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GSBS Dissertations and Theses Graduate School of Biomedical Sciences
2018-08-06
SERINC5: Its Sensitivity to Nef and Restriction of HIV-1
Weiwei Dai University of Massachusetts Medical School
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SERINC5: ITS SENSITIVITY TO NEF AND RESTRICTION OF HIV-1
A Dissertation Presented
By
Weiwei Dai
Submitted to the Faculty of the
University of Massachusetts Graduate School of Biomedical Sciences, Worcester
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
August 3rd, 2018
Immunology and Microbiology Program
SERINC5: ITS SENSITIVITY TO NEF AND RESTRICTION OF HIV-1
A Dissertation Presented By Weiwei Dai The signatures of the Dissertation Defense Committee signify completion and approval as to style and content of the Dissertation
Heinrich Göttlinger, M.D., Thesis Advisor
Celia Schiffer, Ph.D., Member of Committee
Timothy Kowalik, Ph.D., Member of Committee
Maria Zapp, Ph.D., Member of Committee
Markus Thali, Ph.D., External Member of Committee
The signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee
Trudy Morrison, Ph.D., Chair of Committee
The signature of the Dean of the Graduate School of Biomedical Sciences signifies that the student has met all graduation requirements of the school.
Mary Ellen Lane, Ph.D.,
Dean of the Graduate School of Biomedical Sciences Immunology and Microbiology Program August 3rd, 2018 Acknowledgments
Throughout my Ph.D. journey there have been many people whose mentorship, inspiration, friendship and support have been greatly appreciated. I am very grateful to my advisor, Dr. Heinrich Göttlinger, for providing the excellent scientific training and opportunity to do my thesis research in his lab. Additionally,
I appreciate support from all the Göttlinger lab members. In particular, I would like to thank Yoshiko Usami, who contributed tremendously to this work, and
Paul Peters, who helps edit and improve my writing.
I would like to offer my heartfelt thanks to the members of my committee,
Trudy Morrison, Celia Schiffer, Paul Clapham, Timothy Kowalik, Abraham Brass,
Maria Zapp, Markus Thali. I thank them all for the patience and interest they showed in my progress and growth. I especially thank Trudy, who believes in me from the very beginning of my graduate school study. She has offered guidance and encouragement at all critical moments. Her expectation in me was once my major motivation during hard time of graduate school.
I would also like to thank the excellent community provided by UMass
Medical School. Dean, Mary Ellen Lane, is the one who really cares about students and our education but not only training. UMass Med alumni are kind to help whenever I reach out. I also thank all my friends on- and off- campus, whose companion and encouragement means a lot to me.
i Finally, this work would not have been possible without the unconditionally love and support of my parents and my husband, who make everything possible.
ii Abstract
The accessory protein Nef of human immunodeficiency virus type 1 (HIV-1) has long been known to enhance the infectivity of HIV-1 progeny virions. The multipass transmembrane proteins serine incorporator 3 (SERINC3) and
SERINC5 were recently identified as novel antiviral proteins that restrict HIV-1 infectivity. Nef enhances HIV-1 infectivity by removing SERINCs from the plasma membrane, which prevents their incorporation into progeny HIV-1 virions. To exploit this potent intrinsic antiretroviral factor for potential therapy development, it is critical to explore the determinants in SERINC5 that govern its downregulation by Nef and its restriction on HIV-1 infectivity. Here I report that the ability to inhibit HIV-1 infectivity is conserved among vertebrate SERINC5 proteins, whereas the sensitivity to downregulation by Nef is not. However, a Nef- resistant SERINC5 became Nef-sensitive when its intracellular loop 4 (ICL4) was replaced by that of Nef-sensitive human SERINC5. Conversely, human
SERINC5 became resistant to Nef when its ICL4 was replaced by that of a Nef- resistant SERINC5. In general, ICL4 regions from SERINCs that exhibited resistance to a given Nef conferred resistance to the same Nef when transferred to a sensitive SERINC, and vice versa. I demonstrate that human SERINC5 can be modified to restrict HIV-1 infectivity even in the presence of Nef. Moreover, by generating chimeras between SERINC5 and SERINC2, which does not exhibit antiretroviral activity, I demonstrate that SERINC5’s inhibitory function, unlike the
iii sensitivity to Nef, requires the participation of more than one region. Helix 4 and extracellular loop 5 (ECL5) of SERINC5 are both required for the potent restriction of HIV-1 infectivity. In contrast, a large amino-terminal portion of
SERINC5 is not required for its antiretroviral activity of SERINC5. The determinants in ECL5 disperse throughout the loop. Furthermore, the ECL5 of
SERINC5 is a hotspot region that determines the Env-dependent antiretroviral activity of SERINC5.
iv Table of Contents
Acknowledgments ...... i Abstract ...... iii LIST OF FIGURES ...... vii LIST OF TABLES ...... ix LIST OF COPYRIGHTED MATERIALS PRODUCED BY THE AUTHOR ...... x LIST OF THIRD PARTY COPYRIGHTED MATERIAL ...... xi LIST OF ABBREVIATIONS ...... xii Chapter I: Introduction ...... 1 HIV-1 genomic structure and virion structure ...... 2 HIV-1 replication and antiretroviral drug therapy...... 5 HIV-1 entry ...... 8 Reverse transcription ...... 13 DNA integration ...... 18 Viral gene expression...... 22 Viral particle production ...... 27 HIV-1 pathogenesis ...... 33 HIV restriction factors ...... 36 Discovery of HIV-1 restriction factors ...... 37 APOBEC3G ...... 37 TRIM5a ...... 39 Tetherin ...... 40 Newly defined restriction factors ...... 43 The role of Nef in HIV-1 replication and pathogenesis ...... 44 Functional domains in Nef ...... 45 Nef-mediated downregulation of surface proteins ...... 48 Nef modulates signal transduction pathways ...... 50 Nef enhances the infectivity of progeny virions ...... 51 Summary of dissertation ...... 53 Chapter II: A long cytoplasmic loop governs the sensitivity of SERINC5 to Nef...... 54 Introduction ...... 55 The discovery of SERINC5...... 55 SERINC protein family...... 56 Nef-mediated downregulation of surface proteins...... 58 Downregulation of SERINC5 by Nef...... 60 Summary ...... 61
v Results ...... 62 Discussion ...... 83 The highly conserved antiviral activity of SERINC5...... 85 The mechanism and machinery of the counteraction of SERINC5 by Nef...... 85 The effect of Nef polymorphism on the counteraction of SERINC5...... 86 The role of SERINC5 and its counteraction in viral replication...... 88 Materials and methods ...... 90 Chapter III: The determinants in SERINC5 for its Env-dependent antiretroviral activity ...... 96 Abstract ...... 96 Introduction ...... 97 The mechanism by which SERINC5 blocks HIV-1...... 97 Env-dependent antiretroviral activity of SERINC5...... 98 SERINC5 incorporation enhances HIV-1 sensitivity to neutralizing antibodies. .... 102 Summary ...... 103 Results ...... 104 Discussion ...... 124 The determinants of SERINC5 that governs the restriction...... 124 The impact of SERINC5 on conformational changes in Env...... 126 The antagonism of SERINC5 by Env...... 127 How does SERINC5 possibly affect HIV-1 pathogenesis?...... 129 Materials and methods ...... 130 Chapter IV: Discussion ...... 136 Counteraction of SERINC5 by Nef...... 136 Unknown functions of SERINC proteins ...... 140 Conditional inhibition of HIV-1 by SERINC5 ...... 142 Conclusions ...... 146 Bibliography ...... 147
vi LIST OF FIGURES
Figure 1.1 HIV-1 genomic structure and virion structure. 4 Figure 1.2 Distinct classes of antiretroviral drugs target different steps of the HIV-1 replication cycle in a CD4+ T cell. 7 Figure 1.3 Steps of HIV-1 entry. 12 Figure 1.4 Mechanisms of actions of NRTIs and NNRTIs. 17 Figure 1.5 DNA cutting and joining steps of integration. 20 Figure 1.6 Early and late phases of HIV-1 mRNA expression. 26 Figure 1.7 The Gag polyprotein coordinates assembly, budding and maturation. 31 Figure 1.8 Time course of typical HIV-1 infection 35 Figure 1.9 Interplay between restriction factors and accessory proteins. 42 Figure 1.10 Sequence alignment and protein-protein interaction domains in HIV-1 and SIV Nef. 47 Figure 1.11 The activities of Nef in infected cells. 52 Figure 2.1 Anti-HIV-1 activity but not responsiveness to Nef is conserved among SERINC5 orthologs. 64 Figure 2.2 Transfer of human SERINC5 ICL4 is sufficient to confer Nef responsiveness to frog SERINC5. 67 Figure 2.3 The ICL4 of human SERINC5 confers sensitivity to widely divergent Nefs. 69 Figure 2.4 The ICL4 of frog SERINC5 confers resistance to Nef. 71 Figure 2.5 The physical interaction between Nef and the ICL4 of human SERINC5. 73
Figure 2.6 The ICL4 of human SERINC2 confers resistance to NefSF2 but not Nef97ZA012. 75 Figure 2.7 Swapping the ICL4 of human SERINC5 for that of human SERINC2 confers resistance to down-regulation by NefSF2. 76
vii Figure 2.8 Human SERINC3 acquires sensitivity to NefSF2 in the presence of the ICL4 of human SERINC5. 78 Figure 2.9 Point mutations in the ICL4 of human SERINC5 confer resistance to Nef. 80 Figure 2.10 Nef poorly counteracts inhibition of HIV replication by a SERINC5 chimera harboring frog SERINC5 ICL4. 82 Figure 2.11 A long cytoplasmic loop governs the sensitivity of SERINC5 to Nef. 84 Figure 3.1 In contrast to other N-proximal cysteines of SERINC5, C13 is critical for the potent antiviral activity of SERINC5. 106 Figure 3.2 The incorporation of chimeras between SERINC5 and SERINC2. 110 Figure 3.3 The role of SERINC5’s helices in the restriction of HIV-1 infectivity. 113 Figure 3.4 SERINC5 bearing the ECL5 of SERINC2 fails to inhibit the infectivity of Nef- HIV-1 virions. 116 Figure 3.5 Chimeric SERINC2 proteins fail to inhibit HIV-1 infectivity. 119 Figure 3.6 Deletions within the ECL5 of SERINC5 only partially impaired its ability to restrict HIV-1 infectivity. 121 Figure 3.7 ECL5 mutations proportionally disrupts the antagonism of SERINC5 by Env. 123 Figure 4.1 The significance of each domain of SERINC5 in the inhibition of HIV-1. 143
viii LIST OF TABLES
Table 3.1 The effects of fusion inhibitors and neutralizing antibodies on SERINC5’s function. 101 Table 3.2 The phenotype of chimeras between SERINC5 and SERINC1 108 Table 3.3 The phenotype of chimeras between SERINC5 and SERINC2 111 Table 3.4 The primary sequences of chimeras of SERINC5 and SERINC1. 133 Table 3.5 The primary sequences of chimeras of SERINC5 and SERINC2. 133 Table 3.6 The primary sequences of chimeric SERINC5 proteins with exchanged helices and ECLs of SERINC2. 134 Table 3.7 The primary sequences of chimeric SERINC2 proteins. 134
ix LIST OF COPYRIGHTED MATERIALS PRODUCED BY THE AUTHOR
Chapter II is reprinted and adapted from the co-authored with permission from Elsevier. License #: Creative Commons Attribution – NonCommercial – NoDerivs (CC BY- NC-ND 4.0) Dai, W., Usami, Y., and Gottlinger, H. (2018). A Long Cytoplasmic Loop Governs the Sensitivity of the Anti-viral Host Protein SERINC5 to HIV-1 Nef. Cell Rep. 22, 869–875.
x LIST OF THIRD PARTY COPYRIGHTED MATERIAL
Figure # Publisher License # Figure 1.1 Springer Nature 4380800455330 Figure 1.2 Cold Spring Harbor Laboratory Press Open resource Figure 1.3 Cold Spring Harbor Laboratory Press Open resource Figure 1.4 Springer Nature 4380801373929 Figure 1.5 Cold Spring Harbor Laboratory Press Open resource Figure 1.6 Annual Reviews, Inc 4392041427456 Figure 1.7 Cold Spring Harbor Laboratory Press Open resource Figure 1.8 Cold Spring Harbor Laboratory Press Open resource Figure 1.9 Cold Spring Harbor Laboratory Press Open resource Figure 1.10 American Society for Microbiology Open resource Figure 1.11 Elsevier 4372550955431
xi LIST OF ABBREVIATIONS
6HB Six-helical Bundle 7SK snRNP 7SK Small Nuclear Ribonucleoprotein AIDS Acquired Immune Deficiency Syndrome ALCAM Activated Leukocyte Cell Adhesion Molecule AP Adaptor Protein APOBEC3G Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G ART Antiretroviral Therapy ASK1 Apoptosis Signaling Regulating Kinase 1 BlaM b-lactamase CA Capsid CDK9 Cyclin-dependent Kinase 9 cryo-EM Cryo-electron Tomography CTL Cytotoxic T Lymphocytes DIS Dimer Initiation Site DKO Double Knockout ECL Extracellular Loop EIAV Equine Infectious Anemia Virus Env Envelope ESCRT Endosomal Sorting Complexes Required for Transport FDA US Food and Drug Administration GFP Green Fluorescence Protein glycoGag Glycosylated Gag protein HAART Highly Active Antiretroviral Therapy HDAC Histone Deacetylases HDACi Histone Deacetylase Inhibitors HIV-1 Human Immunodeficiency Virus type 1 HLA Human Leukocyte Antigen HR Heptad Repeat ICL Intracellular Loop IFITM Interferon Induced Transmembrane protein IN Integrase LEDGF/p75 Lens Epithelium-derived Growth Factor LTR Long Terminal Repeat LRA Latency Reversing Agent MA Matrix MHC-I Major Histocompatibility Complex class 1 MLV Murine Leukemia Virus MPER Membrane-proximal External Region
xii MVB Multivesicular Bodies MX2 Myxovirus resistance 2 Nef Negative Factor NC Nucleocapsid Nef Negative Factor NFAT Nuclear Factor of Activated T cells NSI Nonsyncytium Inducing NNRTI Non-nucleoside-analog Reverse Transcriptase Inhibitor NRTI Nucleoside-analog Reverse Transcriptase Inhibitor P-TEFb Positive Transcription Elongation Factor b Pak P21-activated Kinase pbs primer binding site PIC Preintegration Complex ppt polypurine track PR Protease RRE Rev-Responsive Element RT Reverse Transcriptase RTC Reverse Transcription Complex SAMHD1 SAM Domain HD Domain-containing protein 1 SERINC Serine Incorporator SP Spacer Peptide SP1 Specificity Protein 1 SU Surface TAR Transactivation-responsive Region TCR T-cell Receptor TGN Trans-Golgi Network TM Transmembrane TRIM5a Tripartite Motif 5-a Tsg101 Tumor susceptibility gene 101 UEV Ubiquitin E2 Variant VS Virological Synapse VSV-G Vesicular Stomatitis Virus G protein
xiii Chapter I Introduction
Since the first case of acquired immune deficiency syndrome (AIDS) in the
US was reported in 1981, HIV-1 has infected worldwide more than 70 million people and caused the death of about 35 million people (WHO 2017). Thanks to the development of antiretroviral therapy (ART), the HIV-1 infection can be controlled and is considered as a clinically controllable chronic disease. Potent combination ART provides remarkable control of viral loads, and restores immunity in patients. Even with dramatic reduction in morbidity and mortality, there were still 36.7 million people living with HIV/AIDS at the end of 2016. 1.0 million people died of HIV-1-related illnesses in 2016 (UNAIDS). There is no cure or vaccine yet. If ART treatment is interrupted, viral rebound occurs due to the establishment of latently infected resting CD4+ T cells (Chun et al., 1997; Finzi et al., 1999). Taken together, a better understanding of host-viral interactions and immune defenses is required. In addition to innate and acquired immune responses, humans have evolved specific antiviral factors that are constitutively expressed and already active at the first virus-cell interaction. This dissertation investigates the inhibition of HIV-1 by a novel host antiretroviral factor, SERINC5, and its counteraction by the HIV-1 viral protein Nef. This introductory chapter prepares the background on HIV-1 and the logic for this thesis research.
1 HIV-1 genomic structure and virion structure
HIV is a lentivirus containing two copies of positive-sense single-stranded viral RNA. The HIV-1 genome encodes the major structural and non-structural proteins shared with other replication-competent retroviruses (Figure 1.1A)
(Freed, 2001). The long terminal repeats (LTR), composed of U3, R and U5 regions, are at the 5’ and 3’ end. The gag, pol and env genes, from the 5’ to 3’ ends of the genomes, encode the essential structural and enzymatic proteins.
Their translation products are initially synthesized as polyprotein precursors, which are subsequently processed into viral proteins incorporated into the mature virions. The gag gene encodes the precursor protein Pr55Gag, which is cleaved by the viral protease (PR) into the mature Gag proteins matrix (MA or p17), capsid (CA or p24), nucleocapsid (NC or p7), p6, as well as two spacer peptides
(SP), p1 and p2. The 160-kd Gag-Pol precursor is cleaved by the viral PR and gives rise to three individual pol-encoded enzymes, which are PR, reverse transcriptase (RT), and integrase (IN). The envelope (Env) glycoprotein precursor, gp160, is encoded by the env gene. Unlike the Gag and Pol precursors, gp160 is processed by a cellular protease, which result in two glycoproteins. One is the surface (SU) Env glycoprotein gp120, and the other is the transmembrane (TM) glycoprotein gp41. They are organized into trimeric spikes displayed on the surface of the virion. They are non-covalently associated. Gp120 contains five relatively conserved domains (C1–C5) and five
2 variable regions (V1– V5). Gp41 is composed of the ectodomain, transmembrane domain and the cytoplasmic tail.
In addition to the gag, pol and env genes, HIV-1 encodes the essential regulatory proteins Tat and Rev, as well as the accessory proteins Vif, Vpr, Vpu, and Nef. Tat is essential for transcriptional elongation from the HIV-1 promoter, while Rev is required for the transport of viral RNA from the nucleus to the cytoplasm. Vif, Vpr, Vpu, and Nef were named “accessory”, because they are not required for virus replication in tissue culture. However, they are critical elements of combating host intrinsic immunity. The function of accessory viral proteins will be discussed in the following part of this chapter.
The virion packages all of the components required for infectivity. MA forms the discontinuous outer matrix shell, lying underneath the lipid membrane of mature virions. It attaches to the lipid membrane via an amino-terminal myristoylated and positively charged segment. Env forms trimeric spikes on the surface of the virion. The CA protein forms the conical mature capsid, also called the core. The capsid approaches the matrix closely at both ends. In the center of the core is the NC protein, which covers the RNA genome. RT, PR and IN are essential enzymatic proteins that are incorporated into virions as well. tRNAs and other small host RNAs are not shown in the model (Figure 1.1B).
3
A
B
Figure 1.1. HIV-1 genomic structure and virion structure. A. Organization of the HIV-1 genome. B. Model for a mature HIV-1 virion. Reprinted and adapted with permission Springer Nature (Freed, 2001).
4 HIV-1 replication and antiretroviral drug therapy.
In addition to its own viral proteins, HIV-1 exploits the cellular factors to promote its replication in the target cell. HIV-1 enters susceptible cells by utilizing the cell surface receptors, which determine the viral host range. HIV-1 encodes the enzymatic proteins to reverse transcribe and integrate the viral genome into host chromosomal DNA, but this process also depends on cellular transport machinery. The essential regulatory proteins Tat and Rev together with host transcription factors and spliceosomes regulate viral gene expression at the transcriptional and posttranscriptional level.
An understanding of the molecular biology of the viral life cycle provides a basis for antiretroviral drug development. HIV-1-specific antiretroviral drugs were initially given as monotherapy. The administration of a cocktail of antiretroviral drugs revolutionized the treatment of HIV-1 infection (reviewed in Arts and
Hazuda, 2012). Current combination therapy, known as HAART (highly active antiretroviral therapy), effectively suppresses the plasma HIV-1 viral load to undetectable levels, which also restores the immune system in advanced HIV- infected patients (Autran et al., 1997; Lederman et al., 1998). HAART avoids the selection of virus clones bearing drug-resistance mutations that preexist in the viral quasi-species before initiating therapy (Coffin, 1995). The challenge for future antiretroviral drug therapy is to simplify the regimen to fewer drugs with higher potency and longer duration.
5 A better understanding of the virus replication cycle has advanced mechanistic-based drug discovery. Based on their molecular mechanism, current
US Food and Drug Administration (FDA)-approved drugs can be divided into six classes, which are fusion inhibitors, coreceptor antagonists, nucleoside-analog reverse transcriptase inhibitors (NRTIs), non–nucleoside reverse transcriptase inhibitors (NNRTIs), integrase inhibitors and protease inhibitors (Arts and
Hazuda, 2012). Figure 1.2 illustrates the replication cycle of HIV-1 and the targets of antiretroviral drugs from various classes. In this chapter, I will review the molecular mechanism of each step in viral life cycle, with the focus on the targets for ART.
6
Figure 1.2. Distinct classes of antiretroviral drugs target different steps of the HIV-1 replication cycle in a CD4+ T cell. The numbered steps and inhibitors targeting each step are as followed: 1. Entry—attachment inhibitors, chemokine receptor antagonists, and fusion inhibitors 2. Reverse transcription—NNRTI, NRTI 3. Integration—integrase inhibitors 4. Transcription—inhibitors of Tat-TAR interaction 5. Virus assembly and production—inhibitors to block virion maturation 6. Protease processing—protease inhibitors Reprinted and adapted with permission from Cold Spring Harbor Laboratory Press (Arts and Hazuda, 2012).
7 HIV-1 entry
The process of HIV-1 entry into a susceptible cell represents the first step in the viral replication cycle. It is characterized by a series of conformational changes of the viral surface glycoproteins upon engagement of the receptor CD4 and of a coreceptor. A clearer understanding of the molecular mechanism of HIV-
1 entry has advanced the development of neutralizing antibodies and of antiretroviral drugs that inhibit viral entry. The mechanism and categories of neutralizing antibodies will be discussed in Chapter III.
The molecular mechanism of entry.
Following adhesion of the virus to the host cell, the entry of HIV-1 into host cells involves the binding of the gp120 Env glycoprotein to its primary receptor, the CD4 glycoprotein (Maddon et al., 1986; McDougal et al., 1986) (Figure 1.2).
A starting point leading to the discovery of the HIV-1 receptor CD4 was the observation that AIDS is primarily related to a defect of the helper-inducer T lymphocytes (Schroff et al., 1983). Further studies showed that HIV-1 preferentially infects CD4+ T cells in vitro (Klatzmann et al., 1984). Incubation of
CD4+ T cells with certain monoclonal antibodies against CD4 blocks HIV-1 binding, and the HIV-1 Env glycoprotein can be coprecipitated with CD4
(McDougal et al., 1986). The normal function of CD4 is to enhance T-cell receptor (TCR)-mediated signaling. The structure of CD4 in complex with the core of gp120 has been solved. Env interacts with the CD4 binding site in gp120
8 (Kwong et al., 1998), causing rearrangements of V1/V2 and subsequently V3.
The variable loops V1-V3 are lying at the apex of the Env trimer, and play critical roles in viral entry and immune evasion (Hartley et al., 2005). The conformational changes within gp120 upon CD4 binding result in the exposure of a binding site for the chemokine coreceptors, CCR5 or CXCR4 (Kwong et al., 1998).
The first coreceptor for HIV-1 entry to be identified was the G protein- coupled chemokine receptor CXCR4, which is preferentially used by laboratory- adapted HIV-1 isolates (Feng et al., 1996). Soon thereafter, several groups identified CCR5, a receptor for the b-chemokines RANTES, as the coreceptor for primary HIV-1 strains (Alkhatib et al., 1996; Deng et al., 1996; Doranz et al.,
1996; Dragic et al., 1996). A 32 bp deletion within the coding region of ccr5 results in a frameshift and generates a non-functional receptor (Dean et al.,
1996; Liu et al., 1996; Samson et al., 1996). Individuals homozygous for the deletion are profoundly resistant to R5-tropic primary HIV-1 viruses (Dean et al.,
1996; Liu et al., 1996; Samson et al., 1996). The coreceptor usage of HIV-1 classifies HIV-1 strains (Berger et al., 1998). Isolates that use CCR5 are termed
R5 viruses. These include the majority of primary HIV-1 isolates, including so- called macrophage-tropic strains of HIV-1. Strains that solely use CXCR4 are termed X4 viruses, but these are relatively rare in infected individuals. More common are isolates able to use both co-receptors, which are termed R5X4
(Berger et al., 1998). Isolates of HIV-1 from early in the course of infection predominantly use CCR5 for infection (Connor et al., 1997; Keele et al., 2008).
9 R5 are also found to be dominant in the transmission between individuals (Keele et al., 2008). However, in patients with disease progression, the virus expands its coreceptor use to include CXCR4, in addition to CCR5 (Connor et al., 1997;
Schuitemaker et al., 1992). The primary difference between the sequences of R5 and X4 viruses is within the V3 region of Env (Jensen et al., 2003). The mechanism of coreceptor switch during disease progression remains unclear though it has important consequences for therapy with HIV entry inhibitors.
Coreceptor binding triggers the membrane fusion potential of Env. The last step of viral entry is membrane fusion mediated by gp41. The conformational changes induced by coreceptor binding expose the hydrophobic, glycine-rich fusion peptide region of gp41. The fusion peptide anchors into host cell membrane, and eventually forms a six-helical bundle (6HB) (Chan et al., 1997;
Weissenhorn et al., 1997). Within the 6HB, three peripheral carboxy-terminal helices (HR2 domains) pack onto a central trimeric coiled-coil formed by heptad repeat 1 (HR1 domain) in an anti-parallel manner (Wilen et al., 2012) (Figure
1.3). The conformational changes leading to HIV-1 entry are reminiscent of the low-pH-induced conformation of influenza hemagglutinin (Chan et al., 1997;
Weissenhorn et al., 1997). Carboxy-terminal to the gp41 HR2 domain is the membrane-proximal external region (MPER) , which is accessible to the neutralizing antibodies 2F5 and 4E10 on the native Env trimer (Finnegan et al.,
2002; de Rosny et al., 2004). In contrast to the exposure of HR1 and HR2 upon
10 interaction with receptor and coreceptor, the MPER loses the accessibility as fusion progresses (Pancera et al., 2017).
Membrane fusion mediated by viral proteins follows the general model for lipid bilayer fusion (Chernomordik et al., 1987; Melikyan, 2008). The outer monolayers of two membranes first join and initiate the fusion. The distal monolayers follow to come into direct contact and form a hemifusion diaphragm.
Dilation of fusion pores to allow viral nucleocapsid delivery (~50 nm) is critical for effective infection, though the mechanism of pore enlargement is not clear. The expansion of viral pores to a size sufficient for viral entry are less favored than hemifusion or pore formation due to the higher energy cost (Melikyan, 2008).
11
Figure 1.3. Steps of HIV-1 entry. The transmembrane, gp41, and surface, gp120, subunits (1) of HIV-1 Env mediate viral entry. The binding of Env to CD4 causes the exposure of V3 loop (2), which mediates the co-receptor binding (3). Fusion peptides anchor themselves to the target membrane via their hydrophobic segments and forms six-helix bundle. It brings the viral and cellular membranes together and forces membrane to merge (4). Reprinted and adapted with permission from Cold Spring Harbor Laboratory Press (Wilen et al., 2012).
Entry inhibitors.
There are two major classes of entry inhibitors targeting either fusion or co-receptor binding. The HR1 and HR2 domains in gp41 have to interact with each other for conformational changes that ultimately drive membrane fusion.
Synthetic peptides that mimic HR2 competitively inhibit the packing of HR2 against HR1, and thus the formation of the 6HB. T-20 (enfuvirtide) was designed with this rationale, and shows potent antiviral activity in vivo (Kilby et al., 1998;
Lalezari et al., 2003). CCR5 antagonists bind to a cavity within CCR5, thus
12 inhibiting membrane fusion by blocking the interaction between gp120 and CCR5
(Dragic et al., 2000; Tsamis et al., 2003). The CCR5 antagonist Maraviroc was approved by the FDA for clinical use. It prevents the binding of CCR5 to the V3 loop of gp120 (Dragic et al., 2000).
Reverse transcription
Following the delivery of the viral capsid into the cytoplasm of the host cell, the single-stranded viral RNA genome needs to be converted into linear double-stranded DNA for the integration process. Reverse transcription and integration are the hallmarks of retroviruses. Howard Temin and David Baltimore first discovered reverse transcriptase (RT) from RNA sarcoma viruses, which synthesizes DNA from an RNA template (Baltimore, 1970; Temin and Mizutani,
1970). An arsenal of FDA-approved antiretroviral drugs target HIV-1 RT.
The molecular mechanism of reverse transcription
Two enzymatic activities in RT that carry out reverse transcription are a
DNA polymerase that copies either an RNA or DNA template and an RNase H that degrades RNA in a duplex of RNA-DNA. A host tRNA initiates the synthesis of the first minus-strand DNA from the primer binding site (pbs) towards the 5’ end of viral RNA. DNA synthesis forms an RNA-DNA duplex, which is a substrate for RNase H. Subsequently, RNase H digests the 5’ end of the viral RNA and leaves the newly-synthesized minus-strand DNA. The ends of the viral RNA are
13 repeat sequences, R. They allow the newly-synthesized DNA to be transferred from the 5’ end to 3’ end, which results in the synthesis of the full-length genome.
A polypurine tract (ppt) is the only region resistant to RNase H cleavage, and acts as the primer for the initiation of the plus-strand DNA.
HIV-1 genomic RNAs are often nicked. The second copy of viral RNA allows the synthesis of minus-strand DNA to transfer to the second RNA template and bypass the nick. The template switching accounts for recombination happening during reverse transcription. Both ends of the DNA product formed during this process contains U3-R-U5, which comprise the LTRs at the ends of the provirus. In infected cells, DNA synthesis occurs in a reverse transcription complex (RTC), which consists of multiple viral proteins (Fassati and Goff, 2001).
Genetic events during reverse transcription
The diversity of viruses within an infected individual suggests large genetic variation in patients (Keele et al., 2008). The high rate of virus turnover in infected individuals, in addition to a high mutation rate, drives the development of genetic variation (Coffin, 1995; Ho et al., 1995). The high frequency of mutations in part results from an error-prone RT lacking a proofreading mechanism, though there is no good way to exclude the contribution from host RNA polymerase II
(Hu and Hughes, 2012). The total mutation rate for HIV-1 replication is approximately 2 X 10-5 per nucleotide per replication cycle, a rate that is close to
14 other retroviruses (Abram et al., 2010; Pathak and Temin, 1990). In addition to
RT and RNA polymerase II, the host restriction factor APOBEC3G affects the fidelity and efficiency of reverse transcription by altering the synthesized minus- strand DNA (Harris et al., 2003; Mangeat et al., 2003). This will be discussed later in this chapter.
Another genetic event happening during reverse transcription is recombination. Frequent HIV-1 recombination raises genetic diversity in the viral population and contributes to high-level multiple-drug resistance (Moutouh et al.,
1996). Recombination under immune selection pressure is a strategy of viral escape (Streeck et al., 2008). Template switching during the synthesis of minus- strand DNA can generate a recombinant genetically distinct from the parental viral genomes. Such recombination requires two heterogenous RNA templates packaged into the same virion (Hu and Temin, 1990). The original model for template switching is that a break in one RNA template causes a switch to the other RNA copy (Coffin, 1979). A recently modified model suggests that the steady state between the rates of polymerization and RNA degradation determines in vivo template switching (Hwang et al., 2001).
RT Inhibitors
RT is the target for two distinct classes of antiretroviral agents: the NRTIs, which are analogs of native nucleoside substrates, and the NNRTIs, which bind in a site near the polymerase catalytic domain of RT (Kohlstaedt et al.,
15 1992). NRTIs lacking 3’-OH moieties prevent the formation of 3’-5’ phosphodiester bonds between the elongating DNA chain and incoming 5’- nucleoside triphosphates (Mitsuya et al., 1985; Richman, 2001). Currently, there are eight FDA-approved NRTIs: abacavir (ABC), didanosine (ddI), emtricitabine
(FTC), lamivudine (3TC), stavudine (d4T), zalcitabine (ddC), zidovudine
(AZT), Tenofovir disoprovil fumarate (TDF) (Arts and Hazuda, 2012). The binding of NNRTIs changes the conformation of the substrate-binding site and reduces the enzymatic activity of RT (Kohlstaedt et al., 1992). Four approved NNRTIs are etravirine, delavirdine, efavirenz, and nevirapine. Figure 1.4 summarizes the mechanisms of actions of NRTIs and NNRTIs.
Administration of the above drugs causes the emergence of drug-resistant viruses. Two mechanisms mediate resistance to NRTIs. One is caused by ATP- dependent pyrophosphorolysis, which reverses chain termination via removal of
NRTIs from the 3’ end of the nascent chain (Boyer et al., 2001). The other mechanism of NRTI resistance is the failure of NRTI incorporation into the nascent chain caused by mutations in RT (Garcia-Lerma et al., 2003; Margot et al., 2002; Quan et al., 1996). NNRTI resistance is due to mutations in the
NNRTI-binding pocket of RT (Arts and Hazuda, 2012).
16
Figure 1.4. Mechanisms of actions of NRTIs and NNRTIs. A. NRTIs incorporate themselves into newly synthesized DNA nucleoside chain, and stop attachment of nascent nucleosides and thus blocking ongoing viral DNA synthesis. B. NNRTIs bind to and alter the configuration of RT, thus affecting its enzymatic activity. Reprinted and adapted with permission from Springer Nature (Richman, 2001).
17 DNA integration
Integration of a DNA copy of the viral genome into host cell DNA is another hallmark of retroviruses. Once integration is completed, the proviral DNA is replicated during cycles of cell division. The provirus provides the template for transcription of viral RNA genomes and viral mRNAs for producing the viral proteins. The mechanism and biochemistry of integration have been extensively studied are well understood, which contributed to the identification of IN inhibitors. The first IN inhibitor received FDA approval in 2007 (Sato et al., 2006).
Identification of the integration sites provides a footprint to trace the expansion of infected cells during viral persistence (Maldarelli, 2016) and attracts attention for efforts towards a cure (Lusic and Siliciano, 2017).
The mechanism of integration
The DNA copy produced by reverse transcription together with several viral proteins form the preintegration complex (PIC) (Bowerman et al., 1989). The
DNA breakage and joining steps of integration (Fujiwara and Mizuuchi, 1988) are illustrated in Figure 1.5. First, two nucleotides at the 3’ ends of each linear viral
DNA need to be removed from the blunt ends (Figure 1.5A, B). These recessed
3’ ends attack target DNA and generate a 5’ overhang of target DNA. The 5’ ends of this target DNA are joined to the 3’ends of the donor DNA to form the integration intermediate (Figure 1.5C). The distance of the sites of attack on the two target DNA strands is five nucleotides. The single-stranded gap and the 5’
18 end overhang of viral DNA are repaired by cellular enzymes (Figure 1.5D, E), which forms the short duplication of target DNA sequences that flank integrated proviruses.
The mechanism of nuclear localization during integration has not been fully addressed. Several genome-wide siRNA analysis carried out in the efforts to discover host factors required for HIV-1 replication identified cellular proteins required for integration step (Brass et al., 2008; König et al., 2008). Among them, nuclear pore proteins transportin 3 and Nup358 have been shown to be required for nuclear translocation and integration. After the PIC enters the nucleus, it is also possible that the ends of the viral DNA are ligated to each other and form 2-
LTR circles.
IN binds to host lens epithelium-derived growth factor (LEDGF/p75) and catalyzes HIV DNA integration (Llano et al., 2006). Before the essential role of
LEDGF/p75 in integration was established, it was well known that IN is tethered to chromatin by LEDGF/p75 (Cherepanov et al., 2003; Llano et al., 2004). The functional modules for the tethering mediated by LEDGF/p75 are the amino- terminal PWWP, A/T-hook elements binding to chromatin, and a carboxy- terminal integrase-binding domain (Cherepanov et al., 2004). However, changes in overall levels of HIV integration and replication in LEDGF/p75-knockdown cells have been modest (Ciuffi et al., 2005; Llano et al., 2004). The reason is that fractionally minute levels of endogenous LEDGF/p75 are sufficient for integration (Llano et al., 2006). Binding sites on chromosomes for LEDGF/p75 lie
19 preferentially in active transcription units, which are in agreement with HIV-1 integration sites. The interaction site for LEDGF/p75 with integrase is a promising target for antiretroviral therapy (Christ et al., 2010).
Figure 1.5. DNA cutting and joining steps of integration. A. The linear viral DNA copies with blunt ends. B. 3’ end processing by removing two nucleotides from 3’ end of each viral DNA copy. C. DNA-strand transfer. The processed 3’ ends of viral DNA are joined to the 5’ ends of the target DNA. Steps B and C are catalyzed by IN. D. Cellular enzymes removes the 5’ overhang of viral DNA and fill the gap between viral and target DNA. Steps C and D are referred as strand transfer. E. The integrated provirus. Reprinted and adapted with permission from Cold Spring Harbor Laboratory Press (Wilen et al., 2012).
20 Integration site
Integration site selection by retroviruses has been of great interests because of its significance for gene therapy. Recently, this field has attracted more attention since the integration profile of HIV-1 is useful to characterize latently infected cells (Lusic and Siliciano, 2017; Siliciano and Greene, 2011).
The analysis of the DNA sequences at junctions between proviral DNA and host
DNA shows that integration occurs within actively transcribed host genes in resting CD4 T cells of infected individuals (Han et al., 2004). Active transcription units are characterized by high G/C content, high CpG island density, high frequencies of Alu repeats, and low frequencies of LINE repeats. Open chromatin is not the sole reason accountable for targeting active transcription units, since other retroviruses favor different target sites in chromosomes (Wu et al., 2003).
IN is the principal viral determinant of target site preference (Lewinski et al.,
2006).
Analysis of HIV proviruses in CD4+ lymphocytes from individuals after prolonged combination ART reveals that 40% of the integrations are in clonally expanded cells and proviruses are integrated into genes associated with regulation of cell growth (Maldarelli et al., 2014). Another group using a modified translocation-capture sequencing method identified hot spots for integration near human Alu sequences (Cohn et al., 2015). With the development of long-read sequencing technologies such as Pacific Biosciences sequencing, it
21 is possible to directly examine longer sequences of HIV with the junction sequence and host genome sequence in a high-throughput fashion.
Integrase inhibitors
IN is the most recent target for antiretroviral drug development (Hazuda et al., 2004). All integrase inhibitors in development target the joining of viral and cellular DNAs. The mechanism of action of IN inhibitors involves binding to the complex between IN and the viral DNA, as well as interactions with the cofactor
Mg2+ in the active site of IN (Grobler et al., 2002). Mutations at the IN active site that coordinate the essential cofactor Mg2+ can cause the resistance to IN inhibitors.
Viral gene expression.
After being integrated into the host genome, the HIV-1 provirus serves as the template for transcription. HIV-1 gene expression is orchestrated by two viral regulatory proteins, Tat and Rev. Tat activates viral transcription by stimulating elongation from the 5’ LTR. Rev transports mRNAs encoding the structural viral proteins from the nucleus to the cytoplasm.
Regulation by Tat
The HIV-1 5’ LTR acts as a promoter and enhancer. Tat was first discovered as a transacting factor required for viral gene expression under the
22 control of the viral LTR (Sodroski et al., 1985). Tat functions through the transactivation-responsive region (TAR), a regulatory element located downstream of the initiation site. The specific stem-loop secondary structure of
TAR is required for its biological activity (Berkhout et al., 1989). Tat recognizes
TAR via a U-rich region at the loop of TAR and initiates the conformational changes of TAR RNA (Feng and Holland, 1988).
Positive transcription elongation factor b (P-TEFb) is a cofactor essential for Tat-mediated transactivation. P-TEFb was identified by Tat-affinity chromatography and consists of cyclin T1 and cyclin-dependent kinase 9 (CDK9)
(Wei et al., 1998; Zhu et al., 1997). Tat requires some transcription before HIV gene expression is initiated. Thus, Tat regulates the elongation of all viral transcripts that contain TAR, rather than initiating the transcription (Kao et al.,
1987). The binding of Tat to P-TEFb activates CDK9. The regulation of P-TEFb itself is critical for HIV transcription. The majority of P-TEFb is sequestered and inactive in a 7SK small nuclear ribonucleoprotein (7SK snRNP) complex. 7SK snRNP inhibits the kinase activity of CDK9 and prevents recruitment of P-TEFb to the HIV-1 promoter (Nguyen et al., 2001; Yang et al., 2001). Tat overcomes this sequestration by disrupting 7SK snRNP complexes, and induces the release of free P-TEFb.
The LTR
23 The U3 region at the 5’ end of the HIV-1 LTR contains binding sites for transcription factors including nuclear factor of activated T cells (NFAT), NF-kB and specificity protein 1 (SP1) (Kinoshita et al., 1998; Nabel and Baltimore,
1987). The “enhancer region” contains two NF-kB binding motifs.
HIV-1 integration favors active transcription units. Thus, integrated proviruses in actively transcribed genes can be silenced by heterochromatin
(Cary et al., 2016). A well-known mechanism of maintaining latency is the recruitment of histone deacetylases (HDACs) to the HIV-1 5’ LTR, forming a repressive chromatin environment (Margolis et al., 2016). Given that proviral latency is the principal barrier to a cure, reactivation of latent resting CD4+ T cells is the current strategy to clear the viral reservoir. Latency reversing agents
(LRAs) are tools to be studied and further developed for this purpose (Margolis et al., 2016). So far, the most widely investigated LRAs are histone deacetylase inhibitors (HDACis). Though direct proof-of-concept of HDACi has been achieved in clinical studies, the potency and effectiveness of these inhibitors need further improvement (Archin et al., 2012).
Rev
HIV-1 primary transcripts undergo extensive alternative splicing to produce all the mRNAs to encode viral proteins. In addition to generating full- length gag, gag-pol, and env, HIV-1 produces a complex pattern of spliced RNAs to encode the essential regulatory genes and accessory genes. Unspliced and
24 incompletely spliced transcripts from cellular genes are degraded in the cellular environments. To survive in the host, the viral accessory protein Rev promotes the transportation of such viral RNAs out of the nucleus. Rev binds to viral RNAs that contain the Rev-responsive element (RRE), located in the env gene (Malim et al., 1989; Sodroski et al., 1986).
The expression of viral proteins in either the early or late phases of the
HIV-1 life cycle is determined by the level of Rev (Figure 1.6) (reviewed in Karn and Stoltzfus, 2012). Full-length unspliced ~9-kb, incompletely spliced ~4-kb mRNA and completely spliced ~1.8-kb mRNAs are produced at both early and late times. At the early phase of HIV-1 mRNA expression, there is no Rev or only a low-level of Rev. Unspliced or incompletely spliced mRNAs fail to be transported out of the nucleus and are either spliced or degraded by cellular enzymes (Figure 1.6A). When the level of Rev in the nucleus exceeds the threshold necessary for function, all viral mRNAs are transported to the cytoplasm and translated (Figure 1.6B).
25
Figure 1.6. Early and late phases of HIV-1 mRNA expression. A. In the absence of Rev or with low-level Rev, only Rev, Tat, and Nef are expressed. B. When Rev accumulates, unspliced and incompletely spliced mRNAs are transported out of the nucleus. Reprinted and adapted with permission from Annual Reviews, Inc (Karn and Stoltzfus, 2012; Pollard and Malim, 1998).
26 Viral particle production
The last step of the HIV-1 life cycle is assembly and budding. Before the progeny virions become infectious and can infect the next target cell, they undergo maturation. The Gag glycoprotein and its proteolytic products drive the process. There are three stages based on virion morphogenesis: assembly, budding, and maturation.
Assembly
HIV-1 particle production occurs in a series of steps promoted by the viral
Gag protein (Ono and Freed, 2001). Gag domains are separated by PR cleavage sites (Figure 1.7A). MA is the amino-terminal domain of Gag. It binds to the plasma membrane and recruits the Env protein. Genetic and biochemical evidence suggested the direct interaction of the HIV-1 gp41 cytoplasmic tail with
MA, which is required for HIV-1 maturation (Murakami and Freed, 2000; Wyma et al., 2000). The central domain of Gag, CA, mediates protein-protein interactions during assembly and forms the conical core of mature virions. The NC domain functions to capture the viral genome during assembly. P6 at the carboxy- terminal contains late assembly domains, which bind to the cellular ESCRT membrane fission machinery to promote viral budding from the plasma membrane. The p6 domain also contains binding sites for the HIV-1 accessory protein Vpr.
27 The viral membrane is derived from the cellular plasma membrane.
However, viral lipid composition differs from that of the producer cell (Ono,
2009). The HIV Gag and Gag-Pro-Pol polyproteins specifically associate with cholesterol-enriched, detergent-resistant microdomains (“rafts”) at the plasma membrane (Ono and Freed, 2001). Mass spectrometry provided a quantitative analysis of the lipid constituents of HIV and of the difference from the host membrane (Brugger et al., 2006).
The dimer initiation site (DIS) in the 5’ UTR of the genomic viral RNA mediates the dimerization of two copies of the RNA genome. RNA dimerization is required for RNA packaging. RNA-Gag interactions facilitate the aligning of Gag molecules at the plasma membrane (Kutluay and Bieniasz, 2010). The carboxy- terminal domain of CA stabilizes the interactions of RNA and Gag. The packing signal (Y site) at the 5’ UTR comprises four stem loops. The splice donor site within the Y site contributes to the selection of unspliced RNA for efficient packaging. In addition to the viral genome RNA, virions also package tRNA primers required for the initiation of reverse transcription.
The application of imaging technologies and structural analyses further advanced our understanding of HIV-1 assembly and budding. Total-internal- reflection fluorescent microscopy in living cells allows the study of the genesis of individual virions in real time (Ivanchenko et al., 2009; Jouvenet et al., 2008). The accumulation of Gag protein in cells accelerates the membrane assembly rate of virions. The average time for assembly is 5-6 min. Cryo-electron tomography
28 (cryo-EM) of budding viruses showed that the immature Gag lattice is established as the virus assembles (Carlson et al., 2010). High-resolution cryo-EM allowed the positioning of individual protein domains and defined the three-dimensional structure of the immature Gag lattice in greater detail (Briggs et al., 2009; Schur et al., 2015).
Budding
To facilitate the release of virions from the plasma membrane, retroviruses usurp the host ESCRT (endosomal sorting complexes required for transport) machinery, which normally creates vesicles that bud into late endosomal compartments called multivesicular bodies (MVB), and also separates two daughter cells during cytokinesis (Bieniasz, 2009; Morita and Sundquist, 2004).
Two late assembly domains in p6 recruit early-acting ESCRT factors. First, PTAP motif in p6 binds to the ubiquitin E2 variant (UEV) domain of tumor susceptibility gene 101 (Tsg101) (Garrus et al., 2001; Martin-Serrano et al., 2001). Tsg101 is a subunit of the ESCRT-I complex. The second late domain in p6 is a YPXL motif which binds the ESCRT factor ALIX (Strack et al., 2003). Tsg101 and ALIX both function by recruiting ESCRT-III and VPS4 complexes, which in turn mediate membrane fission (Bieniasz, 2009; Morita and Sundquist, 2004). Tetherin is a host restriction factor that can block a later step of HIV-1 release, which will be discussed in detail below.
29 Maturation
Upon budding from producer cells, HIV-1 progeny virions are in an non- infectious, immature form. The Gag polyprotein first assembles into spherical immature particles and radially points toward the virion interior (Figure 1.7 B, C left). Upon the PR-mediated cleavages of the Gag and Gag-pol polyproteins, the immature virions transits into its mature infectious form, resulting in the conical core of the infectious virus (Figure 1.7 B, C right) (Gross et al., 2000). Gag processing also affects Env-mediated fusion (Murakami et al., 2004).
Unprocessed Gag interacts with the gp41 cytoplasmic tail, and suppresses Env- mediated viral fusion (Murakami et al., 2004). The conical core of viral particles is composed of ~1200 copies of CA, while MA remains associated with the inner side of the viral membrane (Briggs et al., 2003). Intersubunit CA interactions stabilized the capsid lattice.
The viral capsid is crucial for host-viral interactions during the early stages of HIV-1 replication. It interacts with TRIM5a and cyclophilin A (Keckesova et al.,
2006; Stremlau et al., 2006). The stabilization of the capsid matters for reverse transcription (Forshey et al., 2002). Mutational analyses of CA suggests that the capsid plays an important role in nuclear import of the PIC (Lee et al., 2010;
Yamashita et al., 2007).
HIV-1 PR cleaves the viral Gag and Gag-pol polyprotein precursors during maturation, and it is an effective drug target. Ten PR inhibitors are currently
30 approved by FDA. However, ~ 20 substitutions are known to be associated with resistance to PR inhibitors (Shafer et al., 2001).
Figure 1.7. The Gag polyprotein coordinates assembly, budding and maturation. A. Domain composition of the HIV-1 Gag protein. Arrows denote the five sites that are cleaved by the viral PR during maturation. B. Schematic model showing the organization of the immature (left) and mature (right) HIV-1 virion. C. Central section from a cryo-EM tomographic reconstruction of an immature (left) and mature (right) HIV-1 virion. Reprinted and adapted with permission from Cold Spring Harbor Laboratory Press (Sundquist and Krausslich, 2012).
31 Cell-to-cell transmission
It was first reported in 1992 that dendritic cells can transfer infectious HIV-
1 to uninfected CD4+ T cells without themselves becoming infected, so called trans-infection (Cameron et al., 1992). The HIV receptors CD4, CCR5, and
CXCR4 on the T cell are recruited to the dendritic cell/T cell interface while the viruses are concentrated in the same region (McDonald et al., 2003). However, the mechanism of trans-infection and its importance in vivo within distinct anatomical compartments is not clear. A recent study of murine leukemia virus
(MLV) infection in mice shows that robust infection of MLV in lymph nodes and spleen requires CD169-dependent trans-infection (Sewald et al., 2015).
In T-lymphocyte cultures, the effective cell-cell transmission of HIV-1 is via the virological synapse (VS) (reviewed in Alvarez et al., 2014). HIV-1 release is polarized and accumulated at the cell-to-cell contact (Haller et al.,
2014). Polarization and formation of VS are dependent on an intact microtubule and actin cytoskeleton (Jolly et al., 2007). The cellular adhesion molecules
ICAM-1 and LFA-1 are suggested to stabilize VS (Jolly et al., 2007). Recent study provided evidence for existence of in vivo VS using three-dimensional electron tomography (Ladinsky et al., 2014). Although cell-to-cell transmission and cell-free transmission exhibit vastly different efficiencies, they seem to involve fundamentally similar molecular mechanisms of viral assembly, budding and release (Sundquist and Krausslich, 2012).
32 HIV-1 pathogenesis
The in vivo replication of HIV-1 viruses causes the loss of CD4+ T cells. In the absence of ART, HIV-1 infection leads to AIDS, characterized by numerous opportunistic infections and symptoms like dementia and wasting. Figure 1.8 shows the typical disease progression marked by the reduction of CD4+ T cells and viral loads in infected individuals without treatment (Coffin and Swanstrom,
2013).
During the first 1-2 weeks, the transmitted virus, now suggested to be a single virion (Keele et al., 2008), starts to replicate and spread from the initial infection site. Neither viremia nor immune responses are detectable. From 2-4 weeks, a high level of viremia (up to 107 or more copies of viral RNA per milliliter of blood) develops, accompanied by “flu-like” symptoms. At the peak of viremia, antibodies are produced and cytotoxic T lymphocyte (CTL) responses take place.
A recent study of acutely infected patients suggests that anti-HIV-1 specific CTL responses during hyperacute infection impact the viral set point (Ndhlovu et al.,
2015). High-level viremia is due to the absence of an early immune response and a large number of activated CD4+ T cells, which are the target cells for HIV-1 replication. The end of this phase is characterized by the drop in the level of viremia, and a transient drop in the number of CD4+ T cells. The following 1 to 20 years is clinical latency. The HIV-1 viral set point refers to the stable or slowly increasing level of viral load during this phase. The number of CD4+ T is near
33 normal or gradually falling. Plasma viral load is a better predictor of progression to AIDS than the number of CD4+ T cells (Mellors et al., 1996). Finally, the clinical hallmark of AIDS is that the number of CD4+ T cells drops below 200 cells/µL). Opportunistic infections start to appear.
ART has changed the fate of HIV-1-infected individuals. The majority of patients on suppressive therapy have low levels of viremia. However, once ART is interrupted, viral rebound occurs. The reason is that HIV-1 can establish a state of latent infection at the level of individual T cells (Siliciano and Greene,
2011). Activated CD4+ T cells are highly susceptible to HIV-1 infection and subject to rapid turnover (Ho et al., 1995). However, some activated CD4+ T cells may become infected and then survive long enough to revert back to a resting state (Finzi et al., 1999). Latent virus persists even in patients on potent antiretroviral therapy since it resides in long-lived memory T cells (Finzi et al.,
1997). This latent reservoir is now the major barrier to the cure of HIV-1 infection.
34
Figure 1.8. Time course of typical HIV-1 infection. Patterns of CD4+ T cell decline and viremia vary greatly from one patient to another. Reprinted and adapted with permission from Cold Spring Harbor Laboratory Press (Coffin and Swanstrom, 2013).
35 HIV restriction factors
In addition to conventional innate and adaptive immune responses, humans and other mammals have evolved specific antiviral factors. These host- encoded restriction factors serve as effective inhibitors of retroviral replication.
Some of the restriction factors are constitutively expressed, while some of them are induced by interferons as part of the innate immune response. Their advantage is that they do not have to ‘‘learn’’ to combat viruses but are already active at the first virus-cell interaction (reviewed in Kirchhoff, 2010; Malim and
Bieniasz, 2012). HIV-1 evades the defense of antiviral factors in human cells, thus allowing efficient viral replication. In contrast, the ability of HIV-1 to replicate in nonhuman cells is often severely compromised by restriction factors (Malim and Emerman, 2008). Thus, restriction factors are important determinants of viral host range and cross-species transmission (Gaddis et al., 2004; Keckesova et al., 2006).
This chapter discusses three well-defined restriction factors that potently inhibit HIV-1: namely, APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G), TRIM5a (tripartite motif 5-a), and tetherin (BST-2 or CD317). This introduction focuses on the mechanisms by which they inhibit viral replication, how HIV-1 evades their protection, and their potential contributions to viral pathogenesis and cross-species transmission.
36 Discovery of HIV-1 restriction factors
Studies of ecotropic MLV initiated the field of “restriction factor.” The isoforms of mouse-encoded gene Fv1, Fv1n and Fv1b, determines the tropism of MLV (Best et al., 1996). Mice carrying Fv1n are susceptible to N-tropic MLVs, but resistant to B-tropic MLVs (Best et al., 1996).
Although the approaches for discovering restriction factors have evolved over the decades as novel technologies emerge, the general strategy remains the same. The potent restriction factor APOBEC3G, which is counteracted by
HIV-1 Vif, was identified by comparing cDNAs from the cell lines that are either permissive or nonpermissive for Vif-deficient viruses (Sheehy et al., 2002). An analysis of differential expression via microarray among cell lines with distinct phenotypes induced by type I interferon revealed the role of tetherin in the inhibition of HIV-1 virion release in the absence of Vpu (Neil et al., 2008).
TRIM5a, which targets incoming HIV-1 capsids, was discovered by exploiting the transfer of resistance to HIV-1 between permissive and nonpermissive cells.
Isolation of a cDNA conferring resistance uncovered the restriction factor
TRIM5a (Stremlau et al., 2004).
APOBEC3G
APOBEC3G is a cytidine deaminase, which mediates the editing of cytidine residues to uridines. In the absence of Vif, APOBEC3G exerts its inhibitory activity by being selectively packaged into HIV-1 particles by binding to
37 the NC domain of Gag (Bogerd and Cullen, 2008; Zennou et al., 2004). After being transferred into target cells, APOBEC3G catalyzes the deamination of cytidine to uridine in nascent single-stranded negative-strand cDNA (Harris et al.,
2003), and causes lethal guanosine-to-adenosine hypermutation within the viral plus-strand (Mangeat et al., 2003; Zhang et al., 2003). In addition, APOBEC3G inhibits viral DNA synthesis during reverse transcription (Bishop et al., 2008;
Miyagi et al., 2007).
The antiretroviral activity of APOBEC3G is antagonized by the HIV-1 accessory protein Vif. Vif serves as an adaptor protein to recruit APOBEC3G to ubiquitin ligase complexes, resulting in polyubiquitination and proteasomal degradation of APOBEC3G, thereby preventing its packaging into progeny virions (Marin et al., 2003; Sheehy et al., 2002; Stopak et al., 2003; Yu et al.,
2003). In addition to Vif-mediated regulation, type I interferon can induce
APOBEC3G proteins (Koning et al., 2009). The contribution of APOBEC3G- mediated hypermutation to the HIV-1 landscape is not clear (reviewed in Hu and
Hughes, 2012). The mechanism of APOBEC3G-mediated restriction and its counteraction by Vif is summarized in Figure 1.9. A small molecule RN-18, that antagonizes Vif and functions only in the presence of APOBEC3G, is under development (Nathans et al., 2008).
38 TRIM5a
TRIM5a was initially identified as the factor responsible for the resistance of rhesus macaque cells to HIV-1 infection (Stremlau et al., 2004). Subsequent studies revealed that TRIM5a from a variety of primates possess similar antiretroviral activities (Hatziioannou et al., 2004; Keckesova et al., 2004; Perron et al., 2004; Yap et al., 2004). TRIM5a proteins are poor inhibitors of retroviruses derived from the same host species, but effectively restrict retroviruses from other species. TRIM5a is a ~500 amino acid cytoplasmic protein characterized by the TRIM (tripartite) motif, which is composed of RING, B-box 2, and coiled- coil domains. A SPRY domain at the carboxy-terminus governs the species- specific restriction by each TRIM5a protein. An analysis of the evolutionary history of TRIM5a revealed positive selection within the SPRY domain (Sawyer et al., 2005; Song et al., 2005). The SPRY domain also governs the binding of
TRIM5a to HIV-1 capsid (Stremlau et al., 2006).
The mechanism by which TRIM5a blocks HIV-1 infection is not fully understood (Figure 1.9). Studies suggest that TRIM5a can specifically recognize the capsid and promote its rapid disassembly (Stremlau et al., 2006). Higher- order TRIM5a oligomerization contributes to the efficiency of capsid recognition, and thus promotes antiviral potency (Diaz-Griffero et al., 2009). A very recent study suggests that in addition to providing a cross-species barrier, human
TRIM5a is a cell-specific restriction factor dependent on C-type lectin receptor
39 function (Ribeiro et al., 2016). It potently restricts HIV-1 infection of Langerhans cells but not that of subepithelial DC-SIGN+ dendritic cells.
Tetherin
Vpu is required for efficient HIV-1 particle release in certain human cells, while it is dispensable in some other human cells. The requirement for Vpu during virion release is inducible by IFN-a treatment (Neil et al., 2007). A comparison of the genetic profiles of cells with distinct requirements for Vpu for virion release identified tetherin as the restriction factor counteracted by Vpu
(Van Damme et al., 2008; Neil et al., 2008).
In the absence of Vpu, tetherin traps the budding virions at the surface of infected cells, and their accumulation causes nascent virion endocytosis (Neil et al., 2006). Tetherin is a type II single-pass transmembrane protein with a transmembrane anchor at the amino-terminus and a glycophosphatidylinositol lipid anchor at its carboxyl-terminus. These membrane anchors are linked by an extracellular domain with a coiled-coil structure (Hinz et al., 2010; Yang et al.,
2010). Mutational analyses suggest that the configuration of tetherin rather than its primary sequence is required for antiviral activity (Perez-Caballero et al.,
2009). Both of the anchor domains are essential for tethering function (Perez-
Caballero et al., 2009). One model of tetherin action proposes that one transmembrane anchor is inserted into the cell membrane and the other into the virion envelope (Figure 1.9).
40 HIVs and SIVs have independently evolved antiviral tools to antagonize tetherin. HIV-1 Vpu colocalizes with and binds to tetherin, and reduces the level of tetherin at the cell surface (Van Damme et al., 2008; Neil et al., 2008). HIV-1
Vpu antagonizes tetherin by causing its internalization or trapping it in a perinuclear compartment (Dubé et al., 2010; Mitchell et al., 2009). The precise mechanism is not yet entirely clear. In contrast, SIVs use Nef to antagonize tetherin (Jia et al., 2009; Zhang et al., 2009). Nef-binding proteins are not required for the antagonism of tetherin, and the mechanism by which Nef counteracts tetherin is not clear. SIV Envs were shown to antagonize tetherin as well (Gupta et al., 2009).
41
Figure 1.9. Interplay between restriction factors and accessory proteins. HIV-1 RNA is shown in light blue, HIV-1 cDNA in dark blue, and restriction factors in red. The sites of Vif, Vpu, and Nef antagonism are indicated. Reprinted and adapted with permission from Cold Spring Harbor Laboratory Press (Malim and Bieniasz, 2012).
42 Newly defined restriction factors
Though many restriction factors targeting distinct steps of the HIV-1 replication cycle have been identified and well-characterized, perhaps more restriction factors remain to be discovered (Malim and Bieniasz, 2012). Rapidly evolving genetic approaches and high-throughput technologies make it possible to explore novel restriction factors on a larger scale. Park et al. used a genome- wide CRISPR-based screen to identify host factors in a physiologically relevant cell system (Park et al., 2017). They repetitively infected a pool of knockout mutants with R5 viruses, followed by an analysis of surviving cells compared with parental cells. Among five factors they identified, four of them facilitated viral entry, while only one, activated leukocyte cell adhesion molecule (ALCAM), was required for cell-to-cell HIV transmission. No host factors involved in late stages of HIV-1 replication were identified, which is perhaps not surprising, since their approach tends to select for host factors required for early events in HIV-1 replication.
Starting with unexplained inhibitory effects of type I interferon on early steps of the HIV-1 replication cycle, two groups identified myxovirus resistance 2
(MX2) as an interferon-inducible inhibitor of HIV-1 replication (Goujon et al.,
2013; Kane et al., 2013). A comparison of gene expression profiles in cell lines that differ in their ability to support the inhibitory action of interferon-a at early steps of the HIV-1 replication cycle resulted in the identification of MX2. The block to infection occurs at a late post-entry, preintegration step of HIV-1
43 replication. MX2 blocks HIV-1 by inhibiting CA-dependent nuclear import of subviral complexes (Goujon et al., 2013; Kane et al., 2013).
An unknown restriction factor imposes a quite formidable barrier to HIV-1 infection in human dendritic and myeloid cells (Goujon et al., 2008; Hirsch et al.,
1998). Two groups identified SAMHD1 (SAM domain HD domain-containing protein 1) as the dendritic- and myeloid-cell specific restriction factor (Hrecka et al., 2011; Laguette et al., 2011). HIV-2/SIVsm accessory protein Vpx counteracts
SAMHD1 by loading it onto an ubiquitin ligase, resulting in proteasome- dependent degradation of the protein.
The role of Nef in HIV-1 replication and pathogenesis
The negative factor (Nef) of HIV-1 is a ~27 kDa myristoylated protein that resides both in the cytoplasm and in association with cytoplasmic membranes.
Nef is abundantly expressed early during the viral life cycle. Though initially Nef was thought to be a negative factor that inhibits viral replication (Cheng-Mayer et al., 1989), subsequent studies on Nef demonstrated its key role in disease progression and pathogenesis. Individuals infected with Nef-deficient HIV-1 maintained low viral loads and normal CD4 lymphocyte counts in the absence of antiretroviral treatment (Deacon et al., 1995; Kirchhoff et al., 1995). In rhesus monkeys infected with SIVmac, a functional Nef is required for maintaining high
44 virus loads and for full pathologic potential (Kestier et al., 1991). Rhesus monkeys vaccinated with live attenuated SIVmac with a deletion in nef are protected against live, pathogenic SIVmac (Daniel et al., 1992). However, subsequent studies have shown that the live attenuated SIV vaccine with deletions in nef can cause AIDS in infant and adult macaques (Baba et al., 1995,
1999). Nef performs a striking number of activities by modulating signal transduction cascades and protein trafficking. Some key functions of Nef are summarized in Figure 1.11.
Functional domains in Nef
Nef consists of an array of potential protein-protein interaction domains
(Geyer et al., 2001; Roeth and Collins, 2006). The structure fragments and functional domains were assembled into the full-length Nef polypeptide (Geyer and Peterlin, 2001). The Nef structure is partitioned into three segments: a myristoylated membrane anchoring region at the amino-terminus, followed by an a-helical core domain, and a carboxy-terminal flexible loop (Figure 1.10). An amino-terminal MGxxx(S/T) motif mediates the myristoylation of Nef. Several motifs are involved in signaling. For instance, a proline-rich (PxxP)3 sequence cluster interacts with SH3 domains of Src family kinases. It mediates the interactions between Nef and signaling proteins like Hck and Vav, which is required for cellular activation by Nef (Fackler et al., 1999). A di-arginine at 105,
106 is required for the activation of p21-activated kinase (Pak) by Nef (Fackler et
45 al., 1999). Several motifs are reported to be involved in the downregulation of host proteins from the plasma membrane by Nef. An endocytic (E/D)xxxLL signal
(dileucine motif) is best preserved. In addition, two aspartic acid residues (D174,
D175) participate in the downregulation of CD4 (Aiken et al., 1994). Motifs involved in the Nef-mediated CD4 and MHC-I downregulation are located in flexible regions of Nef, suggesting the recognition of primary Nef sequences by endocytic machinery. In contrast, interaction sites for SH3 domains are located within the well-folded core domain, indicating the recognition of highly structured protein surfaces.
In addition to HIV-1, HIV-2 and SIVs also encode Nef. Though several regions are highly conserved, there are many differences (Figure 1.11). The core domain of Nef is relatively conserved. The amino- and carboxy- termini containing extended loop sequence are less conserved. These sequences are enriched in motifs for cellular trafficking (Roeth and Collins, 2006).
46
.
1 and SIV Nef. Nef. SIV and 1 - underlined. Roeth and Collins, 2006)and Roeth ( crobiology for protein interactions are protein for interactions
protein interaction domains in HIV in domains interaction protein - American MiSociety for Nef sequences aremotifs their proteinand aligned. Boxes indicate cellular
mac239 Nef and SIV
Sequence alignment and protein 1NL4 - 3 - binding Residues(in partners parentheses). important Figure 1.10. 1.10. Figure HIV The Reprintedand permission adaptedfrom with
47 Nef-mediated downregulation of surface proteins
In HIV-1-infected T cells, Nef downregulates critical immune receptors on the cell surface including CD4, MHC-I, CD28, and CXCR4 (Bell et al., 1998; Guy et al., 1987; Haller et al., 2014; Kirchhoff, 2010; Schwartz et al., 1996). These receptors are recruited to the endocytic machinery or rerouted to lysosomes for degradation (Figure 1.11).
Removing the antigen-presenting molecule MHC-I is one of the immune evasion strategies of HIV-1. Downregulation of MHC-I in HIV-1-infected CD4+ T cells was reported years before the mechanism was identified (Scheppler et al.,
1989). HIV-1 Nef prevents the exposure of viral antigens on the surface of HIV- infected cells by reducing the surface expression of MHC-I (Schwartz et al.,
1996). This function allows HIV-infected cells to escape recognition and lysis by antiviral CTLs in primary T cell models (Collins et al., 1998). Interestingly, HIV-1 selectively downregulates some allotypes of MHC-I while sparing others on the surface. Nef downregulates human leukocyte antigen (HLA)-A and HLA-B without reducing HLA-C and HLA-E surface levels, which would diminish inhibitory signals for NK cells (Cohen et al., 1999). In this way, Nef protects infected cells from both adaptive and innate immunity. A recent study demonstrated that Vpu in primary HIV isolates downregulates HLA-C, which leads to the downregulation of all major MHC-I molecules by HIV-1 (Apps et al.,
2016).
Nef also down-regulates the viral receptor CD4. This activity is highly
48 conserved among SIV and HIV-1 Nefs (Aiken et al., 1994; Benson et al., 1993).
The presence of CD4 on the surface of infected cells reduces the efficiency of virus budding and viral infectivity (Spina et al., 1994). Moreover, complex formation between CD4 and the HIV-1 Env glycoprotein disrupts the trafficking of both proteins (Roeth and Collins, 2006). The downregulation of CD4 by Nef also prevents viral superinfection (Aiken et al., 1994; Benson et al., 1993).
Nef disrupts the trafficking of MHC-I and CD4. Nef has been shown to bind to the early forms of MHC-I molecules with unphosphorylated cytoplasmic tails, which are newly loaded with foreign peptides (Kasper et al., 2005). In the presence of Nef, MHC-I molecules accumulated in the trans-golgi network (TGN)
(Roeth et al., 2004), where the clathrin adaptor protein complex 1 (AP-1) normally functions and binds to sorting signals in the cytoplasmic tails of proteins.
Nef recruits AP-1 to reroute MHC-I from the TGN to lysosomes in infected T cells
(Roeth and Collins, 2006). Nef also binds to the cytoplasmic tail of CD4 and recruits AP2 to transport CD4 from the cell surface to lysosomes for degradation
(Cluet et al., 2005; Ross et al., 1999).
A growing list of surface proteins is shown to be affected by Nef, including
CD28, CXCR4, DC-SIGN, CD8, MHC-II and the invariant chain (Haller et al.,
2014; Schindler et al., 2003; Sol-Foulon et al., 2002; Stumptner-Cuvelette et al.,
2001; Swigut et al., 2001; Venzke et al., 2006). CD3 is only downregulated by
HIV-2 and SIV Nefs. But this function is missing in HIV-1 and its simian precursors (Schindler et al., 2006). A complete understanding of the mechanism
49 and selectivity of Nef downregulation needs further investigation.
Nef modulates signal transduction pathways
Nef interacts with various cellular kinases and modulates the responses of
HV-1 infected T cells to TCR-mediated stimulation. Nef together with Lck is recruited to cell-cell contact interfaces after TCR stimulation (Fenard et al.,
2005). Nef promotes the induction of cellular transcription factors, such as NF-AT and NF-kB (Fortin et al., 2004; Varin et al., 2003). Notably, NF-kB activation is essential for robust proviral transcription (Nabel and Baltimore, 1987). The majority of HIV-1 and SIV Nefs were shown to increase NF-kB activity early during the viral life cycle, which is from viral entry to the expression of Tat, Rev and Nef (Sauter et al., 2015). Nef promotes NFAT-dependent gene expression, and requires an intact SH3 binding domain for this activity (Manninen et al.,
2001).
Nef modulates the apoptosis of infected cells and bystander cells. HIV-1 infection leads to enhanced apoptosis predominately in bystander cells but not in productively infected cells (Finkel et al., 1995). Nef promotes the killing of bystander cells through the induction of Fas ligand (FasL) expression on the surface of HIV-1 infected cells (Xu et al., 1997, 1999). The subsequent interaction of FasL with Fas (CD95) expressed on neighboring cells, including on
CTLs, leads to bystander cell killing, which is another mechanism of immune evasion by HIV-1. By inhibiting apoptosis signaling regulating kinase 1 (ASK1),
50 Nef inhibits both Fas- and TNFa-mediated apoptosis in the HIV-1-infected host cell (Geleziunas et al., 2001).
Nef enhances the infectivity of progeny virions
Nef enhances the specific infectivity of progeny virions, but the mechanism remained mysterious for decades. Reduction of CD4 expression on the producer cell by Nef is reported to elevates viral infectivity (Aiken and Trono,
1995; Chowers et al., 1994; Miller et al., 1995). However, effects of Nef on viral infectivity can be observed in even CD4-negative producer cells. Thus, Nef must serve another role (Pandori et al., 1998).
The recent identification of the antiviral host factors SERINC5 and
SERINC3 addresses this problem. In the absence of Nef, the multipass transmembrane proteins SERINC3 and SERINC5 are incorporated into progeny virions and restrict HIV-1 infectivity (Matheson et al., 2015; Rosa et al., 2015;
Usami et al., 2015). Nef enhances HIV infectivity by removing SERINCs from the plasma membrane, which prevents their incorporation into progeny HIV-1 virions
(Usami et al., 2015). SERINC3 and SERINC5 will be discussed in detail in
Chapter II.
51
Figure 1.11. The activities of Nef in infected cells. Nef downmodulates MHC-I, CD4, CD28, CXCR4, and CD3 (only HIV-2 and SIVs) from the surface of infected CD4+ T cells by recruiting them to the endocytic machinery via interactions with adaptor protein complexes or by rerouting them to endosomes. Furthermore, Nef interacts with cellular kinases and induces downstream signaling events to modulate T cell activation; to activate NF-AT, NF-kB, and AP-1; and to induce the transcription of death receptors. Nef also directly enhances the infectivity of progeny virions. Reprinted and adapted with permission from Elsevier (Malim and Bieniasz, 2012).
52 Summary of dissertation
The work presented in the following chapters addresses critical gaps in our understanding of the newly identified antiretroviral host factor SERINC5 and of its interplay with Nef. First, in Chapter II, the antiviral activity of SERINC5 orthologs and their counteraction by Nef are elucidated. These studies demonstrate that the anti-HIV-1 activity is a conserved property of widely divergent SERINC5 proteins. However, their sensitivity to Nef is not conserved. Nef sensitivity can be transferred by exchanging an intracellular loop region. Human SERINC5 can be modified to restrict HIV-1 even in the presence of Nef. Next, Chapter III focuses on determinants required for the anti-HIV activity of SERINC5. Work presented in this chapter demonstrates that the determinants in SERINC5 governing the antiretroviral activity are not confined to one domain.
Extracellular loop 5 in SERINC5 is a hotspot region that determines the Env- dependent antiretroviral activity of SERINC5. Chapter IV provides an in-depth discussion of these results, their relevance, and outlines future experiments that will continue to illuminate molecular mechanisms of intrinsic antiviral immunity.
53 Chapter II. A long cytoplasmic loop governs the sensitivity of SERINC5 to Nef
Abstract
Recent studies have identified the multipass transmembrane proteins
SERINC3 and SERINC5 as antiviral proteins that are incorporated into Nef- deficient HIV-1 virions and counteracted by Nef (Rosa et al., 2015; Usami et al.,
2015). SERINC5, in particular, can dramatically inhibit the infectivity of Nef- deficient HIV-1. HIV-1 Nef removes SERINC3 and SERINC5 from the cell surface and prevents their incorporation into progeny virions. In contrast, Nef increases the cell surface expression of SERINC1, indicating that SERINC3 and
SERINC5 are selectively downregulated (Matheson et al., 2015). We now report that the anti-HIV-1 activity but not the sensitivity to Nef is conserved among vertebrate SERINC5 proteins. However, a Nef-resistant SERINC5 became Nef- sensitive when its intracellular loop 4 (ICL4) was replaced by that of Nef-sensitive human SERINC5. Conversely, human SERINC5 became resistant to Nef when its ICL4 was replaced by that of a Nef-resistant SERINC5. In general, ICL4 regions from SERINCs that exhibited resistance to a given Nef conferred resistance to the same Nef when transferred to a sensitive SERINC, and vice versa. Importantly, our results establish that human SERINC5 can be modified to restrict HIV-1 infectivity even in the presence of Nef.
54 Introduction
The discovery of SERINC5.
Nef was noted to enhance the specific infectivity of progeny virions more than 20 years ago (Aiken and Trono, 1995; Chowers et al., 1994; Miller et al.,
1995). This function of Nef requires its presence during virus production, but Nef has no detectable effect on the encapsidation of the genomic viral RNA, the incorporation of Env, or the morphology or stability of the mature core (Aiken and
Trono, 1995; Forshey and Aiken, 2003; Schwartz et al., 1995). Nevertheless, Nef increases the efficiency of reverse transcription in target cells (Aiken and Trono,
1995; Chowers et al., 1995; Schwartz et al., 1995). The effects of Nef on HIV-1 infectivity are determined by variable HIV-1 Env regions (Usami and Gottlinger,
2013). It also involves the interaction of Nef with the GTPase Dynamin-2, a critical regulator of clathrin-mediated endocytosis (Pizzato et al., 2007; Usami et al., 2014). In addition, glycosylated Gag protein (glycoGag) of MLV shares the ability of enhancing HIV-1 infectivity in a similar manner to Nef, despite sharing no sequence homology with Nef (Pizzato, 2010).
Several groups identified the host factors attributable to the enhancement of viral infectivity by Nef, which are SERINC3 and SERINC5 (Matheson et al.,
2015; Rosa et al., 2015; Usami et al., 2015). Usami and colleagues conducted a proteomic analysis of purified virions produced by T lymphoid cells infected with wild-type or Nef-deficient HIV-1NL43 (Usami et al., 2015). They found that
55 SERINC3 was the only protein that was identified in Nef-deficient virions but not in wild-type virions, or in virions produced in the presence of MLV glycoGag.
Furthermore, they found that the incorporation particularly of SERINC5 into progeny virions dramatically inhibited their infectivity, and that Nef counteracted these effects. Rosa et al. and colleagues compared the transcriptomes of producer cell lines in which NefNL4-3 caused a differential enhancement of viral infectivity (Rosa et al., 2015). The rationale for this approach was that the effects of Nef were expected to correlate with the levels of expression of a potential anti- viral factor. They found that the levels of the SERINC5 transcript correlated most closely with the enhancement of viral infectivity by Nef. Both of the groups used knockout/knockdown and reconstitution approaches to show that SERINC5 is in particular accounts for the infectivity enhancement function of Nef (Rosa et al.,
2015; Usami et al., 2015).
SERINC protein family.
There was limited information about the function of SERINC proteins before the identification of its restriction of HIV-1 infectivity. SERINC proteins were reported to facilitate the incorporation of serine in the biosynthesis of sphingolipids and phosphatidylserine when ectopically expressed in Escherichia coli and yeast (Inuzuka et al., 2005). It is legitimate to hypothesize that SERINC5 inhibits HIV-1 by altering the composition of lipid membranes of progeny virions.
However, a recent study disputed this model by showing no significant alterations
56 in the steady-state lipid composition of producer cells and HIV particles bearing or lacking SERINC5 (Trautz et al., 2017). Chapter III will discuss the function of
SERINC5 and how it inhibits HIV-1 infection in more detail. In addition to the function, the atomic-level structure of SERINC proteins is so far poorly studied.
The putative secondary structure of SERINC proteins indicates that they contain
10-12 transmembrane domains, which poses significant challenges for structural studies.
There are five members in SERINC protein family, SERINC1-5. Only
SERINC3 and SERINC5 exhibit effective antiretroviral activity, of which the latter inhibits the infectivity of HIV-1 to a considerably larger extent (Rosa et al., 2015;
Schulte et al., 2018; Usami et al., 2015). SERINC3 and SERINC5 are both counteracted by Nefs from different clades, with an exception that SERINC3 is immune to the downregulation by NefSF2 (Usami et al., 2015). The ability to inhibit
HIV-1 infectivity is conserved among primate and rodent SERINC5 proteins
(Heigele et al., 2016). In contrast to most of the host restriction factors, such as
APOBEC3F/G (Sawyer et al., 2004), TRIM5α (Sawyer et al., 2007), SAMHD1, and BST-2, primate SERINC5 orthologs do not display the signatures of an evolutionary arms race with viral pathogens (Murrell et al., 2016), which is consistent with their highly conserved primary sequences.
57 Nef-mediated downregulation of surface proteins.
Nef downregulates important immune receptor proteins by recruiting them to the endocytic machinery or by rerouting them to lysosomes for degradation
(reviewed in Roeth and Collins, 2006). Nef hijacks the clathrin-associated complex AP-2 to induce the internalization of the viral receptor CD4 (Aiken et al.,
1994; Garcia and Miller, 1991; Greenberg et al., 1997), while it exploits AP-1 to downregulate MHC-I and tetherin (Le Gall et al., 1998; Zhang et al., 2009). The heterotetrameric clathrin-associated complexes (AP-1, AP-2, AP-3, and AP-4) are components of vesicle coats that mediate intracellular trafficking. APs selectively recruit proteins by binding to canonical motifs in the cytoplasmic domains. The AP-2 complex harbors a recognition site for the tyrosine-based sorting motif Yxxf in the µ2 subunit and a recognition site for the dileucine-based sorting motif ExxLf in the s2/a unit (in which x is any amino acid, and f represents a large hydrophobic amino acid, either L, I, V, M or F). In addition, the specificity of AP recruitment relies on the subcellular distribution of AP complexes. AP-1 localizes to the TGN, while AP-2 localizes to the inner side of the plasma membrane. A dileucine motif and two diacidic motifs in the C-terminal flexible loop of Nef are required for the downregulation of CD4 (Craig et al.,
1998). In contrast, the dileucine motif is dispensable for the downregulation of
MHC-I (Mangasarian et al., 1999).
Specific motifs in the cytoplasmic tails of surface proteins downregulated by Nef determine the selectivity and efficiency of Nef function. Nef selectively
58 downregulates HLA-A and HLA-B without affecting HLA-C or HLA-E (Cohen et al., 1999; Le Gall et al., 1998), which protects the infected cells from NK cell- mediated killing (Cohen et al., 1999), and CTL recognition. The cytoplasmic tail of MHC-I governs responsiveness to the downregulation by Nef, and specific cytoplasmic tail residues (Y321, D328 and A325) are required for Nef activity
(Cohen et al., 1999). These required residues are present in HLA-A and HLA-B allotypes. However, HLA-C and HLA-E that do not respond to Nef lack one or more of these residues and fail to bind Nef (Cohen et al., 1999; Le Gall et al.,
1998).
Structures of Nef in complex with target proteins and AP complexes help to understand the mechanism of downregulation mediated by Nef (Manrique et al., 2017; Shen et al., 2015). For instance, a recent structural study illuminates why only HIV-2 and SIV Nefs, but not HIV-1 Nef, can downregulate CD3
(Manrique et al., 2017). In particular, the study shows that HIV-1 Nef lacks an N- terminal tyrosine motif that is required for the internalization of CD3 by Nef
(Manrique et al., 2017). Another structural study shows that HIV-1 Nef hijacks clathrin by stabilizing a complex between AP-1 and the small guanosine triphosphatase Arf1 (Shen et al., 2015).
Despite the use of different molecular mechanisms, Nef and Vpu possess overlapping functions of regulating cell surface proteins (Haller et al., 2014). Most of the SIVs and the O group of HIV-1 use their Nefs to counteract tetherin, while
HIV-1 M strains use Vpu for the same function (Sauter et al., 2009; Zhang et al.,
59 2009). A recent study concluded that Vpu does not counteract SERINC5
(Heigele et al., 2016).
Downregulation of SERINC5 by Nef.
The machinery of SERINC5 downregulation requires the interaction of Nef with the clathrin adaptor complex AP-2 (Pizzato et al., 2007; Rosa et al., 2015).
In particular, the highly conserved dileucine-based sorting motif (ExxxLL) in the
C-terminal loop of HIV-1 Nef is required for the downregulation of SERINC5
(Heigele et al., 2016; Manrique et al., 2017; Rosa et al., 2015; Trautz et al.,
2016). In addition to the dileucine motif, three additional charged residues,
ERE177, in the C-terminal loop of Nef are indispensable for SERINC5 antagonism (Heigele et al., 2016). These residues are also critical for the downregulation of CD4, but not of MHC-I (Sauter et al., 2015). In general, Nef residues essential for SERINC5 antagonism are also essential for CD4 downregulation by Nef (Trautz et al., 2016). The findings are perhaps not surprising, since the downregulation of CD4 and of SERINC5 by Nef both dependent on the adaptor complex AP-2.
Interestingly, not all SIV Nefs require a ExxxLL motif for downregulating
SERINC5. The SIVcol Nef, which lacks a canonical ExxxLL motif, potently antagonizes SERINC5, which suggests a convergent evolution of SERINC5 antagonism (Heigele et al., 2016). Interestingly, a phylogenetic study indicates a
60 correlation between the potency of SERINC5 antagonism by Nef and the prevalence of SIVs in their natural hosts (Heigele et al., 2016).
Nef selectively downregulates SERINC3 and SERINC5 but not SERINC1 from the cell surface (Matheson et al., 2015). Furthermore, the effects of HIV-1
Nef proteins on SERINC3 and SERINC5 are strain-dependent. For instance, the
Nef proteins of the primary HIV-1 isolates 97ZA012 and 93BR020 strongly inhibit the incorporation of SERINC3 and SERINC5 into virions; in contrast, the Nef protein of the T cell line-adapted strain SF2 is only active against SERINC5
(Usami et al., 2015). Thus, Nef proteins can discriminate between different
SERINC family members. This is reminiscent of the selective recognition of distinct MHC-I allotypes (Cohen et al., 1999). Thus, it is important to understand which domains in SERINC5 determine its recognition and downregulation by Nef.
Summary
Recent studies have identified the multipass transmembrane proteins
SERINC3 and SERINC5 as antiviral proteins that are incorporated into Nef- deficient HIV-1 virions. Nef counteracts the antiretroviral activity of SERINC5 by removing it from the cell surface, and thus prevents its incorporation into progeny virions (Rosa et al., 2015; Usami et al., 2015). It has been shown that the downregulation of SERINC5 requires the interaction of Nef and clathrin- associated AP-2 complex (Pizzato et al., 2007; Rosa et al., 2015). SERINC5 downregulation requires a dileucine motif in Nef, and other Nef residues that are
61 recognized by AP-2 (Heigele et al., 2016; Manrique et al., 2017; Rosa et al.,
2015; Trautz et al., 2016). Moreover, Nef selectively downregulates some
SERINC proteins but does not affect others. However, the determinants that govern sensitivity to Nef have not been identified.
We now show that an intracellular loop of human SERINC5 confers sensitivity to widely divergent Nefs in the context of a Nef-resistant vertebrate
SERINC5, and also confers sensitivity to NefSF2 in the context of human
SERINC3, which is normally resistant to this particular Nef. Importantly, human
SERINC5 acquires resistance against Nefs from different HIV-1 clades when this intracellular loop is replaced by the corresponding region from a Nef-resistant
SERINC5. Taken together, our results identify a major determinant of Nef responsiveness, and establish that human SERINC5 can be made resistant to
Nef-induced downregulation.
Results
The anti-viral activity of SERINC5 proteins is conserved, but their responsiveness to Nef differs.
Most of the host antiretroviral factors are important determinants of viral host range and cross-species transmission (Gaddis et al., 2004; Keckesova et al., 2006). HIV-1 evades their potent inhibitory activity in human cells. But the ability of HIV-1 to replicate in non-human cells is often severely compromised
62 due to the host factors. To test whether the ability to inhibit HIV-1 infectivity is conserved among SERINC5 proteins, we used 293T cells to produce HIV-1 virus in the presence of exogenous SERINC5 proteins. Importantly, 293T cells express very little endogenous SERINC5. To generate progeny virus capable of only a single cycle of replication, we used an Env-deficient HIV-1 mutant, and Env was provided in trans from a separate plasmid.
As little as 100 ng of a relatively weak (pBJ5-based) SERINC5 expression plasmid is sufficient to potently inhibit the single cycle infectivity of Nef− HIV-1 virions (Usami et al., 2015). Under the same conditions, mouse SERINC5 reduced the specific infectivity of Nef− HIV-1 virions produced in 293T cells to a similar extent (40- to 50-fold) as human SERINC5 (Figure 2.1A). Zebrafish
(Danio rerio) SERINC5, which is only 61% identical to human SERINC5, reduced progeny virus infectivity 13- or 21-fold when 100 or 500 ng expression vector were used, respectively (Figure 2.1A).
Next, we examined the sensitivities of mouse and zebrafish SERINC5 to
NefSF2, which selectively inhibits the incorporation of human SERINC5 into HIV-1 virions (Usami et al., 2015). While the inhibitory effect of mouse SERINC5 on
− Nef HIV-1 produced in 293T cells was clearly counteracted by NefSF2 expressed in trans, the effect of zebrafish SERINC5 was unaffected by NefSF2 (Figure
2.1B). We infer that widely divergent SERINC5 proteins share the ability of human SERINC5 to inhibit HIV-1 infectivity, but not necessarily its sensitivity to
Nef.
63
Figure 2.1. Anti-HIV-1 activity but not responsiveness to Nef is conserved among SERINC5 orthologs. (A) Human, mouse, and zebrafish SERINC5 proteins share the ability to inhibit the specific infectivity of HIV-1 progeny virions. The amount of transduced plasmids of SERINC5 for the infectivity assay was labeled in the figure. (B) NefSF2 expressed in trans in virus producer cells counteracts the effect of exogenous mouse but not zebrafish SERINC5 on Nef‒ HIV-1 progeny virion infectivity. Bar graphs represent the mean + SD from 3 biological replicates. The amount of transduced plasmids of SERINC5 for the infectivity assay was 100 ng.
Transfer of Nef sensitivity to a Nef-resistant SERINC5.
The differential responsiveness of vertebrate SERINC5 proteins to Nef provided a potential tool to identify determinants that confer sensitivity to Nef.
Because zebrafish SERINC5 was somewhat less active against Nef− HIV-1 than
64 human SERINC5, we also tested the SERINC5 protein of the Western clawed frog (Xenopus tropicalis). Frog SERINC5 expressed from pBJ5 inhibited the single cycle infectivity of Nef− HIV-1 progeny virions with a potency comparable to that of human SERINC5 (Figure 2.2A). However, whereas human SERINC5 is efficiently counteracted by NefSF2 (Usami et al., 2015), the inhibitory effect of
− frog SERINC5 expressed from pBJ5 on Nef HIV-1 was unaffected by NefSF2
(Figure 2.2C). Even when expressed from pBJ6, which lacks an SV40 origin required for episomal amplification as well as SV40 enhancer sequences (Rosa et al., 2015), frog SERINC5 reduced the specific infectivity of Nef− HIV-1 more than 40-fold, and this inhibitory effect largely persisted in the presence of NefSF2 expressed in trans (Figure 2.2C). In the absence of exogenous SERINC5, the
− effect of NefSF2 on the infectivity of Nef HIV-1 virions was quite modest in these experiments (Figure 2.2C), perhaps because 293T cells express only low levels of endogenous SERINC5 (Usami et al., 2015).
We subsequently used frog SERINC5 as a recipient of human SERINC5 sequences to identify determinants that confer sensitivity to NefSF2. The TMHMM membrane protein topology prediction method (Krogh et al., 2001) predicts that
SERINC5 has 10 transmembrane helices, and our previous findings indicate that the loop connecting helices 7 and 8 is surface-exposed (Usami et al., 2015). This in turn suggested that the loop connecting helices 8 and 9 represents the fourth and longest intracellular loop (Figure 2.2B). To examine the role of this region, we replaced the ICL4 of frog SERINC5 by that of human SERINC5 (Figure
65 2.2B). The resulting fS5/hS5-ICL4 chimera largely retained the ability of frog
SERINC5 to inhibit the specific infectivity of Nef− HIV-1 progeny virions, but lost much of its resistance to NefSF2 expressed in trans (Figure 2.2C). This observation applied to SERINCs expressed both from pBJ5 and from the very weak pBJ6 expression plasmid. Thus, the transfer of the ICL4 of human
SERINC5 was sufficient to confer substantial sensitivity to NefSF2.
As previously reported (Usami et al., 2015), the incorporation of haemagglutinin (HA)-tagged human SERINC5 into progeny virions was strongly inhibited by NefSF2 (Figure 2.2D, lanes 1 and 2). In contrast, the incorporation of frog SERINC5 was only slightly affected by NefSF2 (lanes 3 and 4). Finally, the effect of NefSF2 on the incorporation of the fS5/hS5-ICL4 chimera resembled its effect on human SERINC5 (lanes 5 and 6). We conclude that the transfer of the
ICL4 was sufficient to allow NefSF2 to significantly inhibit both the anti-HIV-1 effect and the virion incorporation of frog SERINC5.
66
Figure 2.2. Transfer of human SERINC5 ICL4 is sufficient to confer Nef responsiveness to frog SERINC5. (A) Frog SERINC5 is as potent as human SERINC5 in inhibiting the single-round infectivity of Nef‒ HIV-1 progeny virions. The amounts of SERINC5 plasmids differed in left and right panels. (B) Schematic illustration of the parental SERINC5 proteins and of the chimera examined. (C) The effect of frog SERINC5 on Nef‒ HIV-1 progeny virion infectivity is largely resistant to NefSF2 but becomes sensitive upon replacement of its ICL4 by that of human SERINC5. The expression plasmids of SERINC5 used for the left panel were pBJ5-based, which the ones for the right panel were pBJ6-based. (D) Western blots showing the effects of NefSF2 on the incorporation of human, frog and chimeric SERINC5 into Nef‒ HIV-1 virions. Bar graphs represent the mean + SD from 3 biological replicates.
67 The ICL4 of human SERINC5 confers sensitivity to widely divergent Nefs.
Unlike NefSF2 (clade B), which selectively inhibits the incorporation of human SERINC5 into HIV-1 virions, Nef97ZA012 (clade C) also inhibits the incorporation of human SERINC3 (Usami et al., 2015). Nevertheless, the inhibitory effect of frog SERINC5 on HIV-1 infectivity was essentially unaffected by Nef97ZA012 (Figure 2.3A), as was the incorporation of frog SERINC5 into HIV-1 virions (Figure 2.3B). In contrast, Nef97ZA012 enhanced the specific infectivity of
Nef− HIV-1 produced in the presence of the fS5/hS5-ICL4 chimera about 13-fold
(Figure 2.3A), and strongly inhibited the incorporation of the chimeric protein
(Figure 2.3B). Thus, the ICL4 of human SERINC5 conferred sensitivity to HIV-1
Nefs from different subtypes.
Because the ability to counteract SERINC5 is conserved among all primate lentiviral lineages (Heigele et al., 2016), we additionally examined the
− effects of SIV Nefs. SIVmac239 Nef increased the specific infectivity of Nef HIV-1 produced in the presence of frog SERINC5 or of the fS5/hS5-ICL4 chimera 17- and 27-fold, respectively (Figure 2.3C). Thus, SIVmac239 Nef counteracted even unmodified frog SERINC5. In contrast, unmodified frog SERINC5 was largely resistant to the Nef proteins of SIVagm155 and SIVagm677, and the transfer of the
ICL4 of human SERINC5 substantially increased its sensitivity to these Nefs
(Figures 2.3D and 2.3E). Overall, these findings demonstrate that the ICL4 of human SERINC5 can confer sensitivity to Nefs from widely divergent primate lentiviruses.
68
A B C Nef- Nef 97ZA012 Nef : fS5/ 1.8 *** 97ZA012 + + fS5 hS5-ICL4 A 1.6 B C Nef - Nef- Nef S5-HA LAI 1.4 97ZA012 fS5/ HA *** Nef97ZA012: fS5 1.8 ty + + hS5-ICL4 1.2 Virus Virus 1 2 ivi 1.6 t 1 2 3 4 Nef - 1 S5-HA LAI 1.4 f ec **** HA
in HIV-1 HIV-1 ty 0.8 Virus 1 2 1.2 Virus gag ivi CA
t 1 2 3 4 Pr55
ive 1 2 3 4 1 t 0.6 1 2 f ec ****
in HIV-1 HIV-1 0.8 0.4 Rela 1 2 3 4 CA S5-HA Pr55gag ive Cells t 0.6 0.2 NS 1 2 Cells NefLAI- 0.4 Rela 0 Cells S5-H1 A2 3 4 HA 0.2 NSVector fS5/ fS5 Cells NefLAI- 1 2 (pBJ5) hS5-ICL4 fS5/ 0 1 2 3 4 fS5 HA Vector fS5/ hS5-ICL4 fS5 1 2 (pBJ5) hS5-ICL4 100 ng each fS5/ fS5 hS5-ICL4 100 ng each
Nef- Nef Nef- Nef Nef- Nef D SIVmac239 E SIVagm155 F SIVagm677 D Nef- Nef E Nef- Nef ** F Nef- Nef C 5 SIVmac2391.6 D* * 5SIVagm155 1.2 E SIVagm6775 0.8 ** ** ** ** 5 1.6 5 1.2 5 0.8 ** 4.5 4.5 ** 4.5 0.7 4.5 1.4 4.5 4.5 41.4 4 1 0.7 4 4 4 1 4 ty 1.2 0.6
ty 3.51.2 3.5 0.6 3.5 3.5 ivi ** 3.5 0.8 3.5 *** ivi ** 0.8 *** 3 1 1 3 0.5 3 0.5
3 ec t 3 3 ec t f f in in 2.5 2.50.8 0.8 2.5 0.6 2.5 0.6 2.5 0.4 2.5 0.4
ive 2 2 2 ive 2 2 2 t 0.6 0.3 t 0.6 0.3 1.5 1.5 0.4 0.4 1.5 1.50.4 1.5 0.2 1.5 Rela ** 0.4 0.2
1 Rela 1 1 1 ** 0.2 1 1 0.5 0.2 0.5 ** 0.2 0.5 0.1 ** 0.5 0.2 0.5 ** 0.5 0.1 ** 0 0 0 0 0 0 Vector fS5/ Vector fS5/ Vector fS5/ 0 fS5 0 0fS5 0 fS5 0 0 (pBJ5) Vector hS5-ICL4 fS5/ (pBJ5) VectohS5-ICr L4 (pBfSJ55/) hS5-ICLVecto4 r fS5/ fS5 fS5 fS5 (pBJ5) hS5-ICL4 (pBJ5) hS5-ICL4 (pBJ5) hS5-ICL4
Figure 2.3. The ICL4 of human SERINC5 confers sensitivity to widely divergent Nefs. (A) The inhibitory effect of frog SERINC5 on HIV-1 infectivity is resistant to HIV-1 subtype C Nef97ZA012 but becomes sensitive in the presence of the ICL4 of human SERINC5. (B) Western blots showing that Nef97ZA012 does not affect the incorporation of frog SERINC5 into Nef‒ HIV-1 virions but strongly inhibits the incorporation of a chimeric version that harbors the ICL4 of human SERINC5. (C-E) Effects of SIV Nef proteins on the inhibition of HIV-1 infectivity by frog SERINC5 and by a version that harbors the ICL4 of human SERINC5. In all cases, the histograms represent the mean + SD from three measurements.
69 The ICL4 of frog SERINC5 confers resistance to Nef.
The results described above showed that the fS5/hS5-ICL4 chimera gained sensitivity to various Nefs. We next made the reciprocal hS5/fS5-ICL4 chimera bearing the ICL4 of frog SERINC5 in the context of human SERINC5
(Figure 2.4A), in order to test the hypothesis that the ICL4 of human SERINC5 is necessary for its Nef responsiveness. The hS5/fS5-ICL4 chimera inhibited the
− infectivity of Nef HIV-1 virions carrying EnvHXB2 as potently as authentic human
SERINC5 (Figures 2.4B and 2.4C). Although the infectivity of Nef− HIV-1 virions carrying the relatively SERINC5-resistant EnvJRFL was inhibited to a lesser extent as expected (Rosa et al., 2015), the hS5/fS5-ICL4 chimera again was at least as potent as human SERINC5 (Figure 2.4D), confirming that it retained full anti-HIV activity.
Crucially, whereas NefSF2 increased the infectivity of progeny virions carrying EnvHXB2 about 37-fold in the presence of exogenous human SERINC5, only a 2-fold increase was observed in the presence of the hS5/fS5-ICL4 chimera
(Figure 2.4B). Furthermore, similar results were obtained with Nef97ZA012 (Figure
2.4C). Consistent with these observations, the incorporation of the hS5/fS5-ICL4 chimera into HIV-1 virions was not inhibited by NefSF2, and only moderately reduced by Nef97ZA012 (Figure 2.4E). As expected, both Nef proteins strongly inhibited the incorporation of authentic human SERINC5 (Figure 2.4E). We conclude that the hS5/fS5-ICL4 chimera exhibits marked resistance to HIV-1 Nef proteins.
70
Figure 2.4. The ICL4 of frog SERINC5 confers resistance to Nef. (A) Schematic illustration of the parental SERINC5 proteins and of the chimera examined. (B) Human SERINC5 bearing the ICL4 of frog SERINC5 remains fully capable of inhibiting the infectivity of Nef‒ HIV-1 virions but exhibits strong resistance to a clade B HIV-1 Nef (NefSF2). (C) The SERINC5 chimera also exhibits resistance to a clade C HIV-1 Nef (Nef97ZA012). (D) The SERINC5 chimera inhibits the infectivity of Nef‒ HIV-1 virions carrying a relatively resistant HIV-1 Env at least as well as the wild-type human SERINC5.
71 (E) Western blots showing the effects of clade B and C HIV-1 Nefs on the incorporation of human SERINC5 and of the SERINC5 chimera into Nef‒ HIV-1 virions. Bar graphs represent the mean + SD from 3 biological replicates.
Evidence for a physical interaction between Nef and the ICL4 of human SERINC5.
A pronounced tendency to aggregate makes it challenging to examine whether SERINC5 and Nef interact via co-immunoprecipitation. Thus, we tested whether SERINC5 affects the virion-association of the LL164,164AA AP-2 binding site mutant of NefLAI (Figure 2.5A), which does not counteract SERINC5 (Rosa et al., 2015). The mutant Nef was more abundant in HIV-1 virions produced in the presence of the fS5/hS5-ICL4 chimera rather than of frog SERINC5 (Figure
2.5B). Since frog SERINC5 and the fS5/hS5-ICL4 chimera were incorporated about equally in the absence of a functional Nef (Figure 2.3B), this finding provides indirect evidence for a physical interaction between Nef and the ICL4 of human SERINC5.
72
Figure 2.5. The physical interaction between Nef and the ICL4 of human SERINC5. (A) Schematic illustration of NefLAI mutant which is disabled of binding to AP2. (B) Western blots showing that a NefLAI mutant unable to counteract SERINC5 (LL164,165AA) is more abundantly incorporated into HIV-1 virions produced in the presence of frog SERINC5 bearing the ICL4 of human SERINC5. The virions lacked protease to prevent the cleavage of Nef.
ICL4 of human SERINC2 confers resistance to NefSF2, but not Nef97ZA012.
To further investigate the role of the ICL4 in other members of SERINC family, we replaced the ICL4 of human SERINC5 by that of human SERINC2, which exhibits no sequence similarity in this region. Human SERINC2 does not exhibit antiretroviral activity, and is resistant to downregulation by NefSF2.
The resulting hS5/hS2-ICL4 chimera (Figure 2.6A) inhibited the specific infectivity of Nef− HIV-1 virions, albeit less potently than authentic human
73 SERINC5 (Figure 2.6B). In this experiment, NefSF2 increased virion infectivity more than 50-fold in the presence of exogenous human SERINC5, but less than
2-fold in the presence of the chimera. Furthermore, the incorporation of the hS5/hS2-ICL4 chimera into HIV-1 virions was essentially resistant to NefSF2
(Figure 2.6C).
To directly test the surface expression of SERINC5 chimeric proteins and how Nef regulates it, we used SERINC5(iHA), which contains an internal HA tag within an extracellular loop (Usami et al., 2015). Flow cytometry revealed that the surface expression of SERINC5(iHA) was not compromised when its ICL4 was replaced by that of SERINC2, and that the ICL4 swap conferred resistance to down-regulation by NefSF2 (Figure 2.7). Together, these results establish that the
ICL4 swap markedly reduced the sensitivity of human SERINC5 to NefSF2.
During the course of these experiments, we observed that the incorporation of human SERINC2 into Nef-deficient HIV-1 virions, although unaffected by NefSF2, is inhibited by Nef97ZA012 (Figure 2.6D). Similarly, the incorporation of the hS5/hS2-ICL4 chimera, while unaffected by NefSF2, was clearly inhibited by Nef97ZA012 (Figure 2.6E). Since the hS5/fS5-ICL4 chimera, which differs only in the ICL4, was resistant to Nef97ZA012 (see Figure 2.4C), these findings raise the possibility that Nef97ZA012 recognizes a determinant within the ICL4 of SERINC2. Additionally, these findings suggest that only ICL4 regions derived from SERINCs that exhibit resistance to a given Nef confer resistance to the same Nef when transferred to a sensitive SERINC.
74
Figure 2.6. The ICL4 of human SERINC2 confers resistance to NefSF2 but not Nef97ZA012. (A) Schematic illustration of the parental human SERINC proteins and of the chimera examined. (B) The inhibitory activity of human SERINC5 bearing the ICL4 of human ‒ SERINC2 on Nef HIV-1 progeny virion infectivity is largely resistant to NefSF2. Bar graphs represent the mean + SD from 3 biological replicates. (C) Western blots showing that the incorporation of the SERINC chimera into ‒ Nef HIV-1 virions is largely unaffected by NefSF2 expressed in trans. The incorporation of the chimera was analyzed in duplicate to document reproducibility. (D) Western blots showing that the incorporation of human SERINC2 into Nef‒ HIV-1 virions is unaffected by NefSF2 but inhibited by Nef97ZA012.
75 (E) Western blots showing that Nef97ZA012 inhibits the incorporation of human SERINC5 bearing the ICL4 of human SERINC2 into Nef‒ HIV-1 virions.
V SERINC5(iHA) hS5/hS2-IL4(iHA) +NefSF2FS +NefSF2FS C S F
SERINC5(iHA) hS5/hS2-IL4(iHA)
+NefSF2 +NefSF2 C S F
HA-APC
Figure 2.7. Swapping the ICL4 of human SERINC5 for that of human SERINC2 confers resistance to down-regulation by NefSF2. JurkatTAg S5 -/- cells stably expressing SERINC5(iHA) or the S5/hS2-ICL4(iHA) chimera together with NefSF2 were surface-stained with anti-HA antibody and analyzed by flow cytometry. Per cent fractions of cells expressing SERINC5(iHA) or the S5/hS2-ICL4(iHA) chimera on the surface are indicated.
76 Transfer of sensitivity against NefSF2 to SERINC3.
SERINC3 is another member of the SERINC family that exhibits antiretroviral activity, though this activity is much weaker than that of SERINC5.
Unlike other HIV-1 Nefs, NefSF2 has no effect on the incorporation of SERINC3 into HIV-1 virions (Usami et al., 2015). To examine whether the ICL4 of
SERINC5 confers sensitivity to NefSF2 in the context of human SERINC3, we generated the hS3/hS5-ICL4 chimera, which harbors the ICL4 of human
SERINC5 in a human SERINC3 background (Figure 2.8A). While the incorporation of authentic human SERINC3 was unaffected by NefSF2 as previously reported (Usami et al., 2015), the incorporation of the chimera was strongly inhibited (Figure 2.8B), confirming the role of the ICL4 in determining
Nef sensitivity.
77 A B NefSF2: + + + S3-HA
1 2 3 4 5 6 Virus hS3 ICL4 HIV-1 CA 1 2 3 4 5 6
hS5 Cells S3-HA
1 2 3 4 5 6 hS3/hS5-ICL4 hS3 hS3/hS5-ICL4 (0.5 μg) 0.1 μg 0.2 μg
Figure 2.8. Human SERINC3 acquires sensitivity to NefSF2 in the presence of the ICL4 of human SERINC5. (A) Schematic illustration of the parental human SERINC proteins and of the chimera examined. (B) Western blots showing that NefSF2 inhibits the incorporation of human SERINC3 into Nef‒ HIV-1 virions only upon replacement of its ICL4 by that of human SERINC5.
78 Point mutations in the ICL4 of human SERINC5 confer resistance to Nef.
Although Nef typically targets dileucine- or tyrosine-based sorting motifs, such motifs are not evident within the ICL4 of human or mouse SERINC5
(Figure 2.9A). To examine which region of the ICL4 determines sensitivity to
Nef, we generated versions of Nef-resistant frog SERINC5 that have relatively poorly conserved segments replaced by the corresponding human SERINC5 sequences (Figure 2.9A). We observed that the transfer of ICL4 residues 9-26 but not of ICL4 residues 26-41 of human SERINC5 significantly increased the sensitivity of frog SERINC5 to NefSF2 (Figure 2.9B). Within the 9-26 segment, a leucine residue (L350 in human SERINC5) is present in Nef-sensitive but not in
Nef-resistant SERINC5 proteins (Figure 2.9A). Point mutations that targeted this residue (L350A) or another small hydrophobic residue nearby (I353A) reduced the sensitivity of human SERINC5 to NefSF2 about 8- and 3-fold, respectively
(Figure 2.9C). Both mutations together (L350A/I353A) reduced the sensitivity of human SERINC5 more than 12-fold, and an 8-fold reduction was observed when
L350 and I353 were replaced by the corresponding residues in frog SERINC5
(L350T/I353M) (Figure 2.9C). We also observed that the L350A and
L350A/I353A mutations rendered the incorporation of human SERINC5 into HIV-
1 virions largely resistant to NefSF2 (Figure 2.9D). These findings confirm the crucial role of the ICL4 in Nef sensitivity.
79
350 A 334 350 353 390 AAA 334334 350350 353353 390390 Human HumaHumaHumann Mouse MousMousMousee Frog FroFrogg Zebrafish ZebrafisZZebrafisebrafishh
fS5/hS5-ICL4(9-26) Q G - R Y A A P E L E I ARC CFC F fS5/hS5-IfS5/hS5-IfS5/hS5-ICLCL4(49(9-2--26)6)6) QQQGGG--RRYYAAAAPPPEEELLLEEEIIIARARARCCCCFCFCFCCCFFF fS5/hS5-ICL4(26-41) F S P - G G E D T E E QQPGKE fS5/hS5-IfS5/hS5-ICLCL4(42(62-64-41)1) FFFS SP P- -G G EG DETDETEEQQPGKE QQPGKE EE
BBB CC D Nef- Nef D Nef- NefSF2 NefSF2: + + + NNeef-f- NNeef fSF2 ** Nef- NefSF2 NeSF2ff :: + + + 7 * SFSF22 1.4 ** NNeeff-- NNeeSFff 2 SFSF22 ++ ++ ++ 7 77 ** 11.4.4 **** SF2 6 1.2 S5-HA 6 66 11.2.2 S5-HS5-HAA 5 1 Virus ty 5 1 Virus 5ty 5 11 Viruss 1 2 3 4 5 6 ty ty 1 2 3 4 5 6 ivi 11 22 33 44 55 66 ivi
ivi ivi 4 0.8
ec t 4 0.8 f
4ec t 4 00.8.8 f ec t ec t HIV-1 f f in HIV-1 in 3 0.6 CA HIV-1HIV-1 in in ** 3 33 00.6.6 ** 1 2 3 4 5 6 CA CCAA ive NS **** 1 2 3 4 5 6 t
ive NS 11 22 33 44 55 66 t
ive ive NSNS t t 2 0.4 2 2 NS 00.4.4 Rela NSNS Rela
Rela Rela 1 0.2 1 0.2 * **** * * 1 1 00.2.2 ** ******** ** * Cells S5-HA CellCells ss S5-HS5-HAA 0 0 0 0 hS5- hS5- 00 Vector L350A/ L350T/ hS5IChS5L-4- hS5IChS5L-4- VectoVectorrr WT L350A I353A L350AL350AL350A/ L350T// L350T/ / 1 2 3 4 5 6 WT ICL4 ICL4 (pBJ5) WWWTTT L350L350L350AAAI353II353353A AAI353A I353M 1 112 223 334 445 556 66 WTWT IC(9ICL-426)L4 (26-41ICICL4L4) (pB(pB(pBJJ5J5)5)) I353II353353A AI353II353M M L350A/ (9(-926)--26) (26-41(26-41(26-41))) hSERINC5: WT L350A L350A/ hSERINC5 hSERINC5: WT L350A I353L350AAL350A// pBJ6-fS5 (1 μg each) (100hSERINChSERINCng each)55 hSERINC5: WTWT L350L350AAI353AI353I353AA pBpBJ6J-fS56-fS5(1(1(1μμgggeacheacheach))) (100(100(100ngngngeacheacheach) ))
Figure 2.9. Point mutations in the ICL4 of human SERINC5 confer resistance to Nef. (A) Alignment of the ICL4 regions of Nef-sensitive (human, mouse) and Nef- insensitive (frog, zebrafish) SERINC5 proteins, and schematic illustration of intra- ICL4 chimeras examined. (B) The transfer of human SERINC5 ICL4 residues 9-26 but not of residues 26- 41 to the ICL4 of frog SERINC5 increases its sensitivity to NefSF2. (C) Point mutations targeting small hydrophobic amino acids within the ICL4 of human SERINC5 reduce the sensitivity of its effect on HIV-1 infectivity to NefSF2. (D) Western blots showing the effects of NefSF2 on the incorporation of WT human SERINC5 and of ICL4 mutants into Nef‒ HIV-1 virions. In all cases, the histograms represent the mean + SD from three measurements.
80 Nef poorly counteracts inhibition of HIV replication by a SERINC5 chimera.
To investigate the role of SERINC5 and its ICL4-mediated counteraction by Nef in HIV-1 replication and spreading, we reconstituted SERINC3+SERINC5 double-knockout (DKO) JTag cells with human SERINC5 and the chimera bearing the ICL4 of frog SERINC5 (hS5/fS5-ICL4) (Figure 2.4A). HIV-1NL43- based viruses encoding either wild-type or a Nef-deficient version of NefNL43 were used to infect cells. Both Nef+ and Nef- HIV-1 were able to replicate in the double-knockout cells at comparable rates, while Nef+ virus more robustly replicated in cells reconstituted with human SERINC5 compared to Nef- virus
(Figure 2.10A, B). These observations confirmed that Nef counteracts the inhibition of HIV-1 replication by human SERINC5 (Usami et al., 2015).
DKO cells reconstituted with either human SERINC5 or hS5/fS5-ICL4 controlled the viral spreading of Nef-deficient HIV-1 for at least 21 days.
However, Nef+ HIV-1 replication in DKO cells reconstituted with hS5/fS5-ICL4 was delayed compared to that in human SERINC5-reconstituted DKO cells until
19 days post-infection, which indicated that the resistance to Nef conferred by frog SERINC5 ICL4 led to the inhibition of HIV-1 replication (Figure 2.10A, B).
However, Nef+ HIV-1 eventually spread in hS5/fS5-ICL4-reconstituted cells
(Figure 2.10B), which might suggest that additional restriction factor(s) counteracted by Nef can suppress viral spreading.
81
Figure 2.10. Nef poorly counteracts inhibition of HIV replication by a SERINC5 chimera harboring the frog SERINC5 ICL4. (continued on the next page)
82 Effects of Nef on virus spreading in double-knockout JTAg cells lacking SERINC3 and SERINC5, SERINC5-reconstituted double-knockout cells, and hS5/fS5-ICL4-reconstituted double-knockout cells. The spreading of HIV-1NL43- based viruses encoding either wild-type or disrupted version of Nef97ZA012 was examined by western blotting of cell lysates with anti-CA antibody 12 days or 15 days after infection with 2 ng ml-1 p24 (A), or by monitoring p24 accumulation in the supernatants (B).
Discussion
This study shows that widely divergent SERINC5 proteins from different vertebrate species share the ability to markedly inhibit the infectivity of Nef− HIV-
1 virions. However, sensitivity to Nef is not conserved and can be transferred to a
Nef-resistant SERINC5 by exchanging the ICL4 region. In the same manner, human SERINC3 can be made sensitive to a particular Nef, supporting the crucial role of the ICL4 region in Nef sensitivity. Conversely, our results show that the anti-HIV-1 activity of human SERINC5 can be made to persist even in the presence of Nef, which might provide a novel strategy to combat HIV-1
(summarized in Figure 2.11). However, the in vivo significance of the effect of
Nef on SERINC5 remains to be established.
83
Figure 2.11. A long cytoplasmic loop governs the sensitivity of SERINC5 to Nef. Nef downregulates human SERINC5 from cell surface and prevents its incorporation into progeny virions. hS5-free viruses can successfully infect target cells (left panel). In contrast, frog SERINC5 is resistant to counteraction by Nef, and incorporated into virions even in the presence of Nef. Progeny virions bearing frog SERINC5 fail to infect new target cells (middle panel). Human SERINC5 bearing the ICL4 of frog SERINC5 is resistant to Nef downregulation and incorporated into progeny virions even in the presence of Nef.
84 The highly conserved antiviral activity of SERINC5.
The conservation of the anti-HIV activity among vertebrate SERINC5 proteins indicates that this activity is closely related to the biological function of
SERINC5, which remains to be elucidated. The conserved antiretroviral activity suggested by our studies is in agreement with the computational analysis of primary sequences of SERINC5 (Murrell et al., 2016). SERINC5 plays a role in the interaction of the host with another retrovirus, equine infectious anemia virus
(EIAV) (Chande et al., 2016). Similar to HIV-1 Nef counteracting SERINC5, the
S2 protein of EIAV antagonizes the inhibitory activity of SERINC5 (Chande et al.,
2016). SERINCs have been named for their reported function as carrier proteins that facilitate the synthesis of serine-derived lipids (Inuzuka et al., 2005). This activity suggested a model in which SERINCs inhibit HIV-1 infectivity by modifying the lipid content of progeny virions. However, a recent study argues strongly against this hypothesis by showing that SERINC5 does not alter the lipid composition of HIV-1 progeny virions or the exposure of phosphatidylserine on their surface (Trautz et al., 2017).
The mechanism and machinery of the counteraction of SERINC5 by Nef.
Usami et al. (Usami et al., 2015) and Pizzato and coworkers (Rosa et al.,
2015) have presented evidence indicating that Nef counteracts SERINC5 by preventing its incorporation into HIV-1 virions. More recent work confirmed that
Nef excludes SERINC5 from virions, but also suggested that Nef additionally
85 inactivates the antiviral activity of any SERINC5 that becomes virion-associated even in the presence of Nef as a consequence of overexpression (Trautz et al.,
2016). In our study, the ability of Nef proteins to reduce the incorporation of native or chimeric versions of frog or human SERINC5 correlated well with their ability to enhance HIV-1 infectivity, which supports the notion that virion exclusion is the primary mechanism by which Nef counteracts SERINC5.
The ability of Nef to counteract human SERINC5 depends on the AP-2 clathrin adaptor complex (Rosa et al., 2015), whose cargo-binding AP2M1 and
AP2S1 subunits in particular are more than 95% identical in human, frog, and zebrafish. Nef also hijacks clathrin adaptors to downregulate CD4 and MHC-I, and in these cases Nef:AP complexes recognize linear epitopes within the cytosolic domains of CD4 and MHC-I (Jia et al., 2012; Ren et al., 2014). Our results indicate that certain small hydrophobic residues within the ICL4 of human
SERINC5 are critical for its sensitivity to Nef, and it is tempting to speculate that the region containing these residues is also recognized by Nef:AP complexes.
The effect of Nef polymorphism on the counteraction of SERINC5.
Nearly the entire ICL4 region is predicted to be flexible by the PROFbval program (Schlessinger et al., 2006). The apparent flexibility of the ICL4 could conceivably facilitate interactions with widely divergent Nef proteins through a mechanism involving conformational selection (Boehr et al., 2009). Indeed, our data show that the ICL4 of human SERINC5 can confer sensitivity to Nefs from
86 different primate lentiviral lineages. In addition to the canonical sorting motif
ExxxLL of Nef, an ERE177 sequence within Nef plays a role determining
SERINC5 antagonism (Heigele et al., 2016). The participation of multiple motifs within Nef in its effect on SERINC5 illustrates the importance of overcoming this restrictive factor in HIV-1 infection. On the other hand, the mere replacement of two adjacent amino acids within the ICL4 of human SERINC5 completely erased the sensitivity to Nef (Figure 2.9C). Studies examining polymorphisms within
SERINC5 in a large population might reveal the role of SERINC5 in HIV-1 infection.
The downregulation of immune receptors mediated by Nef benefits viral pathogenesis and persistence. Comparison of nef alleles derived from long-term nonprogressors and progressors revealed that sequence variations in Nef are associated with different stages of infections (Kirchhoff et al., 1999). Primary Nef isolates from elite controllers exhibit an impaired ability to downregulate CD4,
MHC-I, and NKG2D (Alsahafi et al., 2017; Mwimanzi et al., 2013; Toyoda et al.,
2015). Elite controllers are a rare group of infected individuals who spontaneously control plasma viremia below 50 RNA copies/ml in the absence of
ART (reviewed in Deeks and Walker, 2007). The mechanism of elite control remains elusive, even though extensive GWAS studies were carried out to address this question (reviewed in Walker and Yu, 2013). HLA-B*27 and HLA-
B*57 are associated with protection against disease progression in HIV-1 infected individuals (Kaslow et al., 1996; Migueles et al., 2000). However, most
87 people with such “protective HLA alleles” nevertheless progress to AIDS. A recent study suggested that the ability of Nef to counteract SERINC5 is modulated by natural sequence variation as well (CROI 2018). Given that the
ExxxLL motif is conserved in nef alleles from elite controllers (Kirchhoff et al.,
1999), it is possible that additional motifs characterized in the Heigele et al. paper disable the ability to redirect of SERINC5 from the cell surface. If a differential downregulation of SERINC5 and of other cell surface molecules by Nef in progressors and elite controllers could be verified, this would raise the possibility that cellular trafficking is altered in elite controllers.
The role of SERINC5 and its counteraction in viral replication.
Previous studies have shown that SERINC5 inhibits HIV-1 replication in tissue culture, and that Nef counteracts this inhibition (Rosa et al., 2015; Usami et al., 2015). We have shown that the Nef-resistant SERINC5 chimera hS5/fS5-
ICL4 inhibits the replication even of Nef+ HIV-1 (Figure 2.10). However, the Nef- resistant SERINC5 failed to block viral spreading completely. (Figure 2.10). This suggests that Nef may serve another, as yet poorly understood role in replication and viral spreading of HIV-1, in addition to counteracting SERINC3 and
SERINC5. Disruption of the actin cytoskeleton was shown to complement the ability of Nef to enhance HIV-1 infectivity (Campbell et al., 2004). Further studies are needed to examine the underlying mechanisms.
88 Given the conservation of Nef’s ability to counteract human SERINC5 in spite of the considerable variability of Nef’s primary sequence, it is legitimate to hypothesize that the counteraction of SERINC5 in vivo plays a critical role in disease progression and viral pathogenesis. However, the importance of
SERINC5 in HIV-1 replication and viral persistence has not attracted too much attention. HIV cure strategies require an understandings of the mechanism of viral persistence and the maintenance of viral reservoir. The intrinsic characters of infected cells among CD4 T cell subsets determine the size and fate of latently infected lymphocytes (Boritz et al., 2016; Buzon et al., 2014). The expression levels of SERINC5 in distinct T cell subsets and tissues have not been examined.
It is possible that differential levels of SERINC5 in various cell subsets and tissues may account for viral persistence.
To address this question, it is necessary to examine the transcriptional levels of SERINC5 in T cell subsets of infected individuals. Since a minor change of SERINC5’s level significantly alters its inhibitory effect on HIV-1 infectivity in in vitro studies (Rosa et al., 2015; Usami et al., 2015), differential expression levels of SERINC5 are potentially of significance. Besides, there is still no effective cell surface marker for HIV reservoirs (Abdel-Mohsen et al., 2018; Descours et al.,
2017). The transmembrane protein SERINC5 may provide a promising index for locating the rare cells contributing to the HIV reservoir. A limitation of the above experimental design is that transcriptional levels do not adequately represent the amount of SERINC5 on the cell surface. Additionally, a recent study suggested
89 that SERINC5 within detergent-resistant membranes is preferentially downregulated by Nef (Schulte et al., 2018). Thus, the downregulation of
SERINC5 in HIV-1 infection might not cause a significant change at the total transcriptional level.
Materials and methods
Cells.
JTAg, 293T were gifts from G. Crabtree, D. Baltimore. TZM-bl cells were obtained from the AIDS Research and Reference Reagent Program, Division of
AIDS, NIAID, NIH. JurkatTAg S5 -/- cells were published (Usami et al., 2015).
Analysis of virus infectivity.
Pseudovirions capable of a single round of replication were produced by transfecting 293T cells using a calcium phosphate precipitation method. The cells were co-transfected with HXB/Env−/Nef− (1 µg in the experiments shown in
Figures 2.1A, 2.1B, and 2.2A; 2 µg in all other experiments), a pSVIIIenv-based plasmid expressing EnvHXB2 or, where indicated, EnvJRFL (0.1 µg in the experiments shown in Figures 2.1A, 2.1B, 2.2A and 2.3D; 0.4 µg in all other experiments), and the indicated amounts of pBJ5- or pBJ6-based plasmids expressing wild type or chimeric SERINC5 proteins, or with equimolar amounts of empty vector. In addition, equal amounts of the nef-deficient pNefFS or of a
90 plasmid expressing an HIV-1 or SIV Nef were co-transfected where indicated (2
µg in the experiments shown in Figure 2.1B and in the left panel of Figure 2.2C;
0.5 µg in all other experiments). Supernatants containing progeny virions were harvested one day post transfection, clarified by low-speed centrifugation, filtered through 0.45 μm pore filters, and then used immediately to infect TZM-bl indicator cells in triplicate in 6-well plates. Aliquots of the virus stocks were frozen for HIV-1 capsid (p24) antigen quantitation by a standard ELISA. Three days post infection, the indicator cells were lysed, and β-galactosidase activity induced as a consequence of infection was measured using a kit (E2000; Promega). Values were normalized for the amount of p24 antigen present in the supernatants used for infection.
Analysis of SERINC incorporation.
293T cells were co-transfected with 1 µg HXB/Env−/Nef−, pBJ5-based expression plasmids for C-terminally HA-tagged SERINCs or SERINC chimeras (0.1 µg, unless indicated otherwise), and the nef-deficient control plasmid pNefFS or a plasmid expressing an HIV-1 Nef (0.5 μg in the experiments shown in Figures
2.6E, 2.3B, and 2.8B; 2 μg in the experiments shown in Figures 2.2D, 2.6C, and
2.6E). Virions released into the medium were pelleted through 20% sucrose cushions, and virus- and cell-associated proteins were detected by western blotting as described (Accola et al., 2000). Since SERINC proteins are highly aggregation-prone (Usami et al., 2015), samples used for the detection of
91 SERINCs were not boiled before loading. The antibodies used were 183-H12-5C against HIV-1 CA, and HA.11 (Biolegend) against the HA epitope.
Expression plasmids.
The env- and nef-deficient HIV-1 provirus HXB/Env−/Nef−, the pSVIIIenv-based plasmids expressing EnvHXB2 or EnvJRFL, the pBJ5-based expression plasmids for the Nef proteins of HIV-1SF2, HIV-197ZA012, and SIVmac239, the nef-deficient control plasmid pNefFS, and the pBJ5-based expression plasmid for human
SERINC3-HA, human SERINC5, and human SERINC5-HA have been described
(Dorfman et al., 2002; Helseth et al., 1990; Pizzato et al., 2007; Usami et al.,
2015). A pBJ6-based expression plasmid for human SERINC5 was kindly provided by M. Pizzato. The nef genes of SIVagm155 and SIVagm677 were amplified from proviral clones and inserted into pBJ5. The coding sequences for mouse, zebrafish, and frog SERINC5 were amplified from BC062121, IMAGE clone 7650941, and BC044159 (Dharmacon). DNAs encoding mutant and chimeric proteins without or with C-terminal HA tags were generated using an overlap extension PCR method (Horton et al., 1989) and inserted into pBJ5 or pBJ6 downstream of a Kozak sequence. The fS5/hS5-ICL4 chimera has residues
342-390 of frog SERINC5 replaced by residues 342-389 of human SERINC5.
The intra-ICL4 chimeras fS5/hS5-ICL4(9-26) and fS5/hS5-ICL4(26-41) have residues 342-359 and 359-375 of frog SERINC5 replaced by the corresponding residues of human SERINC5. The hS5/fS5-ICL4 chimera has residues 342-389
92 of human SERINC5 replaced by residues 342-390 of frog SERINC5. The hS5/hS2-ICL4 chimera has residues 334-389 of human SERINC5 replaced by residues 343-388 of human SERINC2. The hS3/hS5-ICL4 chimera has residues
358-404 of human SERINC3 replaced by residues 335-389 of human SERINC5.
HIV proviral constructs.
NL4-3/Nefstop, the nef-deficient variant of the prototypic HIV-1NL4-3 used in the replication study, has nef codons 31–33 replaced by three consecutive premature termination codons.
Retroviral vectors.
The coding sequence for SERINC5(iHA) (Usami et al., 2015) preceded by a
Kozak sequence was amplified from pBJ5-SERINC5(iHA) and inserted into the retroviral vector MSCVpuro (Clontech). Additionally, a version of the hS5/hS2-
ICL4 chimera that harbors an internal HA tag at the same position as
SERINC5(iHA) was cloned into MSCVpuro. The coding sequence for HIV-1
NefSF2 preceded by a Kozak sequence was cloned into MSCVhyg (Clontech), yielding MSCVhygNefSF2. A nef-deficient version (MSCVhygNefSF2FS) was obtained by generating a frameshift at a unique XhoI site in nef.
Analysis of Nef incorporation.
93 293T cells were co-transfected with 2 µg of the protease-deficient HIV-1 proviral construct HXB/VPR+/PR− (Bukovsky and Göttlinger, 1996), 2 µg of a pBJ5-based plasmid expressing C-terminally HA-tagged NefLAI with a LL164,165AA mutation
(Pizzato et al., 2007), and 2 µg of a pBJ5-based plasmid expressing native frog
SERINC5 (fS5) or a version harboring the ICL4 of human SERINC5 (fS5/hS5-
ICL4). Virions released into the medium were pelleted through 20% sucrose cushions, and virus- and cell-associated proteins were detected by western blotting. The antibodies used for western blotting were 183-H12-5C against HIV-
1 CA, and HA.11 (Biolegend) against the HA epitope.
Flow cytometry.
Ectopic SERINC expression cassettes were introduced into JurkatTAg S5 -/- cells (Usami et al., 2015) by retroviral transduction with
MSCVpuroSERINC5(iHA) or MSCVpurohS5/hS2-ICL4(iHA). Simultaneously, the cells were transduced with the nef-deficient control vector MSCVhygNefSF2FS or with MSCVhygNefSF2. After selection with puromycin and hygromycin for one week, transduced cell lines grew stably in tissue culture. The doubly transduced bulk cultures were surface-stained with anti-HA antibody and APC-conjugated anti-mouse IgG (Jackson ImmunoResearch). Samples were then and analyzed on a BD LSR II flow cytometer.
94 Analysis of HIV-1 replication in tissue culture.
Ectopic SERINC expression cassettes were introduced into JurkatTAg S5 -/- cells (Usami et al., 2015) by retroviral transduction with MSCVpuroSERINC5 or
MSCVpurohS5/fS5-ICL4. After selection with puromycin, transduced bulk cultures were infected with 2ng ml-1 p24.
Statistical analysis.
Statistical analysis was performed using a two-tailed unpaired t-test with Welch’s correction in case of unequal variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P
< 0.0001, NS, not significant (P > 0.05).
95 Chapter III The determinants in SERINC5 for its Env-dependent antiretroviral activity
Abstract
SERINC5 potently restricts HIV-1 infectivity in the absence of Nef. In addition to Nef, some Env glycoproteins derived from neutralizing antibody- resistant HIV-1 strains antagonize SERINC5’s inhibition of HIV-1 infectivity.
However, it has remained elusive how SERINC5 inhibits viral infectivity and why the inhibition is Env-dependent. To investigate the determinants in SERINC5 that govern the inhibition of HIV-1 infectivity, we examined chimeric SERINC proteins between SERINC5 and other non-inhibitory SERINCs. We now show that a large amino-terminal region of SERINC5 is not specifically required for its antiretroviral activity. SERINC5’s inhibitory function, unlike its sensitivity to Nef, requires the participation of more than one region. Helix 4, helix 9, and the extracellular loop 5
(ECL5) of SERINC5 are all required for potent restriction of HIV-1 infectivity. In particular, the replacement of the ECL5 of SERINC5 by that of another SERINC results in a non-inhibitory SERINC5. The determinates within the ECL5 that contribute to the inhibitory activity of SERINC5 are dispersed throughout the loop. Furthermore, the ECL5 is a hotspot region that determines the Env- dependent antiretroviral activity of SERINC5.
96 Introduction
The mechanism by which SERINC5 blocks HIV-1.
SERINC3 and SERINC5 inhibit the infectivity of Nef-defective viruses upon ectopic expression in producer cells (Rosa et al., 2015; Usami et al., 2015).
We show in Chapter II that the antiretroviral activity is conserved among
SERINC5 proteins. Furthermore, SERINC5 can target retroviruses other than lentiviruses, such as gammaretrovirus MLV (Rosa et al., 2015). However, the mechanism by which SERINC5 inhibits viral infectivity is elusive. Studies using two different fusion assays suggest that the block mediated by SERINC5 occurs at the membrane fusion step (Rosa et al., 2015; Usami et al., 2015). One assay is based on the activation of a reporter in the target cells following the delivery of
Cre recombinase. The other fusion assay is based on the cleavage of a fluorescent substrate after the transfer of a chimeric b-lactamase-Vpr (BlaM-Vpr) from virions into target cells. Both assays indicate that the incorporation of
SERINC5 into HIV-1 virions causes a reduction in their ability to fuse with target cells (Rosa et al., 2015; Sood et al., 2017; Usami et al., 2015).
However, it is not clear which stage of viral fusion is blocked by SERINC5.
The observation that the inhibition of HIV-1 infectivity is more pronounced than the inhibition of fusion supports the theory that SERINC5 disrupts the dilation of the fusion pore but not pore formation (Rosa et al., 2015; Usami et al., 2015).
Alternatively, the reason for the discrepancy might be the different sensitivity of
97 assays that measure entry and post-entry events (Foster et al., 2018). A recent study provides evidence supporting the notion that SERINC5 inhibits fusion by imaging the fusion of single virions virus fusion with living cells (Sood et al.,
2017). Their study suggests that SERINC5 inhibits HIV-cell fusion at the stage of small pore formation. They co-labeled the virions with two fluorescent proteins,
YFP and mCherry. Viral fusion was detected by the release of mCherry-labeled
Gag through a fusion pore larger than 4 nm in diameter. Fusion of SERINC5-HA particles was markedly inhibited compared to that of control virions (Sood et al.,
2017).
Env-dependent antiretroviral activity of SERINC5.
In addition to Nef, Env glycoproteins play a crucial role in determining sensitivity to SERINC5 (Chande et al., 2016; Rosa et al., 2015; Usami et al.,
2015). HIV-1 viruses pseudotyped with vesicular stomatitis virus G protein (VSV-
G) or with the Ebola virus glycoprotein are resistant to SERINC5 (Rosa et al.,
2015). Furthermore, the glycoprotein of the lentivirus EIAV confers partial
SERINC5 resistance to viral particles, even though the EIAV protein S2 counteracts SERINC5 in a similar manner as HIV-1 Nef (Chande et al., 2016).
Virions carrying Env glycoproteins from primary HIV-1 isolates are variably affected by SERINC5 (Beitari et al., 2016; Rosa et al., 2015; Usami et al., 2015), which explains why the enhancement of infectivity by Nef varies when HIV-1 is pseudotyped with Envs derived from primary isolates (Usami and Göttlinger,
98 2013). A panel of transmitted founder viruses are less sensitive to inhibition by
SERINC5 (Beitari et al., 2016). A study using chimeras between SERINC5- sensitive and SERINC5-resistant HIV-1 Envs suggests that the V3 stem loop of
Env is critical for its antagonism of SERINC5 (Beitari et al., 2016).
To further dissect the block in viral fusion caused by SERINC5, researchers examined the effects of fusion inhibitors and neutralizing antibodies on SERINC5’s function. I summarized the published studies from two groups in
Table 1.1 (Beitari et al., 2016; Sood et al., 2017). Beitari et al. evaluated the effect of antiretroviral drugs and neutralizing antibodies on the infectivities of
SERINC5-bearing and -free virions, and Sood et al. used a previously described
BlaM fusion assay (Miyauchi et al., 2009) to investigate the effects of SERINC5 on viral fusion (Table 1.1).
While there are discrepancies between their observations, both studies indicate that SERINC5 disrupts the conformational changes of gp41 induced by receptor binding, and possibly the formation of 6HB that drives the fusion reaction (Chan et al., 1997; Weissenhorn et al., 1997). Sood et al. show that the incorporation of SERINC5 into HIV-1 virions sensitizes viral fusion to neutralizing antibodies against gp41, including to anti-MPER antibody 4E10 (Cardoso et al.,
2005; Hessell et al., 2010). Inhibition of HIV-1 infectivity by 4E10 is sensitized by
SERINC5 incorporation, as suggested by the neutralization assays in the Beitari et al. paper (Table 1.1). Furthermore, Sood et al. showed that SERINC5 sensitizes HIV particles containing both SERINC5-sensitive and SERINC5-
99 resistant Envs to T-20, which binds to gp41 and blocks the formation of the 6HB
(Kilby et al., 1998; Lalezari et al., 2003). However, in Beitari et al. study,
SERINC5 failed to sensitize SERINC5-resistant NL(AD8V3) to T-20. This discrepancy could be due to the fact that AD8V3 is a chimeric Env with an exchanged V3 loop, since sensitivity to T-20 is modulated by the V3 loop of gp120 (Derdeyn et al., 2000). Another possible reason for the discrepancy is the different sensitivities between fusion assay and neutralization assay (Sarzotti-
Kelsoe et al., 2014).
100
bearing viruses. - free and SERINC5 free he effects of fusion inhibitors and neutralizing antibodies on SERINC5’s function. fusion ofinhibitorsantibodies effectson SERINC5’s heand neutralizing T able 3 .1. able T *Note: Other intwobutantibodies do these listedmake used studies table not inneutralizing the a - SERINC5 of inhibition in different
101 SERINC5 incorporation enhances HIV-1 sensitivity to neutralizing antibodies.
The trimeric HIV-1 Env spike is highly protected from antibody-mediated recognition. Nevertheless, broadly neutralizing antibodies target sites of Env vulnerability (Kwong et al., 2011). One target site for neutralizing antibodies is the initial site of attachment to the CD4 receptor, which is on the outer domain of gp120 (Chen et al., 2009; Zhou et al., 2007). The recently isolated antibody
VRC03 appears to target the CD4 binding site with exceptional affinity, and shows neutralization breadths of up to 90% of circulating isolates (Wu et al.,
2010). The human monoclonal antibody PG16, which is also exceptionally potent in its neutralizing activity, targets a quaternary epitope formed by the variable loops V2 and V3 of gp120 (Burton et al., 2009; Robinson et al., 2010). The
MPER of gp41 is another target site for neutralizing antibodies because of its accessibility during gp41 folding. Human antibodies 2F5, Z13e, and 4E10 can recognize the MPER and show reasonable breadths of neutralization, although their potency is low (Muster et al., 1993, 1994; Stiegler et al., 2001; Zwick et al.,
2001). 10E8 is a more recently isolated MPER-specific antibody and shows higher potency than 4E10 (Huang et al., 2012). Human antibody 35O22 binds to a novel site of vulnerability on HIV-1 Env within the interface of gp120/gp41
(Huang et al., 2014).
Beitari et al. have shown that SERINC5 sensitizes HIV-1 only to 4E10,
10E8 and 35O22, which target either the interface of gp120/gp41 or the MPER of gp41 (Beitari et al., 2016). On the other hand, none of the tested antibodies
102 targeting the CD4 binding site or the V1/V2/V3 loops inhibited SERINC5-bearing
HIV-1 to a greater degree than SERINC5-free viruses. SERINC5 incorporation also sensitized the HIV-1 fusion to 4E10 in the Sood et al. study (Sood et al.,
2017). Taken together, these studies raise the possibility that SERINC5 blocks
HIV-1 entry by interfering with the folding of gp41.
Summary
The mechanism by which SERINC5 potently blocks HIV-1 is not clear, though accumulating evidence suggests that the inhibition happens during membrane fusion (Rosa et al., 2015; Sood et al., 2017; Usami et al., 2015).
SERINC5 incorporation sensitizes HIV-1 virions to neutralizing antibodies that target the MPER of gp41, which implies that SERINC5 inhibits HIV-1 entry by altering the conformation of gp41 (Beitari et al., 2016; Sood et al., 2017).
SERINC5 incorporation is shown to inactivate Envs that are sensitive to
SERINC5 (Sood et al., 2017). The stage of entry blocked by SERINC5 would further reveal how neutralizing antibody-resistant Envs antagonize SERINC5.
It is critical to dissect the requirement for each domain in SERINC5 to understand how it inhibits HIV-1 infectivity. Among five members in the SERINC family, SERINC3 and SERINC5, which are 31.9% identical (ClustalO), possess the ability to restrict HIV-1. Though SERINC5 and SERINC2 share 32.4% of primary sequence identity (ClustalO), SERINC2 does not inhibit HIV-1 infectivity.
Interestingly, we find that SERINC2 enhances HIV-1 infectivity to various extents
103 in different producer cell lines. The antiviral activity of SERINC3 and SERINC5 is conserved across species (Dai et al., 2018; Heigele et al., 2016; Murrell et al.,
2016). It is particularly important to investigate the determinants in SERINC5 that are responsible for its exceptional antiviral potency.
To this end, we have examined chimeric constructs between SERINC2 and SERINC5. We conclude that a large amino-terminal portion of SERINC5 is not required for its potent inhibitory function. In contrast, the ECL5 of SERINC5 is critical for the inhibition of HIV-1 infectivity. By comparing the effects of ECL5 mutants on virions bearing Envs resistant or sensitive to SERINC5, we report that ECL5 is a hotspot region that determines Env sensitivity.
Results
C13 in the amino terminus of SERINC5 is indispensable for the potent antiviral activity of SERINC5.
Notably, the amino-terminus of SERINC proteins is enriched in cysteines
(Figure 3.1A). Cysteines at position 12, 14, 19 and 23 of SERINC5 are conserved throughout the SERINC protein family. SERINC5 harbors additional cysteines compared to other SERINC proteins. According to the putative secondary structure of SERINC5 (Schlessinger et al., 2006) and our
104 experimental observations, the amino terminus of SERINC5 is at the cytoplasmic side. In the intracellular environment, it is common to find cysteines binding to metals and induce conformational changes or enzymatic activities. To examine whether enriched cysteines alter SERINC5’s restriction of HIV-1 infectivity, we generated C to A point mutations in the amino-terminus of SERINC5 (Figure
3.1A). C12, C19, and C23 are conserved among SERINC1-5. SERINC5 mutants in which these residues were individually change to alanine reduced the infectivity of Nef− HIV-1 virions produced in 293T cells to a similar extent (12-fold) as wild type SERINC5 (Figure 3.1B). C25 and C26 are conserved among
SERINCs except for SERINC4, which is absent in Jurkat E6.1 cells at the transcriptional level (Usami et al., 2015). Mutations at those sites also did not compromise the inhibitory effect of SERINC5 (Figure 3.1B). SERINC5 does not share C5, C6, and C13 with other SERINC proteins. While versions of SERINC5 harboring C5A and C6A mutations reduced HIV-1 infectivity 7.3- and 5.1- fold respectively, SERINC5 with a C13A mutation reduced HIV-1 infectivity only 2.8- fold (Figure 3.1B). We infer that although most of the cysteines in the N terminal of SERINC5 are not required for the inhibition of HIV-1 infectivity, C13 is critical for the potent inhibitory ability of SERINC5.
105
Figure 3.1. In contrast to other N-proximal cysteines of SERINC5, C13 is critical for the potent antiviral activity of SERINC5. (A) Alignment of the amino-termini of human SERINC1-5 proteins. Arrows indicate cysteine residues that were mutated to alanine. (B) Ability of SERINC5 mutants to inhibit the single-round infectivity of Nef- HIV-1 progeny virions. The histograms represent the mean + SD from three measurements.
106 An extensive N-terminal portion of SERINC5 can be replaced without compromising antiretroviral activity.
Since SERINC1 does not restrict HIV-1 infectivity, we used SERINC1 sequences to replace corresponding regions of SERINC5 in order to examine their importance for the inhibition of HIV-1 (Table 3.2). Figure 3.2A illustrates the putative structure of SERINC5, and the numbering of the extracellular loops
(ECLs), helices (Hs) as well as intracellular loops (ICLs). We tested the infectivity of Nef-deficient viral particles by visualizing green fluorescent protein (GFP)- positive cells after exposure to single-cycle Nef- HIV-1-GFP produced in 293T cells. Infected TZM-bl indicator cells, which express GFP after infection with HIV-
1, were detected by fluorescence microscopy. The substitution of the first 143 amino acids (the amino terminus to the third helix) of SERINC5 by the corresponding region of SERINC1 had no effect on the anti-HIV activity of
SERINC5. In contrast, chimeras in which even larger amino-terminal portions of
SERINC5 were replaced by SERINC1 sequences were unable to inhibit HIV-1 infectivity (Table 3.2). This was due to either a loss of inhibitory function, or to poor expression of the chimeric constructs. We conclude that the region from the amino terminus to the third helix is not specifically required for the anti-HIV-1 effect of SERINC5.
107 Table 3.2. The phenotype of chimeras between SERINC5 and SERINC1.
Chimeras between SERINC2 and SERINC5 reveal the regions critical for
SERINC5’s inhibitory function.
SERINC1 is not efficiently incorporated into progeny virions, which may have contributed to the loss of restrictive activity by some of the S5/S1 chimeras.
To circumvent this potential complication, we also used SERINC2 as a parent for the construction of chimeras, since higher levels of SERINC2 than that of
SERINC1 can be detected in progeny HIV-1 virions and since SERINC2 does not inhibit HIV-1 infectivity (Figure 3.3A) (Schulte et al., 2018).
Because the region from the amino terminus to H3 can be replaced without affecting SERINC5’s inhibitory function (Table 3.2), we individually exchanged loops and helices downstream of this region with corresponding
108 SERINC2 sequences (Table 3.3). The S5/S2(151-179) and S5/S2(180-223) chimeras were both expressed in the producer cells and incorporated into progeny virions in the absence of NefSF2 (Figure 3.2C), but exhibited distinct effects on HIV-1 infectivity (Table 3.3). The S5/S2(151-179) chimera lost the ability to inhibit HIV-1 infectivity, suggesting that ECL2 and H4 are critical for
SERINC5’s inhibitory function. In contrast, the S5/S2(180-223) chimera retained the ability to inhibit HIV-1, which suggests that ICL2 and H5 of SERINC5 are replaceable without compromising its inhibitory function. The S5/S2(287-342) chimera did not inhibit HIV-1 even though it was normally expressed, which indicated that the ECL4 and H8 of SERINC5 are required for its inhibitory function.
The S5/S2(1-150) chimera did not exhibit inhibitory function, but was neither detected in the producer cells nor in progeny virions, regardless of the presence or absence of Nef (Figure 3.2B). The same situation was with
S5/S2(224-260), S5/S2(261-286), and S5/S2(390-456) chimeras (Table 3.3).
Thus, these chimeras did not provide information about the importance of the replaced regions for the inhibitory function of SERINC5. Besides, the chimeras shown in Table 3.3 do not address the importance of the ICL4. None of these chimeras were resistant to NefSF2 (Figure 3.2B, C), confirming the importance of the ICL4 of human SERINC5 in its responsiveness to Nef, as shown in Chapter
II. Overall, these findings demonstrated that, unlike the sensitivity of SERINC5 to
Nef, its inhibitory function of SERINC5 requires more than one region.
109
Figure 3.2. The incorporation of chimeras between SERINC5 and SERINC2. (A) Schematic illustration of SERINC5 and the numbering of each domain. (B) Western blots showing that S5/S2(1-150) failed to be expressed in the producer cells and incorporated into progeny virions. (C) Western blots showing the expression and incorporation of S5/S2(151-179) and (180-223).
110 Table 3.3. The phenotype of chimeras between SERINC5 and SERINC2.
Helix4 of SERINC5 is required for potent anti-HIV-1 function.
Helices in SERINC5 determine the overall structure, and thus likely the function, of SERINC5. To examine the role of each helix in the inhibitory function of SERINC5, we replaced each helix of SERINC5 with its counterpart in
SERINC2. Chimeras carrying H5 or H8 of SERINC2 inhibited HIV-1 infectivity
12.0- and 12.5- fold, respectively, which was comparable to the inhibition observed with parental SERINC5 (17.8-fold). In contrast, the exchange of other helices significantly compromised the inhibitory function of SERINC5 (Figure
3.3B). To further characterize the requirement for H4, H6, H7, H9, and H10 in
111 SERINC5’s inhibitory function, we examined the effects of increasing the amounts of chimeric SERINC5 proteins on HIV-1 infectivity. The S5/S2-H6 and -
H10 chimeras exhibited potent inhibition of HIV-1 infectivity when higher amounts of plasmids encoding these constructs were transfected (Figure 3.3C). Thus, helices 6 and 10 do not contain essential determinants for the inhibition of HIV-1.
In contrast, the S5/S2-H4, -H7 and -H9 chimeras exhibited little inhibitory activity even when relatively high amounts of plasmids encoding these constructs were transfected (Figure 3.3C). The lack of activity of the S5/S2-H7 chimera could be attributed to a lack of incorporation into progeny virions (Figure 3.3D). In contrast, the S5/S2-H4 chimera was incorporated at a level comparable to that of parental SERINC5, and the S5/S2-H9 chimera was incorporated at an even higher level (Figure 3.3D). We conclude that helices 4 and 9 are specifically required for the anti-HIV-1 activity of SERINC5. (Table 3.3).
112
Figure 3.3. The role of SERINC5’s helices in the restriction of HIV-1 infectivity. (A) SERINC2 enhances the infectivity of Nef- HIV-1 progeny virions. (B) The effects of exchanging each helix of SERINC5 on the inhibition of HIV-1 infectivity. In (A) and (B), the histograms represent the mean + SD from three measurements, and the amounts of SERINC plasmids were 100 ng per transfection.
113 (C) Not all of the chimeras exhibited potent inhibitory activity when the amounts of plasmids used for transfection were increased 10-fold. The lower amount of plasmids was 100 ng, and the higher one was 1 µg. (D) Western blots showing the incorporation of chimeric SERINC5 proteins into Nef- HIV-1 virions.
The ECL5 is critical for the restriction function of SERINC5.
The observation that SERINC5 exerts more potent inhibition on viral infectivity than on HIV-1 fusion suggests that SERINC5 blocks the enlargement of the fusion pore (Rosa et al., 2015; Sood et al., 2017; Usami et al., 2015).
Thus, it is conceivable that ECLs of SERINC5 may disrupt this step of viral fusion. Furthermore, ECLs are responsible for the overall structure of SERINC5 by connecting the helices. To investigate the role of ECLs in the inhibitory function of SERINC5, we exchanged each ECL in SERINC5 with the counterpart in SERINC2. The exchange of the ECL1 did not compromise the inhibition of Nef-
HIV-1 infectivity by SERINC5 at all. The chimeric SERINC5 proteins with exchanged ECL2 or ECL3 were moderately less potent in inhibiting HIV-1
(Figure 3.4A). Although the S5/S2-ECL4 chimera was significantly less potent than parental SERINC5, it still inhibited the infectivity of Nef- HIV-1 5.3-fold
(Figure 3.4A). However, SERINC5 bearing the ECL5 of SERINC2 completely lost the ability to inhibit HIV-1 infectivity when 100ng expression plasmid was used for transfection (Figure 3.4A). The cellular expression level of the S5/S2-
114 ECL5 chimera, and its incorporation into progeny virions, were reduced compared to parental SERINC5 (Figure 3.4C). However, this is unlikely the reason for the loss of restrictive activity, since the S5/S2-ECL5 chimera did not result in any inhibition of HIV-1 infectivity even when the amount of expression plasmid transfected was increased 10-fold (Figure 3.4B). Overall, we conclude that the ECL5 is critical for the ability of SERINC5 to restrict HIV-1 infectivity.
115
Figure 3.4. SERINC5 bearing the ECL5 of SERINC2 fails to inhibit the infectivity of Nef- HIV-1 virions. (A) Effects of the indicated chimeras on the single cycle infectivity of Nef- HIV-1. The amount of SERINC expression plasmid used per transfection was 100 ng. The histograms represent the mean + SD from three measurements. (B) Transfection of an excess amount of S5/S2-ECL5 does not inhibit HIV-1 infectivity.
116 (C) Western blots showing the expression and virion-incorporation of the S5/S2- ECL5 chimera. The amount of SERINC expression plasmid used per transfection was 100 ng.
The ECL5 and H9 of SERINC5 are not sufficient to transfer restriction activity to
SERINC2.
The ECL5 and H9 are adjacent to each other. Since both the ECL5 and
H9 of SERINC5 are important for the inhibition of HIV-1 infectivity, we next tested whether the combination of the EL5 and H9, or either of them individually, could transfer restriction activity to SERINC2. Thus, we generated chimeric SERINC2 proteins bearing the ECL5, H9, or both from SERINC5 (Figure 3.5A), and tested their effects on HIV-1 infectivity. None of the constructs could suppress the HIV-1 infectivity, at least at the low amounts of plasmid that were transfected (Figure
3.5A). The S2/S5-ECL5 and S2/S5-H9 chimeras were both expressed in the producer cells, and were incorporated into progeny virions at levels comparable to wild-type SERINC2 (Figure 3.5B). When higher amounts of these plasmids were transfected, the chimeric SERINC2 proteins slightly enhanced HIV-1 infectivity, which is similar to the effect of wild type SERINC2 (Figure 3.5A). The
S2/S5-(ECL5+H9) chimera failed to inhibit HIV-1 infectivity, probably because it was not incorporated into progeny virions (Figure 3.5A, B). Together, these findings suggest that the ECL5 and H9 of SERINC5 are not sufficient to confer inhibitory activity to non-inhibitory SERINC protein. It is possible that the
117 determinants responsible for SERINC5’s restriction function cannot be pinpointed to a limited region.
A recent study suggested that the region comprising the ECL3, H6, ICL3,
H7, and the ECL4 of SERINC5 is sufficient to enable SERINC2 to restrict Nef-
HIV-1 virions (Schulte et al., 2018). To verify their observation in our experimental settings, we acquired the relevant plasmids and tested its effect on
HIV-1 infectivity. Even when a very high amount of the plasmid encoding the putative inhibitory chimera (1.5 µg) was transfected, the chimeric SERINC2 merely reduced HIV-1 infectivity 1.3-fold (Figure 3.5C). In addition, the CMV- based construct led to higher SERINC expression levels than the pBJ5-based
SERINC expression plasmids used by us. Thus, we were unable to confirm that the region extending from ECL3 to ECL4 of SERINC5 is sufficient to transfer inhibitory activity to SERINC2.
118
Figure 3.5. Chimeric SERINC2 proteins fail to inhibit HIV-1 infectivity. (A) Schematic illustration of the S2/S5-ECL5, -H9 and -(ECL5+H9) chimeras, and their effects on Nef- HIV-1 progeny virions. The amounts of transfected plasmids were 100 ng and 1.0 µg. The red numbers indicate the exchanged regions. (B) Western blots showing the cellular expression and viral incorporation of chimeric SERINC2 proteins in the absence of Nef.
119 (C) Schematic illustration of the S2/S5-(ECL3--ECL4) chimera (left). The red numbers indicate the exchanged regions. The S2/S5-(ECL3--ECL4) chimera fails to inhibit HIV-1 infectivity (right). In all cases, the histograms represent the mean + SD from three measurements.
The inhibitory determinants within the ECL5 are dispersed throughout the loop.
Given that the ECL5 of SERINC2 is three amino acids shorter than that of
SERINC5, we hypothesized that the length of the ECL5 is one of the factors determining the antiretroviral function of SERINC5. To test this hypothesis, we inserted consecutive 3-amino-acid-deletions into the ECL5 of SERINC5 (Figure
3.6A). In addition, if the hypothesis were disproved, these deletions were expected to allow us to narrow down relevant determinants within the ECL5.
None of the deletions had the same effect as the replacement of the whole ECL5
(Figure 3.4A and Figure 3.6B). Each of the deletion mutants retained restrictive activity, reducing HIV-1 infectivity 3.3- to 7.9-fold, compared to the 30.0-fold inhibition obtained with parental SERINC5 (Figure 3.6B). The deletions within the ECL5 did not affect the levels of expression of SERINC5, or its incorporation into progeny virions (Figure 3.6C). We conclude that the importance of the ECL5 for SERINC5’s antiretroviral activity is not merely due to its length. Furthermore, determinants important for the restrictive activity of SERINC5 are dispersed throughout the ECL5.
120
Figure 3.6. Deletions within the ECL5 of SERINC5 only partially impaired its ability to restrict HIV-1 infectivity. (A) Alignment of the ECL5 regions of human SERINC1-5, and of SERINC5 proteins from other species (frog and zebrafish) that possess the antiretroviral activity. The ECL5 deletions examined are also indicated. (B) ECL5 deletions impair the ability of SERINC5 to inhibit the infectivity of Nef- HIV-1 virions. The histograms represent the mean + SD from three measurements. (C) Western blots showing that a SERINC5 mutant with a deletion in the ECL5 is normally expressed and incorporated into progeny virions.
121 The ECL5 determines the Env-dependent antiretroviral activity of SERINC5.
In the experiments described above, the Env expressed in trans for measuring single-round infectivity was derived from HIVHXB2, a laboratory- adapted X4-tropic strain. Previous studies from several groups had suggested that some of the primary R5-tropic Envs, such as EnvJRFL, can partially overcome
SERINC5 inhibition (Beitari et al., 2016; Rosa et al., 2015; Usami et al., 2015).
To define the role of the ECL5 of SERINC5 in its antagonism by Env, we examined the effects of deletions within the ECL5 on the infectivity of virions carrying EnvJRFL. SERINC5 mutants with deletions in the ECL5 did not inhibit viral infectivity, while parental SERINC5 reduced the infectivity 4.2-fold (Figure 3.7A).
Paradoxically, the S5-ECL5D3 mutant slightly enhanced the infectivity of progeny virions carrying EnvJRFL (Figure 3.7A).
When comparing the specific infectivity of virions incorporating SERINC5 and either EnvJRFL or EnvHXB2, we noticed that SERINC5’s inhibition of virus bearing EnvHXB2 is 7.4-fold stronger compared to the inhibition of virus bearing
EnvJRFL (Figure 3.7B). Notably, each SERINC5 deletion mutant inhibited the infectivity of progeny virions carrying EnvJRFL to a 4.0- to 8.1-fold lesser extent than the infectivity of virions carrying EnvHXB2 (Figure 3.7B). In contrast, the
S5/S2-ECL5 chimera, which has the whole ECL5 replaced, moderately inhibited the infectivity of virions carrying EnvJRFL, but not the infectivity of virions carrying
Env HXB2 (Figure 3.7A and 3.4A). It suggested that ECL5 mutations proportionally disrupts the antagonism of SERINC5 by Env. However, the
122 exchange of the ECL5 loop led to the discordant antagonism of SERINC5. Taken together, we conclude that the ECL5 is a hotspot region that determines the Env- dependent antiretroviral activity of SERINC5.
Env: JRFL NS
Figure 3.7. ECL5 mutations proportionally disrupts the antagonism of SERINC5 by Env. (A) Effects of deletions within in the ECL5 of SERINC5 on its ability to inhibit the - infectivity of Nef HIV-1 virions carrying EnvJRFL. 100 ng plasmids expressing SERINC5 mutants were used per transfection. (B) Fold change indicates the ratio of the relative infectivity of virions carrying EnvJRFL to EnvHXB2, when 100 ng plasmids expressing corresponding SERINC mutants were co-transfected. In all cases, the histograms represent the mean + SD from three measurements.
123 Discussion
Our results indicate that the ECL5 of SERINC5 is crucial its ability to inhibit HIV-1 infectivity. Even at the highest amount of transfected expression plasmids, SERINC5 bearing the ECL5 from SERINC2 did not inhibit HIV-1 infectivity (Figure 3.4A). In addition, our results indicate that helices H4 and H9 of SERINC5 both play an important role in maintaining the full potency of inhibition. However, the ECL5 and H9 of SERINC5 together failed to transfer the ability to restrict HIV-1 to SERINC2 (Figure 3.5A). Determinants throughout the
ECL5 contribute to the restriction of HIV-1 by SERINC5,since all of the deletions within the ECL5 resulted in a partial reduction in the ability of SERINC5 to inhibit
HIV-1 infectivity (Figure 3.6B). Interestingly, the replacement of the ECL5 of
SERINC5 by that of SERINC2 eliminated the ability to inhibit the infectivity conferred by EnvHXB2 but not by EnvJRFL, even though EnvJRFL is far less affected by wild type SERINC5 (Figure 3.7B). Taken together, our findings suggest that the ECL5 determines the Env-specific antiretroviral activity of SERINC5.
The determinants of SERINC5 that governs the restriction.
Schulte et al. suggested that the transfer of an over 100-amino acid region from SERINC5 that begins with the ECL3 and ends with the ECL4 was sufficient to render SERINC2 capable of restricting HIV-1 infectivity (Schulte et al., 2018).
We acquired the relevant expression plasmids from this group and tested it in our
124 experimental setting. Even at the highest amount of transfected plasmid, we only observed a slight reduction of viral infectivity (Figure 3.5C). The clone they used is codon-optimized and in a CMV-driven vector. Thus, the SERINC5 expression level of their plasmids is much higher than the pBJ5-based constructs used in our study. The failure of the chimeric SERINC2 to inhibit HIV-1 infectivity despite high expression levels suggests that the determinants of SERINC5’s restriction function are unlikely to be in ECL3, the ECL4, or the sequences that connect these elements.
We have shown that most of this region, which consists of the ECL3, H6, the ICL3, H7, and the ECL4, is also dispensable for the potent inhibition of HIV-1 infectivity by SERINC5. With merely 100 ng of a weak expression plasmid transfected, SERINC5 bearing the ECL3 or ECL4 of SERINC2 inhibited HIV-1 infectivity 12.0- and 5.3-fold, respectively (Figure 3.4A). 1 µg of the S5/S2-H6 chimera inhibited HIV-1 infectivity as potently as parental SERINC5 (Figure
3.4C). Though the S5/S2-H7 chimera was not as potently as SERINC5 bearing
H6 of SERINC2, it caused a 3-fold reduction in HIV-1 infectivity (Figure 3.4C).
These observations are in agreement with our observation that the ECL3-H6-
ICL3-H7-ECL4 portion of SERINC5 is not sufficient to confer the ability to restrict
HIV-1 to a non-inhibitory SERINC.
At the highest amount of plasmids transfected, the S2/S5-ECL5 chimera was 1.6-fold less effective than wild type SERINC2 in increasing HIV-1 infectivity.
However, the ECL5 of SERINC5 failed to render SERINC2 restrictive for HIV-1
125 infectivity (Figure 3.5A). SERINC2 bearing both the ECL5 and the adjacent H9 helix from SERINC5 also did not acquire the ability to restrict HIV-1, possibly because it was poorly incorporated into progeny virions (Figure 3.5B). One possible reason for the failure to confer inhibition in the context of SERINC2 could be that the determinants responsible for the enhancement HIV-1 infectivity by SERINC2 are unrelated to those that govern the inhibitory effects of
SERINC5.
The impact of SERINC5 on conformational changes in Env.
The effects of SERINC5 incorporation on neutralization sensitivity suggests that SERINC5 inhibits HIV-1 infectivity by altering the conformation of
Env (Beitari et al., 2016; Sood et al., 2017). In this study, we show that the ECL5 of SERINC5 is essential for its inhibition of HIV-1. It is tempting to speculate that this extracellular loop in SERINC5 interferes with conformational changes of Env.
The V3 stem loop of HIV-1 Env has been shown to determine its antagonism of SERINC5 (Beitari et al., 2016). A previous study suggests that the effects of Nef on HIV-1 infectivity are determined by the conformational status of the apex of the Env trimer (Usami and Göttlinger, 2013). Cryo-EM structures reveal that the V1, V2 and V3 variable regions are near the apex of the Env trimer (Liu et al., 2008; White et al., 2010), which acts to stabilize the unliganded
Env spike (Mao et al., 2012). I hypothesize that the ECL5 determines the sensitivity of Env to SERINC5 by altering the Env trimer. An experimental
126 strategy to examine this hypothesis is to compare EnvSF162, an R5-tropic Env sensitive to SERINC5’s restriction, with the SERINC5-resistant EnvJRFL, and how the SERINC5-sensitivity changes when the V1, V2, and V3 of EnvSF162 and
EnvJRFL are exchanged. EnvJRFL becomes considerably more neutralization sensitive upon replacement of its V1/V2 region by that of neutralization-sensitive
EnvSF162 (Pinter et al., 2004). If the proposed model upholds, the extent of inhibition of HIV-1 infectivity by SERINC5 mutants with deletions in the ECL5 of would be switched by exchanging the variable regions of EnvSF162 and EnvJRFL. In contrast, the effects of the S5/S2-ECL5 chimera would not be swapped when the variable regions of Envs were exchanged. Another experiment to test the hypothesis is to examine the effects of the linear peptide of ECL5 on the conformational changes of Env. However, it is worth noticing that peptide binding assays can suggest off-pathway structures corresponding to an inactivated gp41 in addition to conformations leading to viral fusion (Melikyan, 2008).
The antagonism of SERINC5 by Env.
The sensitivity of Env to SERINC5 might be related the number of Env trimers required for entry (Brandenberg et al., 2015). Whether SERINC5 incorporation interferes with Env incorporation is controversial (Sood et al., 2017;
Usami et al., 2015), as is the effect of Nef on the incorporation of Env. Some studies reported an enhancement of Env incorporation by Nef (Day et al., 2004;
Schiavoni et al., 2004), while no effect on Env incorporation was observed in
127 other studies (Miller et al., 1995; Pizzato et al., 2007; Usami and Göttlinger,
2013).
Neutralizing antibody-resistant EnvJRFL can overcome SERINC5 inhibition independent of Nef (Rosa et al., 2015; Usami et al., 2015). Why did both HIV and
EIAV evolve redundant mechanisms to bypass inhibition by SERINC5 in addition to the counteraction by viral proteins (Chande et al., 2016; Usami et al., 2015)?
Similarly, HIV-1 Vpu and Nef also have evolved complementary abilities to remove the HIV-1 receptor CD4 from the cell surface (Lindwasser et al., 2007). It is possible that SERINC5 performs additional functions that restrict virus replication but are not counteracted by Nef (Chande et al., 2016).
The cellular site of viral fusion matters for the antiviral activity of host factors. The glycoproteins of VSV, Ebola and EIAV can overcome the restriction by SERINC5 (Chande et al., 2016; Rosa et al., 2015), which suggests that the use of an endosomal entry pathway can reduce sensitivity to SERINC5 (Chande et al., 2016; Jin et al., 2005). Similarly, the antiviral restriction activity of the interferon induced transmembrane (IFITM) protein family is linked to the cellular site of viral fusion (Foster et al., 2018). The IFITM proteins, including IFITM1,
IFITM2, and IFITM3, have been demonstrated to inhibit HIV-1 replication by blocking virus entry (Li et al., 2013; Lu et al., 2011), however, to a lesser degree compared to their effects on other viruses (Diamond and Farzan, 2013; Foster et al., 2018). IFITM1 is primarily localized at the plasma membrane, while IFITMs 2 and 3 are within endosomal compartments. Virions that utilize CCR5 for entry are
128 more susceptible to inhibition by IFITM1, and CXCR4-using virions were more subject to restriction by IFITMs 2 and 3 (Foster et al., 2018).
How does SERINC5 possibly affect HIV-1 pathogenesis?
The term “transmitted founder virus” refers to the inferred consensus sequence of a viral isolate during acute infection of humans or monkeys that is responsible for establishing productive clinical infection (Keele et al., 2008; Lee et al., 2009). The sensitivity of Envs from various primary isolates to neutralizing antibodies varies. Envs from newly transmitted viruses are sensitive to antibodies from the partner (Derdeyn et al., 2004). Based on the sensitivity to antibody- mediated neutralization, HIV-1 primary isolates are divided into four subgroups: with very high sensitivity (Tier 1A), above-average sensitivity (Tier
1B), moderate sensitivity (Tier 2), or low sensitivity (Tier 3) (Li et al., 2005;
Seaman et al., 2010). The neutralization sensitivity of transmitted founder viruses follows the similar pattern with tier 2 or 3 primary virus strains (Li et al., 2005), whose V3 and coreceptor binding surface regions are occluded from binding by neutralizing monoclonal or polyclonal HIV-1 antibodies (Keele et al., 2008).
Transmitted founder viruses are relatively insensitive to inhibition by
SERINC5 (Beitari et al., 2016). In this study, we show that R5-tropic neutralization-resistant EnvJRFL is more resistant to SERINC5 inhibition compared to the lab adapted strain EnvHXB2 (Figure 3.6), which is in agreement with previous studies (Beitari et al., 2016; Rosa et al., 2015; Usami et al., 2015).
129 It is possible that SERINC5 exerts pressure on Env selection during the primary infection or viral persistence. Thus, it is important to further investigate the relationship between SERINC5 expression levels and the evolution of env during the course of disease progression.
Materials and methods
Cells.
293T cells were gifts from G. Crabtree, D. Baltimore. TZM-bl cells were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS,
NIAID, NIH.
Analysis of virus infectivity.
Pseudovirions capable of a single round of replication were produced by transfecting 293T cells using a calcium phosphate precipitation method. The cells were co-transfected with 2 µg HXB/Env−/Nef−, 0.4 µg pSVIIIenv-based plasmid expressing EnvHXB2 or, where indicated, EnvJRFL, and the indicated amounts of pBJ5-based plasmids expressing wild type or chimeric SERINC5 proteins, or with equimolar amounts of empty vector. In addition, equal amounts of the nef- deficient pNefFS or of 0.5 µg plasmid expressing an HIV-1 Nef were co- transfected where indicated. Supernatants containing progeny virions were
130 harvested one day post transfection, clarified by low-speed centrifugation, filtered through 0.45 μm pore filters, and then used immediately to infect TZM-bl indicator cells in triplicate in 6-well plates. Aliquots of the virus stocks were frozen for HIV-1 capsid (p24) antigen quantitation by a standard ELISA. Three days post infection, the indicator cells were lysed, and β-galactosidase activity induced as a consequence of infection was measured using a kit (E2000; Promega). Values were normalized for the amount of p24 antigen present in the supernatants used for infection.
Alternatively, the HIV-1 vector HIVec2.GFP was co-transfected together with HXB/Env−/Nef−, the Env expression plasmid and a pBJ5-based plasmid expressing wild type or chimeric SERINC5 proteins. After exposure to equal amounts of virus, infected TZM-bl cells were then identified based on GFP expression.
Analysis of SERINC incorporation.
293T cells were co-transfected with 1 µg HXB/Env−/Nef−, pBJ5-based expression plasmids for C-terminally HA-tagged SERINCs or SERINC chimeras (0.1 µg, unless indicated otherwise), and 0.5 µg nef-deficient control plasmid pNefFS or a plasmid expressing an HIV-1 Nef. Virions released into the medium were pelleted through 20% sucrose cushions, and virus- and cell-associated proteins were detected by western blotting as described (Accola et al., 2000). Since SERINC proteins are highly aggregation-prone (Usami et al., 2015), samples used for the
131 detection of SERINCs were not boiled before loading. The antibodies used were
183-H12-5C against HIV-1 CA, and HA.11 (Biolegend) against the HA epitope.
Expression plasmids.
The env- and nef-deficient HIV-1 provirus HXB/Env−/Nef−, the pSVIIIenv-based plasmids expressing EnvHXB2 or EnvJRFL, the pBJ5-based expression plasmids for the Nef proteins of HIV-1SF2, the nef-deficient control plasmid pNefFS, and the pBJ5-based expression plasmid for human SERINC2, human SERINC2-HA, human SERINC5, and human SERINC5-HA have been described (Dorfman et al., 2002; Helseth et al., 1990; Pizzato et al., 2007; Usami et al., 2015). The plasmids for S2/S5-(ECL3--ECL4), S2 and S5 in Figure 3.5C were provided by
F. Diaz-Griffer, and was described (Schulte et al., 2018). DNAs encoding mutant and chimeric proteins without or with C-terminal HA tags were generated using an overlap extension PCR method (Horton et al., 1989) and inserted into pBJ5 downstream of a Kozak sequence. Point mutations in Figure 3.1 were generated using Pfu DNA polymerase (Agilent) via site-directed mutagenesis. For chimeras in Table 3.2, the numbers following S1 indicate the residues of SERINC1 replaced by the counterpart of SERINC5. The residues for the counterpart of
SERINC5 were listed in Table 3.4. Table 3.5 listed the primary sequences of the chimeric constructs in Table 3.3. Table 3.6 listed the primary sequences of the constructs in Figure 3.3 and 3.4. Table 3.7 listed the primary sequences of the constructs in Figure 3.5.
132 Table 3.4. The primary sequences of chimeras of SERINC5 and SERINC1.
Table 3.5. The primary sequences of chimeras of SERINC5 and SERINC2.
133 Table 3.6. The primary sequences of chimeric SERINC5 proteins with exchanged helices and ECLs of SERINC2.
Table 3.7. The primary sequences of chimeric SERINC2 proteins.
134 Statistical analysis.
Statistical analysis was performed using a two-tailed unpaired t-test with Welch’s correction in case of unequal variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P
< 0.0001, NS, not significant (P > 0.05).
135 Chapter IV Discussion
The multipass transmembrane protein SERINC5 is a newly defined antiretroviral host factor. SERINC5 potently inhibits HIV-1 infectivity, and can be counteracted by the HIV-1 accessory protein Nef (Rosa et al., 2015; Usami et al.,
2015). However, the counteraction of SERINC5 by Nef and the mechanism by which SERINC5 blocks HIV-1 are poorly understood. The work presented in this dissertation for the first time describes the role of SERINC5 subdomains in the interplay between Nef and the inhibition of HIV-1 by SERINC5.
Counteraction of SERINC5 by Nef.
In agreement with other studies (Heigele et al., 2016; Murrell et al., 2016), we observed that the antiretroviral activity is conserved among mammalian
SERINC5 proteins. In Chapter II, we demonstrate that mouse SERINC5 is as potent as human SERINC5 in inhibiting HIV-1. A previous analysis of mouse and five primate SERINC5 orthologs showed that all potently inhibited SIV and HIV-1
(Heigele et al., 2016). We have extended these observations by showing that the
SERINC5 proteins of zebrafish and frog also possess potent antiretroviral activity. A computational analysis shows that primary sequences of SERINC5 proteins from various species are highly conserved (Murrell et al., 2016). Unlike
136 the majority of host restriction factors, SERINC5 orthologs do not display the signatures of an evolutionary arms race with viral pathogens. SERINC5 is not interferon-inducible (Usami et al., 2015), which is another trait that separates
SERINC5 from a typical restriction factor.
We demonstrate that SERINC5’s responsiveness to Nef is divergent, though its antiretroviral activity is conserved. HIV-1 Nef does not counteract the inhibitory effects of frog or zebrafish SERINC5. It has been shown in a previous study that the counteraction of various primate SERINC5 proteins by Nef is largely species-independent (Heigele et al., 2016). Thus, SERINC5 probably poses no major obstacle for zoonotic transmission of primate lentiviruses .
The results presented in Chapter II demonstrate that the longest intracellular loop ICL4 of SERINC5 governs its responsiveness to Nef. This conclusion is supported by several key findings: (1) transfer of the ICL4 of human
SERINC5 is sufficient to confer Nef responsiveness to frog SERINC5; (2) the
ICL4 of human SERINC5 confers sensitivity to widely divergent Nefs, including
Nefs from various HIV-1 strains and several SIV Nefs; (3) though parental human
SERINC3 is resistant to downregulation by NefSF2, it acquires sensitivity to NefSF2 in the presence of the ICL4 of human SERINC5; (4) the ICL4 of frog SERINC5 confers resistance to both clade B and clade C Nefs; (5) the ICL4 of human
SERINC2 confers resistance to NefSF2. In sum, these data demonstrate that Nef responsiveness can be transferred by exchanging the ICL4 of SERINC proteins.
137 While our observations clearly show that the ICL4 of SERINC5 governs
Nef responsiveness, additional studies are desired to understand whether Nef directly binds to SERINC5. We examined the virion incorporation of LL164,165AA
AP-2 binding site mutant of NefLAI, which does not counteract SERINC5 (Rosa et al., 2015). The mutant Nef is more abundant in HIV-1 virions produced in the presence of the fS5/hS5-ICL4 chimera rather than of frog SERINC5. This provides indirect evidence suggesting a physical interactions of SERINC5 with
Nef.
Nef has been shown to bind to the CD4 cytoplasmic tail using yeast two- hybrid assays and purified proteins (Cluet et al., 2005; Harris and Neil, 1994; Jin et al., 2004; Preusser et al., 2001; Ross et al., 1999). Furthermore, HLA-A has been co-immunoprecipitated with Nef (Roeth et al., 2004). Also, Nef has been found to form a complex with MHC-I molecules in the endoplasmic reticulum
(Kasper et al., 2005). A recent study shows that Nef binds to SERINC5 in living cells using a bimolecular fluorescence complementation assay (Shi et al., 2018).
Yeast two-hybrid assays and assays based on purified proteins can be applied to test the binding of the ICL4 of SERINC5 to Nef as well. However, unlike CD4 and
MHC-I, SERINC5 is a multipass transmembrane protein. The ICL4 likely forms a higher-order structure with other intracellular loops or helices. Thus, it may not be informative to examine the binding of Nef to SERINC5 using only a linear polypeptide of ICL4.
138 Furthermore, an analysis of complexes containing SERINC proteins bearing distinct ICL4 loops may reveal novel cellular machinery involved in the regulation of SERINC5. However, it may be challenging to perform co- immunoprecipitation studies with SERINC5 given that (1) it is a large hydrophobic protein prone to aggregation and (2) a specific antibody for
SERINC5 is lacking.
We report that the ICL4 of human SERINC5 confers sensitivity to widely divergent Nefs, including SIVmac239, SIVagm155, and SIVagm677. The endocytic ExxxLL motif is conserved among these SIV Nefs, and mutations of the dileucine within the motif to AA has been shown to disrupt the ability of Nef to counteract SERINC5 (Heigele et al., 2016). The same study, however, reported that the efficiency with which different SIV Nefs counteract SERINC5 varies considerably. Furthermore, the extent of Nef-mediated SERINC5 antagonism correlates with the prevalence of different SIVs in their respective natural hosts, which suggests a role for SERINC5 in viral spreading in vivo.
The effects of Nef polymorphisms on SERINC5 antagonism have not been thoroughly investigated. Nef plays a key role in promoting disease progression and viral pathogenesis, but which Nef activities are crucial for viral replication and pathogenesis in vivo is far from clear (reviewed in Kirchhoff, 2010). Thus, it may be rewarding to investigate the counteraction of SERINC5 by Nefs from elite controllers and chronic progressors. Relatively inefficient SERINC5 antagonism may contribute to the mechanism of the spontaneous control of HIV-1.
139
Unknown functions of SERINC proteins
Nef selectively downregulates some surface proteins but leaves others from the same family unaffected. For instance, Nef downregulates HLA-A and -B molecules from the cell surface, which prevents CTL responses towards the infected cells. On the other hand, Nef does not redirect the cellular trafficking of
HLA-C or -E molecules to avoid the killing of infected cells by NK cells (Cohen et al., 1999; Le Gall et al., 1998). However, the downregulation of HLA-C by Vpu in primary HIV-1 clones suggests new roles of CTLs and NK cells in HIV-1 infection
(Apps et al., 2016).
Similarly, Nef selectively downregulates some of the SERINC proteins from the cell surface but leaves others unaffected (Matheson et al., 2015; Rosa et al., 2015; Usami et al., 2015). Experiments described in Chapter III surprisingly revealed that SERINC2 significantly enhances the infectivity of HIV-1 progeny virions. Thus, leaving SERINC2 on the cell surface could be beneficial for the virus. Further studies are needed to determine the physiological functions not only of SERINC3 and SERINC5, but of other SERINC proteins as well. Their resistance to Nef counteraction might reveal a novel mechanism of immune evasion by HIV-1. Possible questions to be addressed include (1) whether
SERINC proteins engage/activate/inactivate immune receptors; (2) whether the surface expression levels of SERINC proteins alter the recognition of infected cells; (3) how each of the SERINC proteins impacts HIV-1 infectivity in different
140 cell subsets, under distinct conditions; (4) what the cell surface density of each
SERINC protein is in different T cell subsets is.
Importantly, our findings in Chapter II suggest that SERINC2 is resistant to
NefSF2 but not to Nef97ZA012. We further show that both SERINC2 and the hS5/hS2-ICL4 chimera respond to Nef97ZA012. Thus, it is possible that Nef97ZA012 recognizes determinants within the ICL4 of SERINC2. We also show that point mutations of SERINC5 at L350 and I353 cause a loss of responsiveness to
NefSF2. The ICL4 regions of SERINC5 and SERINC2 are poorly aligned given the low similarities. The ICL4 loop of SERINC2 is 12-amino acid shorter than that of
SERINC5. Residues corresponding to L350 or I353 of SERINC5 are not present in the SERINC2 ICL4. The determinants within the ICL4 of SERIN2 that are responsible for its responsiveness to Nef97ZA012 have not been investigated.
Based on our findings concerning the ICL4 of SERINC5, L361 and A363 of
SERINC2 are potential candidates.
In summary, Chapter II demonstrates that the antiretroviral activity of
SERINC5 proteins is conserved among different vertebrate species. However, their responsiveness to Nef is divergent. The ICL4 region of SERINC5 governs its counteraction by Nef. The ICL4 region of human SERINC5 can transfer the responsiveness to widely divergent Nefs. We also show evidence for a potential physical interaction of SERINC5 with Nef. Thus, our studies provide valuable insights into how HIV-1 Nef evades the intrinsic immunity that would otherwise potently restrict viral infectivity.
141
Conditional inhibition of HIV-1 by SERINC5
In Chapter III, we aimed to investigate the determinants of SERINC5 that govern its inhibition of HIV-1. We examined the role of each helix and extracellular loop of SERINC5 in the inhibition of HIV-1 by exchanging them with their counterparts in SERINC2. We show that more than one domain is required for potent inhibition of HIV-1 infectivity. We provide evidence that the exchange of the ECL5 causes a complete loss of function of SERINC5. Furthermore, our results suggest that the ECL5 is a hotspot region carrying out the Env-dependent antiretroviral activity of SERINC5.
The mechanism by which SERINC5 inhibits HIV-1 is far from clear. To investigate which region of SERINC5 is crucial, we generate chimeric SERINC proteins between SERINC5 and other SERINC proteins that do not exhibit antiretroviral activity. We thoroughly examine the requirements for each helix and extracellular loop for the inhibition of HIV-1, and the results are now summarized in Figure 4.1. Our data clearly demonstrate the importance of the ECL5 for the inhibition of HIV-1. SERINC5 bearing the ECL5 of SERINC2 completely lost its effect on HIV-1 infectivity, even when an excessive amount was ectopically expressed. A similar requirement was not seen for any other individual ECL or helix. The exchange of the ECL2 together with helix H4, or of helices H4 or H9, caused a partial loss of SERINC5’s inhibitory function. Other loops and helices were replaceable with no or only a moderate loss of function. Our results also
142 indicate that more than one domain of SERINC5 is needed to confer inhibitory function to a non-inhibitory SERINC protein like SERINC2.
Figure 4.1. The significance of each domain of SERINC5 in the inhibition of HIV-1. The numbers in grey indicate the domains not absolutely required for the fully potent inhibition of HIV-1. The domains numbered in yellow are critical for SERINC5’s function. The domains highlighted in red indicate the significance evidenced by several constructs. The numbers in black indicate domains whose role could not be determined in this study due to the abnormal expression of the resulting constructs.
143 Though we have shown that the ECL5 of SERINC5 is critical for the inhibition of HIV-1, further studies are needed to determine whether the mechanism is dependent on the primary sequence or configuration of the ECL5.
For example, the antiviral activity of tetherin is dependent on the configuration rather than the primary sequence (Perez-Caballero et al., 2009). A completely artificial protein, lacking sequence homology with native tetherin, can mimic its antiviral activity. This finding reveals that tetherin functions autonomously and restricts viral release via its two membrane anchors (Foster et al., 2018; Malim and Bieniasz, 2012; Perez-Caballero et al., 2009).
Here we show that each of the SERINC5 mutants with deletions within the
ECL5 reduces HIV-1 infectivity 3.3- to 7.9-fold, whereas the S5/S2-ECL5 chimera exhibited no inhibitory activity at all. Together, these findings suggest that the determinants in the ECL5 involved in HIV-1 inhibition are dispersed throughout the loop. An alternative explanation is that the conformation of SERINC5 is more important for the antiviral activity of SERINC5 than its primary sequence.
Possible future experiments to address this possibility include the replacement of the ECL5 with artificial peptides with predicted similar structure but lacking sequence homology with the native loop of SERINC5. Such artificial proteins might reveal the mechanism by which SERINC5 inhibits HIV-1.
Furthermore, in agreement with previous studies (Beitari et al., 2016;
Rosa et al., 2015; Usami et al., 2015), we also find that Env glycoproteins can antagonize the antiretroviral activity of SERINC5. SERINC5’s inhibition of viruses
144 bearing EnvHXB2 is 7.4-fold stronger than the inhibition of viruses bearing EnvJRFL.
By comparing the effects of the deletions within the ECL5 of SERINC5 on virions bearing EnvHXB2 with ones bearing EnvJRFL, we show that ECL5 deletions proportionally disrupts the antagonism of SERINC5 by Env. However, the exchange of the whole loop led to the discordant antagonism of SERINC5, which suggests that the ECL5 is a hotspot region carrying out the Env-dependent antiretroviral activity of SERINC5.
A previous study suggests that the V3 loop of Env determines the ability of
HIV-1 Env to counter SERINC5 (Beitari et al., 2016). NL4-3 Env bearing the V3 loop of SERINC5-resistant Envs becomes resistant to ectopic SERINC5 (Beitari et al., 2016). To examine the role of the ECL5 in SERINC5 antagonism by Env, future studies can test the effects of the deletions within the ECL5 on virions bearing wild type EnvNL4-3 or NL4-3 Env bearing the V3 loop from EnvJRFL.
In sum, our study in Chapter III addresses the importance of each domain of SERINC5 in the inhibition of HIV-1 (Figure 4.1). SERINC5 bearing the ECL5 of SERINC2 completely loses its antiviral activity. Moreover, the impaired function cannot be compensated by excessive expression levels. We further show that the ECL5 is a hotspot region carrying out the Env-dependent antiretroviral activity of SERINC5. These results underscore the importance of the ECL5 in the antiviral activity of SERINC5.
145 Conclusions
In summary, the work presented in this dissertation advances our understanding of the novel antiviral factor SERINC5 and its antagonism by Nef and Env. The work provides key mechanistic details that support a model in which the sensitivity of SERINC5 proteins to HIV-1 Nef is not conserved and Nef responsiveness can be transferred by exchanging the fourth intracellular loop of
SERINC5. This information represents a critical improvement in our understanding of the intrinsic antiviral factor SERINC5, and for the first time demonstrates that human SERINC5 can be modified to restrict HIV-1 even in the presence of Nef. This work also reveals a critical role for the fifth extracellular loop in the antiviral activity of SERINC5, and raises several important new mechanistic questions. Overall, this work provides a framework for identifying the determinants of SERINC5 that governs its antiviral activity, and its counteraction by the HIV-1 accessory protein Nef. This in turn may provide molecular targets for the design of new ART regimens that may enhance the host intrinsic immunity to HIV-1 infection.
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