus. I tried testing this hypothesis early on in my research project by engineering ER-retention signal tags to the C-terminus of SERINC5. This resulted in modest increases in the infectivity of

HIV-1 (~20%) at the lowest concentration of SERINC5 co-transfected with the HIV-1 proviral DNA (Fig. 3-1). However, I did not continue to pursue this project to determine whether SERINC5’s antiviral activity occurred on the virus or in the cell, due to the more prominent results I received from the VCAs that I described in Chapter 2. Another idea is that SERINC5 could indirectly inhibit HIV-1 infectivity through altering/inhibiting N- linked glycosylation of HIV-1 Env when both proteins are being post-translationally modified in the cell. However, the pulse-chase results that I explained in Chapter 2 would argue against the latter idea, since we could not detect any significant difference in glycosylation of HIV-1 Env in cell lysates when SERINC5 was present. In further studies, it will be important to determine whether SERINC5 interacts with gp120 directly or indirectly.

The SERINC5 VCA project (Chapter 2) was based on several previous studies investigating the inhibitory effects of SERINC5 on HIV-1 infectivity. The two original

SERINC5 studies from Rosa et al. and Usami et al. provided evidence that the

SERINC5 antiviral effect is Env-dependent, that the protein is expressed in T-cells, and that it inhibits HIV-1 fusion. The authors also discovered that SERINC5 was inhibited by

Nef and other viral proteins (e.g. MLV glycogag). Following these initial findings, there were several other discoveries in the field that led to my initial hypothesis. Beitari et al. discovered that several HIV-1 Envs were resistant to SERINC5 inhibition, and mapped

49 the phenotype to the V3 loop of gp120 (87). Sood et al. discovered that SERINC5 alters

Env clustering, suggesting this can inhibit HIV-1 fusion (90). It was from these initial findings that led us to develop the hypothesis that SERINC5 could be targeting specific epitopes on HIV-1 Env in order to inhibit HIV-1 infectivity.

It was from the initial findings from Beitari, et al. that I postulated that SERINC5 targets gp120 epitopes in addition to the V3 loop. My goal for this project was to more precisely define the antiviral mechanism of SERINC5. I used a total of 25 different antibodies and recombinant proteins to probe the effects of SERINC5 on the corresponding epitopes by testing for the ability of SERINC5 to reduce HIV-1 capture.

The results showed that SERINC5 reduces capture by antibodies recognizing the V3 loop (Figure 2-1), a CD4-induced epitope (Figure 2-1), the HIV-1 Env trimer conformation (Figure 2-6) and N-linked glycans (Figure 2-1). In addition, I was able to determine that CD4 binding seems to be unaffected by SERINC5 with the three different viruses that I used (Figure 2-3). My data provide evidence that HIV-1 fusion is being impaired due to the Env trimer conformation being altered (Figure 2-6), therefore, there are fewer Env trimers on the virus accessible to form a virological synapse. In addition, due to SERINC5 inhibiting the capture of HIV-1 virus to several mAbs, this made the remaining HIV-1 Env particles more sensitive for neutralization as others have previously seen (90, 101). Lastly, my pulse-chase analysis of Env in transfected cells and immunoprecipitation (Figure 2-9) indicates that the 2G12 epitope is formed rapidly upon synthesis and is not affected by SERINC5 when the protein is immunoprecipitated in detergent (RIPA buffer). I therefore suggest that SERINC5 inhibits 2G12 capture of

HIV-1 particles by affecting the Env conformation and occluding the antibody access to the N-linked glycan epitope.

The observations that I made while investigating the epitopes that SERINC5 affects to inhibit HIV-1 capture suggests a model of SERINC5 inhibition where some, but not all, of the HIV-1 Env on HIV-1 particles are conformationally altered (Figure 3-1).

I propose this model from the infectivity data that I described in Figure 1-1 and Figure 2-

1, where even at the highest concentrations of SERINC5, there was still some infectious virus that was able to infect the cells, and from the CD4-induced 17b experiment where

I saw that virus co-transfected with SERINC5 was still able to bind CD4 and increase its binding to the 17b antibody by 5.3x (Figure 2-4). I propose that whether during trafficking or when incorporated into the budding HIV-1 particle, SERINC5 alters the conformation of the HIV-1 Env trimer. From the results from the VCAs that I conducted in chapter 2, I predict that SERINC5 is pulling the HIV-1 Env monomers away from each other to where the PGT145 mAb can no longer bind as efficiently to the Env trimer conformation. More studies will need to be completed in order to develop an atomic structure of how SERINC5 is altering the HIV-1 Env trimer conformation. Other researchers have recently suggested a similar model that correlates with my findings

(104, 117). In my study, I was also able to link that the inhibition of infectivity is directly linked with the inhibition of capture (Figure 2-5).

There are several aspects in my proposed model that provide future directions for this project. I hypothesize that SERINC5 directly interacts with HIV-1 Env in order to inhibit HIV-1 infectivity. Currently, there are no assays that have provided evidence that

SERINC5 directly interacts with HIV-1 Env in order to inhibit HIV-1 infectivity. Therefore,

51 testing this hypothesis will require the development of new technologies and the optimization of biochemical techniques. There are several biochemical techniques that could help answer this hypothesis. Protein-protein interactions can be detected through techniques like the proximity ligation assay, through co-immunoprecipitation, through chemical crosslinking, or by using in vitro binding between recombinantly purified

SERINC5 and purified HIV-1 virions (119). Co-immunoprecipitation is a logical starting point, however, from the initial Co-IP data I collected, I was unable to Co-IP SERINC5 and HIV-1 Env. Others in the field have also tried to Co-IP HIV-1 Env and SERINC5 and have not had any success (90). Therefore, I suggest using a more stringent approach by conducting a set of proximity ligation experiments with SERINC5 + Env and

SERINC2 + Env in order to determine whether SERINC5, and not SERINC2, provides fluorescence activity when in close proximity with HIV-1 Env. The fluorophores that I would engineer on SERINC5 and HIV-1 Env can only fluoresce when the two proteins are 40 nm or less from each other. If SERINC5 and HIV-1 Env fluoresce, but SERINC2 and HIV-1 Env do not, then this would suggest that the two proteins are in close enough proximity to directly interact with one another. This data, along with the previous data that I reported in Chapter 2, would suggest that a direct interaction between SERINC5 and HIV-1 Env is important for SERINC5’s antiviral activity. If the initial results provide evidence that SERINC5 is in close proximity to HIV-1 Env, then I would include the

SERINC5 and SERINC2 internal chimerae made by Schulte et al. to determine which portion of SERINC5 is necessary for the interaction between HIV-1 Env and SERINC5.

In that report, the internal region of SERINC5 (amino acids 203-411) were swapped with

SERINC2 and were shown to be necessary for SERINC5’s antiviral activity. Therefore, I

52 would hypothesize that this region would also be important for a direct interaction to occur if the direct interaction is important for SERINC5’s antiviral activity.

An alternative approach to determine whether SERINC5 and HIV-1 Env directly interact with one another is to use a chemical crosslinker and see if this would stabilize the interaction between SERINC5 and HIV-1 Env. A potential target would be the sulfhydryl groups at the N-terminus of SERINC5. Human SERINC5 has 19 cysteine residues at the N-terminus of the protein, therefore, I would start by using glutaraldehyde as a potential crosslinker because it is known to react with cysteine residues and because others have used it in the field to stabilize gp120 or gp140 proteins (123–125). To test whether the chemical crosslinking works, I will first co-IP western the recombinantly expressed HIV-1 Env trimer, BG505 SOSIP.664, expressed in 293T cells along with recombinantly expressed SERINC5 from Peter Cherepanov’s lab using the mAbs 2G12 and rat α-HA (119). Once I can identify that the chemical crosslinking works, I would attempt to co-immuoprecipitate HIV-1 Env with SERINC5 and SERINC2 to see if by stabilizing the two proteins, I can detect the two proteins together by western blot. One potential limitation for using glutaraldehyde as a chemical crosslinker is that it may occlude some antibodies from being able to bind to HIV-1 Env.

An alternative chemical crosslinker that could be used to stabilize the interaction between HIV-1 Env and SERINC5 is EDC/NHS [1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysuccinimide], which has been shown to be effective in stabilizing the HIV-1 Env trimer conformation (123, 126).

EDC/NHS is a good alternative crosslinker because it is a synthetic crosslinker with better defined specificity and it does not leave linker atoms behind after crosslinking compared to

53 glutaraldehyde. Now that there is a recombinantly expressed SERINC5 protein that is readily accessible to use, I anticipate that using chemical crosslinking and in vitro binding will allow us to determine whether SERINC5 directly interacts with HIV-1 Env in order to inhibit HIV-1 infectivity.

Other aspects of the model that need to be addressed are: 1. What specific amino acids on HIV-1 Env are necessary for SERINC5’s antiviral activity, and 2. Is

SERINC5 perturbing the conformation of a fraction of the Env trimers on the surface of the virus? My results provide evidence that SERINC5 targets the V3 loop, a CD4- induced epitope, an N-linked glycan on gp120, and the Env trimer conformation. To further test which specific epitopes on HIV-1 Env are necessary for SERINC5’s antiviral activity, scanning alanine mutagenesis could be conducted on the aforementioned HIV-

1 Env epitopes to determine whether SERINC5 interacts with specific amino acids on

HIV-1 Env in order to inhibit HIV-1 infectivity. My initial findings provide a good starting point for regions that should be investigated as potentially necessary for SERINC5’s antiviral activity. However, there still could be other regions of HIV-1 Env that the VCAs did not identify that could also be necessary for SERINC5’s antiviral activity and this should be investigated. To determine whether SERINC5 is perturbing the conformation of a fraction of the Env trimers on the surface of the virus, I could repeat the VCAs with

PGT145 and titrate increasing concentrations of SERINC5 to determine whether the capture of PGT145 decreases as the concentration increases. If my hypothesis is correct, then this would suggest that SERINC5 does perturb the conformation of some but not all of the Env on the surface of the HIV-1 particle.

Another potential mechanism for how SERINC5 inhibits HIV-1 infectivity is through altering the glycosylation of the HIV-1 Env, which consequently, alters the conformation of the HIV-1 Env. The pulse-chase experiments that I conducted in chapter 2 suggest that SERINC5 does not detectably alter the rate of Env biosynthesis, processing, glycosylation, or degradation in transfected 293T cells. However, SERINC5 could be affecting glycosylation at other sites on HIV-1 Env, like on gp41, which is also heavily glycosylated. To answer this question, mass spectrometry analysis of the glycosylation of HIV-1 Env could be conducted on HIV-1 Env expressed in the presence or absence of SERINC5.

An alternative model as to how SERINC5 inhibits HIV-1 infectivity could be through reducing the number of HIV-1 Env proteins that are incorporated into the virus particle and SERINC5 altering the conformation of HIV-1 Env. This combination could have a significant impact on the infectivity of the virus, since other studies have previously reported that the number of HIV-1 Env molecules on the virus are important for the infectivity of HIV-1 (127, 128). To test this hypothesis, one should repeat the IP-

Western blot experiments conducted in chapter 2 with more HIV-1 Env mAbs to determine if this result is reproducible. It would be helpful to repeat these experiments side-by-side by western blot analysis to determine if it is reproducible in both assays. If the result is reproducible, then this would suggest that SERINC5 could reduce the number of HIV-1 Env proteins on the surface of the protein. This would be highly unusual, since no one else in the field has reported that SERINC5 reduces the number of HIV-1 Env proteins on the surface of the virus. It is possible that I only observed the co-IP western results when I used 2G12 and 401 and not when I pelleted HIV-1 virions

55 and probed them via western blot due to the initial binding of the antibody to the proteins during the overnight IP. Alternatively, pelleting the virus via western blot, could lose some of the effects caused by SERINC5 because the “heavier” virions at the bottom of the tube are the virions with more Env on the virus, and when I aspirate everything but the virus pellet, I aspirate some of the virus with less Env on the virus that is further up in the sucrose and not at the bottom of the tube. It is also possible that

I see reduced Env on the virus with some mAbs, but not all, due to effects that

SERINC5 has on the conformation of the Env on the virus. By testing each of these variables in additional experiments, I hope to identify whether these results are reproducible. If the results in Figure 2-9 are reproducible, then I will improve upon my model due to new results that will alter how I think SERINC5 is inhibiting the infectivity of HIV-1.

HIV-1 Fusion

1. CD4 Binding 2. Co-receptor Binding 3. Fusion Peptide Insertion 4. Hairpin formation and membrane fusion

HIV-1 + SERINC5 Fusion

1. CD4 Binding 2. Co-receptor Binding 3. Fusion Peptide Insertion 4. Hairpin formation and No/partial membrane fusion

gp41 gp120 Conformationally SERINC5 Env fusion altered gp120 peptide

CD4 CXCR4/CCR5 target cell membrane HIV-1 membrane

Figure 4-1 Model for SERINC5 Inhibition of HIV-1 infection. SERINC5 inhibits HIV-1 infectivity from preventing the virus from fusing to the target cell. SERINC5 alters the conformation of some of the HIV-1 Env trimer on the virus. The conformational change of HIV-1 Env by SERINC5 is linked to the inhibition of infectivity.

Importance

SERINC5 is a host-cell protein that inhibits the infectivity of HIV-1 by a novel and poorly understood mechanism. In my dissertation, I have provided evidence that the

SERINC5 protein alters the conformation of the HIV-1 Env proteins, and this action is correlated with SERINC5’s ability to inhibit HIV-1 infectivity. These results have helped develop a new model for how SERINC5 is inhibiting the infectivity of HIV-1 and will help to continue to grow this booming area of HIV-1 research. With these results and with further defining the specific effects of SERINC5 on the HIV-1 glycoprotein conformation, new antiviral strategies targeting HIV-1 Env can be developed to help treat people who are infected with HIV-1.

Chapter 5

Materials and Methods

Plasmids.

The viruses used in this study were generated from the pNL4-3 and pNL4-3 ΔN proviral clones, which were acquired from the NIH AIDS Reagent Program, Division of

AIDS, NIAID, NIH (129). The pNL4-3ΔE plasmid encodes a frameshift at the Ndel restriction site in env, resulting in Env-deficient particles.

Env-deficient viruses were pseudotyped with the HXB2 and JR-FL-expressing

Env plasmids, pCAGGS-HXB2 and pCAGGS-JR-FL, which were provided by Dr. Greg

Melikyan (90). The SERINC2-HA and SERINC5-HA expressing plasmids, pBJ5-

SERINC2-HA and pBJ5-SERINC5-HA, were provided by Dr. Heinrich Gottlinger (2,

130). The SERINC5-HA expressing plasmid, CMX-SERINC5-HA, was provided by Dr.

Heinrich Gottlinger.

Antibodies and recombinant proteins.

Information on antibodies and recombinant proteins is provided in Table 1. All of the reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS,

NIAID, NIH, except for Goat Anti-Human IgG Fcγ, which was purchased from Jackson

ImmunoResearch Laboratories, Inc.

Cells and viruses.

Human embryonic kidney (HEK293T) cells were purchased from the American

Type Culture Collection. TZM-bl reporter cells for assaying HIV-1 infectivity were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH

(catalog number 8129), from John C. Kappes, Xiaoyun Wu, and Tranzyme Inc. (131–

135). All cells used in this study were cultured at 37°C in 5% CO2 in Dulbecco’s modified Eagle medium (DMEM; Cellgro) supplemented with 10% fetal bovine serum

(FBS), penicillin (50 IU/mL), and streptomycin (50 µg/mL). The virus stocks used for infectivity and the virus capture assays were produced by polyethylenimine (PEI) transfection of HEK293T cells as previously described (136, 137). For viruses produced by co-transfection, the total amount of DNA transfected (10 µg) was kept constant in the transfections. HXB2 HIV-1 particles were produced by co-transfection of 5 µg of NL4-3

ΔEnv and 5 g of pCAGGS-HXB2, whereas JR-FL HIV-1 particles were produced by co-transfection of 7.5 µg of NL4-3 ΔEnv and 2.5 g of pCAGGS-JR-FL. The quantity of

HXB2 and JR-FL Env plasmids used in this study were established by quantifying the expression of Env in cell lysates and concentrated virus particles by immunoblot analysis (data not shown). Viral supernatants were filtered through a 0.45 µm pore-size syringe filters, and the viruses were aliquoted and stored at -80°C. Virus aliquots were thawed only once prior to use and not refrozen. The CA content of the virus stocks were normalized by p24 Enzyme-Linked ImmunoSorbent Assay (ELISA) (138). The ER retention signal, KDEL, was engineered by creating primers that would bind and add the four additional amino acids to the C-terminus of the CMX-SERINC5-HA-signal that was engineered by Dr. Heinrich Gottlinger (2, 130). I engineered a point mutation in the

KDEL ER-retention signal to change KDEL to ADEL in order to inhibit the ER-retention signal (120–122). I used the restriction sites, KpnI and NheI, to anneal the KDEL and

ADEL mutants to the CMX-SERINC5-HA backbone.

Assay of HIV-1 infectivity.

The viral infectivity for each virus was determined by titrating each virus stock onto cultured TZM-bl cells and assaying the expression of luciferase reporter activity in cell lysates as previously described (137).

Virus capture assays.

Viruses binding to antibodies was quantified by assaying particles captured by immobilized antibodies using procedures optimized from previously reported protocols

(105, 107, 109). The virus capture assays were prepared by pipetting 100 µL of 4 µg/mL capture antibody diluted in Phosphate Buffer Saline (PBS) to wells of a 96-well flat- bottom Immunon II plate (Dynex Tech) plate. The plate was sealed and incubated at

37°C overnight. The liquid in the virus capture assay plate was then discarded and the

96-well plate was washed three times with 200 µL PBS. The plate was blocked by preparing 5% calf serum in PBS. 250 µL of the blocking buffer was pipetted into each well, and the plate was incubated at 37°C for 1 hour. The blocking buffer in the plate was discarded and the plate was washed three times with 200 µL of PBS. Virus was diluted to 3.75 ng of virus in 200 µL complete medium and pipetted into each well, and the plate was sealed and incubated at 37°C for 2 hours. The liquid in each well was

61 aspirated and the wells were washed twice with 200 µL complete medium. After two washes, the bound viruses were eluted in Tris-buffer saline (50 mM Tris-HCl [pH 7.8],

130 mM NaCl, 10 mM KCl, 5 mM MgCl2) containing 0.5% Triton X-100 and assayed by p24 ELISA.

For indirect virus capture assay by 2G12 and 2F5 monoclonal antibodies, viruses

(8 ng p24) were incubated for two hours with primary antibodies (4 µg/mL) in 500 µL of complete medium, then pelleted by centrifugation at 45,000 RPM (125,000 x g) for 20 minutes at 4°C in a Beckman TLA 55 rotor. The supernatant was removed, and the pelleted virions were resuspended in 500 µL of 1X PBS. Samples (250 µL) were added to duplicate wells of a 96-well plate that had been precoated with 4 µg/mL of polyclonal

α-Fc antibody and blocked. Plates were incubated and captured virions quantified as in the direct capture assay. A total of 25 mAbs or recombinant proteins were tested for use in the VCAs (Table 4-1).

CD4-virus capture assay.

For the CD4-virus capture assay, we used the direct virus capture assay protocol outlined above, except instead of using a mAb to capture the viruses, we used recombinantly expressed CD4 which was fused to the hinge and Fc regions of human

IgG2 (CD4-IgG2) which was provided by the NIH AIDS Reagent Program, Division of

AIDS, NIAID, NIH: Cat#11780, CD4-IgG2 from Progenics Pharmaceuticals.

Table 4-1 Antibodies and Recombinant Proteins Used in Study Antibody/Protein Epitope Reference(s) Name (95, 112–114, 139, 17b gp120 140) HIV-1 gp41 (epitope: 2F5 ELDKWA) (141–143) 2G12 N-Linked Glycan on gp120 (114, 141, 144–146) 447-52D V3 loop (147) 48D CD4i epitope (112, 148) b12 CD4 binding site (149–152) 10-1074 V3 loop (153) 10e8 MPERα (154) 2191 V3 loop (155) V2/V3 loop quaternary (155–158) 2909 neutralizing epitope 35O22 gp41/gp120 interface (154) 401 gp120 Intracel HIV-1 gp41 (epitope: 4E10 NWFDIT) (159) V3 loop (epitope: 4G10 RIQRGPGRAFVTIGK) (160) 50-69 gp41 (161–164) V3 loop (epitope: 5F7 RIQRGPGRAFVTIGK) (160) A32 CD4i epitope (113, 148) CH58 V2 (165, 166) F425 B4e8 gp120 (167, 168) HG120 V2 loop (165, 166) HIV Ig HIV-1 (169) PG16 V1/V2 loop (170) VRC03 CD4 binding site on gp120 (171) Jackson Fc portion of human IgG ImmunoResearch IgG Fcγ heavy chain Laboratories, Inc. Progenics CD4-IgG2 CD4 binding site Pharaceuticals, Inc. Progenics sCD4 CD4 binding site Pharmaceuticals, Inc. αMPER, Membrane Proximal External Region

Pulse-chase analysis of Env biosynthesis and processing.

Two million 293T cells were seeded in 60 mm dishes and cultured for 24h prior to transfection. Monolayers were transfected with NL4-3.Nef- proviral plasmid (5 µg) and pBJ5 plasmids encoding HA-tagged SERINC2 or SERINC5 (1 µg) using a calcium phosphate procedure(172). After 12 hours of culture, monolayers were washed and replenished with complete medium and cultured for an additional 1-2 hours. Metabolic labeling of proteins was performed essentially as described(173). Cells were starved by culture for one hour in DMEM lacking cystine and methionine (Mediatech, Inc.) supplemented with 10% dialyzed FBS. Cells were pulse-labeled by culture for 20 minutes at 37°C in starvation medium supplemented with 0.2 mCi/mL of EXPRE35S35S

Protein Labeling Mix (1175 Ci/mmol, PerkinElmer). Cultures were washed twice in complete medium, then replenished and cultured for the indicated chase times. Cells were harvested by detachment with PBS containing 1 mM EDTA, pelleted, and lysed in

0.3 mL of PBS containing 1% NP-40, 0.1% sodium dodecyl sulfate [SDS], 0.5% sodium deoxycholate and protease inhibitors (cOmplete Tablets Mini, Roche). Cell debris was removed by centrifugation for 5 min at 16,000 xg in a refrigerated microcentrifuge at

4°C. Prior to immunoprecipitation, extracts were pre-cleared by incubation with 1 microliter of normal human serum and protein A/G+ beads. Proteins were immunoprecipitated by overnight rotation at 4°C in 0.3 mL of solution containing 20 mL of a 1:1 slurry of Protein A/G+ beads (Santa Cruz, Inc.) that had been pre-bound to monoclonal and polyclonal antibodies specific for gp120 and/or gp41. Beads were washed according to the method of Bonifacino et al. (174). Beads were resuspended in

30 mL of Laemmli sample buffer containing 5% 2-mercaptoethanol, and bound proteins

64 were eluted by heating at 95°C for 5 min. Proteins were eluted and digested with endoglycosidase H according to the enzyme manufacturer’s protocol (New England

Biolabs, Inc.) Proteins were separated by electrophoresis on 4-20% polyacrylamide protein gels (Genscript), and gels were fixed and stained with Coomassie blue, dried, and radioactive proteins detected with a FLA7000IP Typhoon Storage Phosphorimager

(GE Healthcare). Immunoblotting of HIV-1 particles was performed by subjecting 1 mL of HIV-1 particles to ultracentrifugation through a 0.2 mL cushion of PBS containing

20% sucrose at 100,000 xg for 30 min in a Beckman TLA-55 rotor. Pellets were dissolved and proteins separated by electrophoresis as described above. For reference, a sample of the Precision Plus Protein Kaleidoscope Prestained Protein

Standards (Bio-Rad) was analyzed on the gel. Proteins were electrophoretically transferred to nitrocellulose and detected by probing with the indicated monoclonal and polyclonal antibodies. Bands were detected by probing with IR dye-conjugated secondary antibodies (LI-Cor) and visualized by scanning in the Li-COR Odyssey instrument.

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