HIV-1 Vpu antagonism of tetherin inhibits antibody-dependent cellular cytotoxic responses by natural killer cells

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1 JVI Accepts, published online ahead of print on 12 March 214 J. Virol. doi:1.1128/jvi Copyright 214, American Society for Microbiology. All Rights Reserved HIV-1 Vpu antagonism of tetherin inhibits antibody-dependent cellular cytotoxic responses by natural killer cells Raymond A. Alvarez 1, Rebecca E. Hamlin 2, Anthony Monroe 1, Brian Moldt 3, Mathew T. Hotta 1, Gabriela Rodriguez Caprio 1, Daniel S. Fierer 1, Viviana Simon 1,2,4 and Benjamin K. Chen 1, 5. 1 Division of Infectious Diseases, Department of Medicine, Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY Department of Immunology and Microbial Science, IAVI Neutralizing Antibody Center, and Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA The Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY Correspondence: ben.chen@mssm.edu Running Title: vpu antagonism of tetherin inhibits ADCC Keywords: HIV-1, vpu, tetherin, Env, ADCC, NK cell, neutralizing antibodies

2 Abstract The type I IFN inducible factor tetherin retains virus particles on the surface of cells infected with vpu-deficient HIV-1. While this mechanism inhibits cell-free viral spread, the immunological implications of tethered virus have not been investigated. We found that surface tetherin expression increased the antibody opsonization of vpu-deficient HIV-infected cells. The absence of Vpu also stimulated NK cell-activating FcγRIIIa signaling, enhanced NK cell degranulation and NK cell-mediated antibody dependent cellular cytotoxicity (ADCC). The deletion of vpu in HIV-1 infected primary CD4 T cells enhanced the levels of antibody binding and Fc receptor signaling mediated by HIV-positive patientderived antibodies. The magnitude of antibody binding and Fc signaling were both highly correlated to the levels of tetherin on the surface of infected primary CD4 T cells. The affinity of antibody binding to FcγRIIIa was also found to be critical in mediating efficient Fc-activation. These studies implicate Vpu antagonism of tetherin as an ADCC evasion mechanism that prevents antibodymediated clearance of virally infected cells. 2

3 Importance The ability of the HIV-1 accessory factor to antagonize tetherin has been considered to primarily function by limiting the spread of virus by preventing the release of cell-free virus. This study supports that a major function of Vpu is to decrease the recognition of infected cells by anti-hiv antibodies at the cell surface, thereby reducing recognition by antibody dependent clearance by natural killer cells. Downloaded from on October 19, 218 by guest 3

4 Introduction Human immunodeficiency virus type 1 (HIV-1) infection activates the innate immune system, but this activation fails to yield viral clearance or sterilizing immunity (1, 2). During the initial phases of acute HIV infection, the induction of type I interferon (IFN) upregulates a host of antiviral factors including the HIV-1 restriction factor tetherin (BST-2/CD317) (3). Tetherin was found to be responsible for the retention of virus particles on the surface of cells infected with HIV-1 that lack the accessory protein Vpu (4, 5). Tetherin is constitutively expressed on several cell types, including mature B-cells, plasma cells and plasmacytoid dendritic cells (pdcs), and tetherin is further upregulated by type I IFN in both macrophages and lymphocytes (6, 7). Within cells, tetherin is predominantly localized to the trans Golgi network (TGN) as well as the plasma membrane and endosomal compartments (7-1). Tetherin inhibits the cell-free release of diverse range of enveloped viruses including nearly all retroviruses tested (11). The ability of tetherin to retain viruses on the cell surface requires a unique structural topology which features two membrane-interacting domains linked by a coiled coil domain and the ability of these molecules to form stable disulfide-linked dimers (12). Studies using fluorescence microscopy and transmission electron microscopy have shown that tetherin accumulates at sites of virus assembly and directly links nascent viruses to the plasma membrane (8, 12-14). To counteract the retention of virus particles, HIV-1 has evolved the accessory protein Vpu, whose expression in infected cells leads to the down- 4

5 modulation of tetherin from the plasma membrane (15). Vpu antagonism of tetherin occurs through ubiquitin-mediated degradation, as well as TGN sequestration, both of which lead to a decrease in tetherin surface expression (16-18). While generally tetherin was proposed to inhibit viral spread, recent studies appear to find that tethered virus may enhance or diminish viral dissemination through cell-cell contact. (19-21). Several studies have shown that Vpu expression can be rapidly lost during in vitro culture (22, 23). The deletion of vpu from HIV-1 does not greatly diminish the spread of virus in infected T-cells lines, implying that viral release is not essential for spread of HIV in cell culture (24). The lack of a strict requirement for Vpu for virus replication in vitro have led some to consider tetherin as a modulator of the mode of infection, (inhibiting cell-free infection but not cell-cell infection) rather than as a strict inhibitor of viral growth (25). In vivo there may be additional reasons to enhance release of cell-free virus particles from the infected cell. A potential advantage to preventing virus retention on the surface of infected cells is for immune evasion. Tethered virus particles may be recognized by circulating antibodies, resulting in the opsonization of infected cells. Antibodyopsonized cells can be cleared through complement-mediated lysis, Fc receptormediated phagocytosis via macrophages or antibody dependent cellular cytotoxicity (ADCC)-mediated killing initiated through FcγRIIIa stimulation on the surface of granulocytes, macrophages or natural killer (NK) cells (26-28). NK 5

6 cells can also mediate the non-cytolytic suppression of viral replication through the secretion of inhibitory chemokines, CCL3, CCL4 and CCL5 (29-31). Non-neutralizing activities of antibodies have been proposed to play an important role in protective immune responses (32). The RV144 HIV vaccine trial found a correlation between the induction of non-neutralizing antibodies and protective immunity (33, 34). Other studies have detected greater ADCC responses in HIV-infected patients with slower disease progression and lower viral loads (35, 36). The primary population that mediates ADCC against virusinfected cells are NK cells, whose functions have also been correlated with slower disease progression and greater immune protection in exposed but uninfected individuals (37). Finally, several studies have demonstrated that elite controllers and Long-Term Nonprogressors (LTNP) have higher ADCC and antibody dependent cytotoxic viral inhibition (ADCVI) antibody titers compared to viremic individuals (38-4). In light of the potential contribution of ADCC in conferring protective immune responses against HIV infection, we examined whether the retention of viral particles by tetherin on the surface of infected cells enhances the detection and clearance of infected lymphocytes via ADCC. We hypothesized that tetherin surface expression enhances anti-hiv antibody binding and modulates the susceptibility of infected CD4+ T-cells to ADCC by NK cells. 6

7 Experimental Procedures Tetherin low and Tetherin high CD4+ Jurkat cells The Jurkat E6 cell line was obtained from Arthur Weiss from the NIH AIDS reagent program (ARP). The cells were stained with tetherin antibody, Clone RS38E (Biolegend) and flow sorted into Tetherin low and Tetherin high populations. These sorted clones maintained a stable tetherin phenotype in culture. To facilitate cell discrimination in cell mixing experiments, these cells were transduced to stably express GFP (MSCV-GFP Puro retroviral vector). Cells were maintained in RPMI containing 1% Fetal Calf Serum (FCS), penicillin streptomycin (PS), glutamine, and puromycin (2 ug/ml). To minimize non-specific killing by primary NK cells, the Tetherin low and Tetherin high Jurkat cells were cocultured for 3 days with primary NK cells from 3 different donors. Viruses and Infection of CD4+ lymphocytes The HIV-1 reporter viruses used were replication competent, full-length, infectious molecular clones derived from the pnl4.3 background. These constructs contain mcherry in place of the HIV-1 accessory protein Nef, and functional Nef expression is restored by the insertion of an internal ribosome entry site (41, 42). The vpu mutant has a stop codon inserted into the start of the vpu gene and the A14L vpu mutant was generated by overlap extension PCR mutagenesis. Viral stocks were generated by transfection of these molecular clones into 293T cells using calcium phosphate methods (43). Viral supernatants were harvested at 48 hours post transfection, passed through.45 micron filters, and stored at -8 C. 7

8 Primary CD4+ T-cells were purified from peripheral blood mononuclear cells obtained from deidentifed HIV-negative blood donors (New York Blood Center) using a Miltenyi Biosciences negative isolation kit, according to manufacturer s instructions and stored in liquid nitrogen prior to use. Primary CD4+ T-cells were thawed and activated using irradiated, allogeneic PBMCs co-cultured at a 2:1 ratio in RPMI containing 1% FCS, 5% human serum (HS), IL-2 (5 IU), and PHA (4ug/ml). CD4+ lymphocytes were infected (MOI.5) by spinoculation methods at a density of 2.5x1 5 cells/well in a flat bottom, 96-well plate. Cells were centrifuged at 12g for 9 minutes at 25 C, and then returned to 37 C (44). Amaxa Nucleofection was used to transfect HIV-1 constructs in Tetherin low and Tetherin high Jurkat cells. 7x1 6 cells were resuspended with proviral expression plasmid in 12ul of Amaxa T-cell line Solution V (Lonza). Nucleofections were performed using program S18 on a Nucleofector 2b (Lonza). Viable cells were purified from transfected populations via ficoll density gradient purification 24 hours post transfection and subsequently used as target cells. Overexpression of tetherin in Tetherin low cells was performed by cotransfection of a mammalian tetherin expression construct, IRAT (Thermo Scientific), along with specified HIV-1 reporter virus constructs using the nucleofection technique described above. Flow Cytometry Analysis Recombinant 4E1 and HIV IG pooled polyclonal patient sera were obtained from the NIH AIDS Reagent Program. The b12 antibody was purchased from 8

9 Polymun Scientific (Vienna, Austria). The b12 mutants with enhanced ADCC capacity (b12 double (S239D/I332E) and triple (S239D/I332E/A33L mutants) along with a control LALA b12 mutant that has a diminished capacity to signal via the FcγRIIIa, were expressed as previously described (45). Indirect surface antibody staining with all anti-hiv antibodies were performed using the primary antibodies at 2ug/ml in PBS 2% FCS for 3 minutes at 4 C. A secondary antibody anti-human IgG alexa 647 (Invitrogen) was used at 2 ug/ml. Cells were fixed in 2% paraformaldehyde PBS prior to analysis by flow cytometry. For tetherin surface staining, an anti-tetherin APC antibody (Biolegend) was used at a concentration of 5ug/ml. Flow cytometry was performed on a BD Fortessa (BD, San Jose CA), and analysis was performed with Flow Jo v8.7.3 (TreeStar, Ashland OR). FcγRIIIa stimulation assay Activation of FcγRIIIa signaling was measured using a Jurkat NFATluc+FcγRIIIa cell line (Jur-γRIIIa; Promega). FcγRIIIa signaling activates the NFAT transcription factor, inducing expression of firefly luciferase driven by an NFAT responsive promoter (46). Tetherin low and Tetherin high CD4+ lymphocytes infected with WT or vpu HIV-1 were purified by Ficoll Hypaque gradient, normalized to 15-2% infection, and pre-incubated for 15 minutes with the indicated concentration of anti-hiv antibodies. Target cells were co-cultured with the Jur-γRIIIa cells at a 5:1 effector to target ratio for 16 hours. The cells were lysed, and firefly luciferase activity was determined with a luciferase assay kit (Promega). Jur-γRIIIa cells co-cultured with the infected target populations in the 9

10 absence of antibody provided background (antibody-independent) luciferase production, and these levels were subtracted from the signal to yield antibodyspecific activation in relative light units (RLU). CD17a degranulation The degranulation of primary NK cells was measured as previously described (47). Briefly, HIV-transduced cells were purified by Ficoll Hypaque gradient to remove cellular debris, normalized to 15-2% infection, and dyelabeled with the CellTrace violet indicator dye (Invitrogen). Infected targets were pre-incubated with the indicated concentrations of antibody for 15 minutes and co-cultured with primary NK cells at 1:1 effector to target ratio at 37 C for 2 hours. After 1 hour, Golgi stop (monensin) (BD Biosciences) was added to the co-culture. Cells were then washed and stained for CD3 (Biolegend; OKT3), CD56 (Biolegend: MEM-188), and CD17a (Biolegend; H4A3) surface expression, and analyzed by flow cytometry. ADCC assay Primary NK cells were purified from PBMC from HIV-negative blood donors (New York Blood Center) using a Miltenyi negative selection kit for CD56+ CD3- NK cells according to manufacturer s protocol and stored in liquid nitrogen until needed. On the day of ADCC assays, NK cells were thawed and allowed to recover for 4 hours in RPMI containing 1% IgG-low FCS, 2IU IL-2, penicillin/streptomycin and glutamine. CD4+ lymphocytes were infected with WT or Δvpu HIV-1 purified by Ficoll Hypaque gradient to eliminate cellular debris, normalized to 15-2% infection, and dye-labeled with 1mM of the CellTrace violet 1

11 indicator dye (Invitrogen). Cells were then re-suspended in assay media (RPMI, 1% IgG-low FCS) and plated at a density of 5x1 4 cells per well in a 96-well, round bottom plate. Following pre-incubation with indicated concentration of antibodies for 15 minutes, the target cells were co-cultured with primary NK cells at 1:1 effector to target ratio at 37 C for 6 hours. After co-culture, the cells were washed in PBS, fixed in PBS with 2% paraformalydehyde, and analyzed by flow cytometry. The specific killing of HIV+ cells was assessed by the loss of cherry high cells as a proportion of the input target cells. Cells co-cultured in the absence of anti-hiv antibodies defined the level of nonspecific or background killing (~3-4%). IgG antibody isolation from HIV-positive and HIV-negative patient sera HIV-positive and HIV-negative plasma samples were collected from patients in the Jack Martin Clinic at Mount Sinai Hospital in New York, NY with an institutional review board approved protocol and provided deidentified with basic clinical profiles. Polyclonal IgG were isolated from 5ul of serum using a NAb spin kit for IgG antibody isolation kit (Thermo Scientific), according to manufacturers protocol. After elution polyclonal IgG were desalted using Zeba Desalting spin columns according to manufacturers instructions and the IgG yields quantified using an Easy-Titer IgG assay kit (Thermo Scientific). Isolated IgG were then aliquotted and stored at -8 C until used in binding and Fc receptor stimulation assays. Statistical analysis 11

12 Statistical analysis of data was performed using Graph Pad PRISM Software, San Diego, CA. Significance between infected populations were calculated using an unpaired two tailed T-test. Correlations between Fc receptor signaling and tetherin surface expression were calculated a Pearson s correlation test and significance of change in signaling comparing WT and vpu infected cells were calculated with the Wilcoxon matched pairs test. P values of less than or equal to.5 were deemed significant. Downloaded from on October 19, 218 by guest 12

13 Results Tetherin model system To examine how the expression of tetherin affects the susceptibility of CD4+ T-lymphocytes to ADCC, we exploited the heterogeneous surface expression of tetherin on the CD4+ Jurkat E6 T cell line. We used flow cytometry to sort these cells into populations expressing high or low levels of tetherin, delineated as Tetherin high and Tetherin low cells, which maintained stable surface expression when propagated in culture (Fig. 1A). We further characterized these populations for surface CD4 and CXCR4 expression and found no significant differences in the expression of either molecule on the two cell lines (data not shown). We infected these cells with an mcherry-expressing wild-type (WT) molecular clone of HIV-1 or an isogenic clone that was deficient for Vpu expression ( vpu). After infection with WT or Δvpu HIV-1, no changes in tetherin surface expression were detected in the Tetherin low cells (Fig. 1B). In contrast, tetherin surface expression was down modulated in the Tetherin high cells infected with WT virus, while high tetherin expression remained unaltered after infection with Δvpu HIV-1 (Fig. 1B). The HIV constructs used in this study encode the mcherry reporter in the nef position, and the accumulation high levels of Cherry fluorescence demarcates cells in the late stages of productive infection. Consistent with vpu being a rev-dependent, gene expressed in the late stages of infection, we observed tetherin down modulation predominantly in the mcherry high cells infected with WT, but not vpu HIV-1 (Fig. 1C). HIV-1 Vpu and Env are expressed from the same bicistronic mrna, so the deletion of vpu could 13

14 potential lead to increased amounts of total Env expression. To examine total Gag and Env expression levels in the Tetherin low and Tetherin high cells infected with WT and Δvpu HIV-1, we performed western blot analysis. The total level of HIV Env expression in the Δvpu vs. WT infected cells was slightly reduced in both the Tetherin low and Tetherin high cells (Fig. 1D). The levels of HIV Gag were similar in the Δvpu vs. WT infected Tetherin low and Tetherin high cells (Fig. 1D) When examined by confocal microscopy, tetherin surface expression was not detected in the Tetherin low population infected with WT or Δvpu HIV-1 (Fig. 1E). However, reduced levels of tetherin surface expression were observed in Tetherin high cells infected with WT virus compared to uninfected cells. Moreover, Tetherin high cells infected with Δvpu HIV-1 maintained similar levels of tetherin expression compared to uninfected Tetherin high cells. In agreement with previous studies, tetherin was observed to accumulate in large puncta on the surface of uninfected and Δvpu HIV-1-infected Tetherin high cells (19). Binding of anti-hiv antibodies to infected T-lymphocytes correlates with tetherin surface expression We next examined the binding of a panel of anti-hiv Env antibodies to the surface of infected cells. The monoclonal anti-hiv antibodies b12, 2G12 and 4E1 are broadly neutralizing antibodies (48, 49). In addition to the monoclonal antibodies, a patient polyclonal anti-hiv immunoglobulin was also used to examine the level of antibody binding to the surface of Tetherin low and Tetherin high cells infected with WT or Δvpu HIV-1 (Fig. 2A). In the Tetherin low cells, no significant differences in the percentages of antibody binding were noted 14

15 (Fig. 2A, B). The binding of monoclonal antibodies may represent the enhanced levels of Env present on the cell surface and/or the exposure of particular conformations of Env. The weak staining of the 2G12, which binds a glycan moiety and is not conformationally dependent, and the pooled patient IgG generally indicate the surface Env levels are not strongly altered by the presence or absence of Vpu in Tetherin low cells. Infection of the Tetherin high cells with WT or Δvpu HIV-1 induced higher levels of anti-hiv antibody binding in comparison to the Tetherin low cells, with the highest levels of antibody binding to the Tetherin high cells infected with Δvpu HIV-1. Similar to the modulation of surface tetherin (Fig. 1B, C), strong anti-hiv antibody binding was only observed in mcherry high HIV-infected cells. We previously observed that the total level of HIV Env expression in Δvpu HIV-1 infected cells was slightly reduced compared to infection with WT HIV-1 (Fig 1D); this suggests that the increase in surface Env detected in Δvpu HIV-1 infected cells is not likely attributable to an increase in total Env expression, but rather to the tetherin-mediated retention of virus particles on the surface of Δvpu HIV-1 infected cells. The efficiency of ADCC is dependent on the concentration and stability of antigen expressed on the surface of target cells (28). Therefore the clearance of infected cells depends not only on the frequency of opsonized target cells, but also on the density of antibody bound to their surface. We therefore calculated a combined measure of anti-hiv antibody binding to the infected populations by multiplying the percentage of infected cells that bound antibody by their mean fluorescence intensity. The total amount of anti-hiv IgG, b12 and 4E1 antibody 15

16 bound to the surface of Tetherin high cells infected with Δvpu was approximately two orders of magnitude higher than the level of binding to the Tetherin low cells infected with WT and/or vpu (Fig. 2D). Moreover, in examining the magnitude of antibody binding, we detected significant differences among all of the infected populations with all the neutralizing Abs tested. Previously the monoclonal antibody b12 has been shown to be more efficient than other anti-hiv antibodies (i.e. 2F5 and 4E1) at mediating antibody dependent cell-mediated viral inhibition (ADCVI) (5). For this reason, we utilized the b12 antibody to examine whether Vpu modulates the efficiency of ADCC against HIV-infected cells. The abundance of surface tetherin expression correlates with Fc receptor signaling, primary NK degranulation, and ADCC. The FcγRIIIa serves as the primary receptor on NK cells, granulocytes, and macrophages to detect antibody-opsonized targets, and it initiates the signaling cascade that leads to ADCC (28). During viral infections, ADCC is mostly mediated via FcγRIIIa stimulation on NK cells. The Jurkat NFAT-luc+ FcγRIIIa (Jur-γRIIIa) effector cell line expresses the FcγRIIIa and an NFAT-sensitive luciferase-reporter that is activated by FcγIIIRa stimulation (Fig. 3A) (46). The Tetherin low and Tetherin high cells were infected with WT or Δvpu HIV-1 and then co-cultured with Jur-γRIIIa cells in the presence of increasing concentrations of the b12 antibody. The Tetherin high cells infected with Δvpu HIV- 1 yielded 3- to 2-fold higher levels of FcγRIIIa stimulation compared to the Tetherin low populations infected with WT or Δvpu HIV-1, respectively (Fig. 3B). The Tetherin high cells infected with Δvpu also yielded 7% higher levels of Fc 16

17 receptor stimulation, as compared to the Tetherin high cells infected with WT HIV-1 (p <.1) (Fig. 3B). The lowest levels of Fc receptor stimulation were observed in response to the Tetherin low cells infected with WT HIV-1 at all b12 antibody concentrations tested (Fig. 3B). Fc receptor signaling stimulated by Tetherin low cells infected with vpu HIV-1 was greater than that observed by infection with WT HIV, at the highest concentration (1ug/ml) of b12 Ab tested (Fig. 3B). Coculturing the infected populations with the Jur-γRIIIa cells in the absence of b12 did not produce luciferase levels above background (Jur-γRIIIa cells cultured alone). To examine the specificity of the Fc receptor signaling, we utilized the LALA b12 antibody that contains a mutation in its constant region, which abrogates Fc receptor signaling while maintaining the ability to bind HIV-1 Env (45). Fc receptor signaling in response to the infected populations in both Tetherin low and Tetherin high cells was completely absent in the presence of the LALA b12 (Fig. 3C). This observation demonstrates that tetherin-mediated FcγIIIR stimulation is dependent on antibody Fc region engagement. A consequence of FcγIIIR signaling is the mobilization and release of lytic granules into antibody-opsonized target cells. During degranulation, LAMP-1 (CD17a), which is normally contained within lytic granules, becomes expressed on the surface of NK cells (51). To examine the ability of vpu/tetherin to influence NK cell degranulation, we co-cultured the infected cell populations with primary CD3-CD56+ NK cells and then measured the accumulation of CD17a on the surface of the NK cells (Fig. 4A). We observed a modest but specific 4-fold 17

18 increase in the level of NK cell degranulation in response to the Δvpu HIV-1 infected Tetherin high cells as compared to WT HIV-infected Tetherin high cells or WT or vpu HIV-infected Tetherin low cells (Fig. 4B, C). Next, we examined if higher levels of Fc receptor signaling and degranulation in response to Tetherin high cells infected with Δvpu rendered these targets more susceptible to NK-mediated ADCC. Cell death was measured using a flow cytometry based killing assay, in which primary NK cells were co-cultured with dye-labeled, HIV-infected target cells (Fig. 4A). The levels of killing were assessed by quantifying the specific loss of cherry high HIV-infected cells as a proportion of the total target cell population (Fig. 4D). The loss of mcherry high cells was measured since late viral gene expression and anti-hiv antibody binding predominantly occurs in the mcherry high expressing cells (Fig. 1A). This approach allowed us to detect preferential killing of the HIV infected cells over a background of 3-4% killing. The Tetherin low and Tetherin high cells infected with WT or vpu HIV-1 were co-cultured with primary NK cells from seven different donors in the absence or presence of the b12 antibody (1, 1,.1, ug/ml). At all b12 antibody concentrations tested, the Tetherin high cells infected with vpu were more susceptible to primary NK-mediated ADCC, as compared to either the Tetherin high or the Tetherin low cells infected with WT HIV-1 (Fig. 4E). At the lowest concentration, the Tetherin high cells infected with Δvpu HIV-1 were 8-times more susceptible to primary NK-mediated ADCC as compared to the Tetherin high cells infected with WT HIV, and approximately 2-fold more susceptible than the 18

19 Tetherin low cells infected with WT HIV-1 (Fig. 4E). These data demonstrate that high tetherin surface expression in conjunction with vpu HIV-1 infection induces primary NK degranulation and enhanced ADCC responses against HIV-infected lymphocytes. Thus far, we observed that Fc receptor stimulation correlated well with the levels of ADCC mediated by primary NK cells. In the following sections, we further exploit the FcγIIIR assay system to further examine if Tetherin is required to increase the susceptibility of vpu-hiv infected lymphocytes to ADCC. The complementation of surface tetherin in HIV-infected T-cell line increases their capacity to signal through the FcγRIIIa. Because the Tetherin low cells represent a common subpopulation of the Jurkat E6 cell line, we wished to test whether the phenotypes in this population could be complemented by overexpression of exogenous tetherin. To examine whether the lack of tetherin was the primary reason that the Tetherin low cells fail to activate FcγRIIIa signaling we cotransfected them with a tetherin expression construct and WT or Δvpu HIV-1. Transfection with WT or Δvpu HIV-1 alone did not induce tetherin surface expression; however, co-transfection with tetherin yielded a 4-fold and 15-fold increase in surface tetherin expression on cell populations in which the WT and vpu HIV-1 were cotransfected, respectively (Fig. 5A). The lower levels of surface tetherin in the WT population reflects Vpu antagonism, whereas the higher levels observed in the Δvpu infected cells represents baseline levels of tetherin expression. 19

20 Anti-HIV antibody binding to these Tetherin low cells cotransfected with HIV WT and tetherin expression vector revealed a 1.7 fold higher percentage of b12 binding to co-transfected cells as compared to cells transfected only with WT HIV-1 (Fig. 5B). The percentage of tetherin-positive cells was 2.8-fold higher in the Tetherin low cells expressing tetherin and Δvpu HIV-1, compared to infection with Δvpu HIV-1 alone (Fig. 5B). The fluorescence index of the antibody-binding cells, calculated as the product of the percentage of HIV antibody-binding cells and the mean fluorescence signal in these cells, showed that the greatest antibody staining was present in the cells cotransfected with the tetherin expression vector and the HIV vpu viral construct (Fig. 5C). When these populations were co-cultured with the Jur-γRIIIa cells, only the tetherintransfected cells, infected with Δvpu HIV-1 stimulated FcγRIIIa signaling in a b12 antibody dose-dependent manner (Fig. 5D). These results suggest that expression of tetherin alone was sufficient to restore greater anti-hiv antibody binding and Fc-stimulating capacity of the Tetherin low cells that express HIV-1 vpu. The specific loss of vpu antagonism for tetherin increases the capacity of HIV-1 infected CD4+ T-cells to signal through the FcγRIIIa (CD16). In addition to down-modulating tetherin, vpu has previously been shown to have effects on regulating cell-surface CD4, CD1d, NK-T- and -B cell antigen (NTB-A), and Poliovirus Receptor (PVR) (52-54). Therefore, we investigated if a mutation in vpu that specifically affects the ability to antagonize tetherin would 2

21 enhance the ability of HIV-infected lymphocytes to stimulate antibody-dependent FcγRIIIa signaling. It has been reported that an alanine to leucine point mutation in Vpu at amino acid 14 (A14L) causes a loss of tetherin antagonism, while maintaining its ability to down modulate CD4 (55). We first examined the effect of this mutation on tetherin and b12 antibody binding. Infection of the Tetherin high cells with the HIV vpu-a14l abrogated the ability of Vpu to antagonize tetherin surface expression to the same level as infection with Δvpu HIV-1 (Fig. 5E). The level of anti-hiv antibody binding to the Tetherin high cells infected with HIV vpu-a14l was significantly higher than that of WT (Fig. 5E). The magnitude of b12 antibody binding was similar to that observed with Δvpu infections (Fig. 5E). In addition, we examined the effect of the A14L mutation on NTB-A and PVR expression. Since CD1d is primarily expressed on DCs and only affects NKT cells, we focused on NTB-A and PVR. Infection of the Tetherin high cells with the HIV vpu- A14L mutant did not affect either NTB-A or PVR surface expression when compared to infection with WT or Δvpu HIV-1 (Fig. 5E). Co-culturing the Vpu-A14L HIV-infected Tetherin high cells with the Jur-γRIIIa cells resulted in a 2-fold increase in the level of FcγRIIIa stimulation compared to WT (Fig. 5F). The level of FcγRIIIa signaling was comparable to that induced by Tetherin high cells infected with Δvpu HIV-1 (Fig. 5F). These results demonstrate that the ability of Vpu to antagonize tetherin is important for the antibody opsonization of HIV-infected cells, which in turn increases FcγRIIIa signaling. 21

22 b12 antibodies with stronger binding to FcγRIIIa (CD16) mediate stronger Fc signaling by HIV-infected cells. Another important factor in mediating the efficiency of ADCC is the capacity of the Fc portion of an antibody to signal through the FcγRIIIa. In particular, b12 antibodies with specific amino acid substitutions in their Fc regions have previously been shown to influence the levels of FcγRIIIa binding and ADCC (45). b12 antibodies with amino substitutions at residues 239 and 332 (double mutant) or 239, 332 and 33 (triple mutant) can mediate greater binding to the FcγIIIR and higher levels of ADCC as compared to WT b12 Ab (45). Tetherin high cells infected with either WT or Δvpu HIV-1 were co-cultured with FcγRIIIaexpressing cells in the presence of WT b12, b12 double mutant (S239D/I332E) or b12 triple mutant (S239D/I332E/A33L). Titrations with these b12 antibodies resulted in higher levels of Fc receptor stimulation in response to the Tetherin high cells infected with Δvpu HIV-1, as compared to WT HIV-1 (Fig. 6A, B, C). Moreover, higher levels of FcγRIIIa stimulation were detected in response to the b12 double and triple mutants compared to the WT b12 antibody (Fig. 6A, B, C). The highest levels of FcγRIIIa signaling were observed in response to Δvpu HIV- 1 infected cells incubated with the b12 triple mutant, with an 18-fold higher level of FcγRIIIa stimulation in response to Δvpu infected cells compared to that of WT HIV-1 (Fig. 6C). Interestingly, the approximately 2-fold difference in FcγRIIIa signaling observed between Tetherin high cells infected with Δvpu vs. WT HIV-1 at the 1ug/ml concentration of WT b12 antibody, was amplified to a 6.5-fold difference at the same concentration of the b12 triple mutant (Fig. 6A, C). 22

23 Moreover, differences in FcγRIIIa signaling were maintained with the triple mutant at lower antibody concentrations (i.e..1 ug/ml) in contrast WT b12 antibody (Fig. 6C). Therefore, we observed that the tetherin-mediated activation of FcγRIIIa signaling was increased by mutations that are known to enhance the binding of antibodies to FcγRIIIa. We next examined the b12 triple mutant for its ability to stimulate FcγRIIIa stimulation in response to infection of primary CD4+ T-cells with Δvpu or WT HIV- 1. Activated primary CD4+ T-cells that expressed uniformly high tetherin levels were infected with either Δvpu or WT HIV-1. Infected cells were then co-cultured with the Jur-γRIIIa cells in the absence or presence of the b12 triple mutant antibody. At all of the antibody concentrations tested, infection with Δvpu HIV-1 yielded 5- to 23-fold higher levels of FcγRIIIa stimulation compared to WT HIV-1 infected primary CD4+ cells (Fig. 6D). Higher surface tetherin levels correlates with enhanced patient antibody binding to infected lymphocytes and FcγIIIRa signaling. We next examined the extent to which the binding of HIV-positive patientderived antibodies to the surface of HIV-1 infected lymphocytes was influenced by tetherin surface expression. Polyclonal IgG was isolated from the sera of three HIV positive and two HIV negative donors and incubated with Tetherin low and Tetherin high cells infected with WT or Δvpu HIV-1 (Fig. 7A). In the Tetherin low cells, no significant differences in either the percentage or MFI of antibody binding were detected with either HIV-negative or HIV-positive patient derived IgG Abs. In the Tetherin high cells, higher levels of antibody binding (% and MFI) 23

24 were detected in HIV-1 Δvpu-infected cells, as compared to cells infected with HIV-1 WT. This effect was observed with all three HIV-positive patient-derived polyclonal IgG, but not from the IgG derived from the two HIV-negative donors (Fig. 7A). To detect the level of FcγRIIIa stimulation induced by the different donorderived antibodies, we co-cultured the Tetherin low and Tetherin high cells infected with WT or Δvpu HIV-1 with Jur-gRIIIa cells in the presence of increasing concentrations of the donor-derived IgG antibodies. Significantly higher levels of FcgRIIIa stimulation were detected in the Tetherin high population infected with Δvpu HIV-1 as compared to the Tetherin high cells infected with WT HIV-1 or Tetherin low populations infected with WT or Δvpu HIV-1, in response to all three HIV-positive patient-derived antibodies when tested at the 1 ug/ml concentration (Fig 7B). Of note, significant stimulation of FcγIIIRa was not observed in response to the infected populations cultured in the presence of antibodies derived from HIV-negative donors (Fig. 7B). Higher surface tetherin levels correlate with enhanced patient antibody binding to infected lymphocytes and FcγIIIRa signaling in primary HIV infected lymphocytes. We next wished to determine if the tetherin expression on HIV-infected primary CD4 T cells may correlate with the ability of these cells to bind polyclonal anti-hiv antibodies and to stimulate Fc-receptor signaling. Primary activated 24

25 CD4+ T cells from three different HIV-negative donors were infected with either WT or Δvpu HIV-1 and the level of antibody binding quantified. None of the control HIV-negative antibodies bound to the surface of the infected primary CD4+ T cells (Fig. 8A, left). All three HIV-positive patient-derived IgG yielded significantly higher levels of antibody binding (% and MFI) to the surface of cells infected with Δvpu HIV-1, as compared to WT HIV-1 (Fig. 8A, right). Interestingly, the differences in IgG antibody binding in both WT and Δvpu HIV-infected cells correlated well with the levels of tetherin surface expression that was retained on the different HIV-infected primary T cells (Fig. 8B). There was a robust linear relationship between tetherin expression and HIV IgG binding on infected cells (Pearson correlation r>.94, R 2 >.9, and p<.5). The level of antibody binding appeared to be influenced both by the baseline expression of surface tetherin, as well as the magnitude of Vpu-mediated down modulation of tetherin from the surface of primary CD4+ T-cells. To examine the level of FcγRIIIa signaling induced by WT and Δvpu HIVinfected primary cells, the infected cells were co-cultured with the Jur-γRIIIa cells in the presence of increasing concentrations of the control HIV-negative or HIVpositive donor-derived antibodies. Neither of the HIV-negative antibodies yielded different levels of FcγRIIIa stimulation in response to any of the infected populations (data not shown). All three HIV-positive patient IgG (Donors 1, 2 and 3) yielded higher levels of Fc receptor stimulation in response to the Δvpu infected as compared to the WT infected cells. Statistically significant differences for over half of the vpu vs WT comparisons were observed at the 1. and 25

26 ug/ml concentrations (Fig. 8C). We employed a matched pair analysis where the different CD4+ donor cells and donor HIV-positive IgGs are considered in aggregate, directly examining the impact of Vpu on Fc receptor signaling, the lack of Vpu expression in the infected cells gave rise to a significant rise in Fc signaling at all concentrations tested (Fig. 8D). Since the levels of antibody binding appeared to correlate with the level of surface tetherin expressed amongst the different infected populations, we plotted the absolute level of Fc receptor stimulation in relation to the level of tetherin surface expression in the WT and Δvpu infected cells (Fig. 8E). Each of the three of the HIV positive patient-derived antibodies tested on different primary cell target cells, we observed a strong positive correlation between the level of Fc receptor stimulation and the level of tetherin surface expression in infected cells. 26

27 Discussion ADCC is postulated to be a critical component of protective immune responses against HIV-1/SHIV infections (34, 35, 38, 56-58). In this study, we show that surface expression of the type I IFN-inducible factor tetherin enhances the presentation of viral antigen on the surface of HIV-infected lymphocytes, thereby increasing HIV-antibody binding. This enhanced opsonization rendered HIV-infected lymphocytes more potent stimulators of Fc signaling with greater susceptibility to NK-mediated killing via ADCC. The ability of Vpu to diminish tetherin expression on the surface of infected cells may protect infected cells from antibody-dependent, FcγR-mediated NK cell killing. A critical component in mediating ADCC is the presentation of viral antigens on the surface of infected cells. During infection with WT HIV-1, Env glycoproteins are expressed at low density on the surface of infected cells due to endocytosis or gp12 shedding (59, 6). In the absence of Vpu, we found that tetherin was able to present higher levels of HIV-1 antigens on the surface of infected cells, which in turn led to enhanced antibody opsonization (Fig 1A). This suggests that tetherin could play a major role in enhancing the otherwise limited expression of HIV Env on the surface of infected cells. Additionally, we observed that surface tetherin induces the formation of large puncta of viral aggregates, which were not present in the absence of tetherin or when tetherin-expressing cells were infected with viruses expressing vpu. Since cross-linking of FcγRIIIa induces signaling (28), the formation of focal aggregates of antibody bound 27

28 tethered virus may further enhance ADCC by acting as a concentrating mechanism for FcγRIIIa signaling. All three broadly neutralizing HIV antibodies tested bound to the greatest extent the Tetherin high cells infected with Δvpu HIV-1. Even though the monoclonal b12, 2G12 and 4E1 antibodies have broad and potent capacity to neutralize cell-free infection (48, 49) (61), these antibodies bound infected lymphocytes to varying degrees, suggesting that the levels of antibody opsonization depends on the epitope recognized. The 2G12 recognizes a largely conformation insensitive glycan based epitope, so may more accurately reflect the total amount of Env expressed on the surface of infected cells. The b12 antibody binds the CD4 binding site while the 4E1 recognizes a fusion induced intermediate in the membrane proximal external region so variations in these antibodies may also reflect some degree of enhanced epitope exposure. The degree to which Env conformations vary between plasma membrane embedded Env versus that on tethered virus particles is still unknown. Since the b12 antibody has previously been demonstrated to mediate efficient ADCVI responses (5), we further tested this antibody. We found that increased b12 antibody binding correlated with higher levels of FcγRIIIa signaling, NK cell degranulation, and NK-mediated ADCC. In the Tetherin low cell line it is possible that deficiencies other than the low tetherin expression may reduce levels of Env and/or virus particles on the surface of the cells. Overexpressing tetherin in the Tetherin low cells along with HIV vpu, further increased antibody binding and FcγRIIIa signaling, suggesting 28

29 that the low tetherin surface expression is a major determinant of the antibody phenotype in these cells. It was also important to determine if the ability of Vpu to antagonize tetherin expression was required to evade ADCC responses. Vpu has many putative functions, but a leucine substitution mutation (A14L) within the transmembrane domain (TMD) of Vpu specifically abrogates tetherin antagonism while maintaining its ability to antagonize CD4 (55). We show here that the vpu- A14L mutant does not modulate NTB-A or PVR expression in the Tetherin high cells, where levels of these proteins were low or undetectable, respectively. In contrast, we observed that high levels of tetherin surface expression were maintained after infection with vpu-a14l HIV-1. Consequently, higher levels of b12 antibody binding were observed in the vpu-a14l HIV-1 infected population compared to those infected with WT HIV-1. Furthermore, FcγRIIIa signaling induced by lymphocytes infected with vpu-a14l HIV-1 were similar to those triggered by cells infected with Δvpu HIV-1. We conclude that in this system the ability of vpu to modulate NTB-A or PVR was not required to evade ADCC responses. Previous studies have demonstrated the importance of antibody Fc regions in dictating the magnitude of ADCC activity. When bound to Tetherin high Δvpu HIV-1 infected cells, the b12 double and triple mutants exhibited enhanced FcγRIIIa stimulation relative to the WT b12 antibody. Furthermore, primary CD4+ T-cells infected with Δvpu HIV-1 in the presence of the b12 triple mutant had a significantly higher capacity to mediate FcγRIIIa signaling in comparison to WT HIV-infected cells. These results indicate that the magnitude of tetherin-mediated 29

30 FcγRIIIa stimulation can be enhanced by antibodies with greater affinity for the Fc receptor. Importantly, the loss of Vpu increased anti-hiv antibody opsonization and Fc receptor signaling by primary infected CD4+ T-cells using IgG antibodies isolated from HIV+ patients. Baseline tetherin surface expression on primary CD4+ T cells and the ability of Vpu to antagonize tetherin varied in different donor cells; however, the presence of vpu decreased surface tetherin expression in all HIVinfected CD4+ T cells. We observed that higher levels of tetherin expression correlated with enhanced Fc receptor stimulation, which further correlated with the level of HIV-patient antibody binding to the surface of HIV-1 infected target cells. Interestingly, these correlations grew more significant as the concentration of antibodies increased. We conclude that Vpu antagonism of tetherin, can inhibit the opsonization and detection of HIV-infected lymphocytes as the concentrations of HIV specific antibodies increase during the course of infection. Two very recent publications propose that CD4 down-regulation by nef and vpu decreases the exposure of CD4 induced (CD4i) epitopes on HIV Env at the cell surface. These studies found that lack of Nef or Vpu increased CD4i antibody binding to HIV-infected cells and ADCC (62, 63). Interestingly, in Pham et al. the deletion of vpu alone did not have a major impact on CD4 down modulation, yet in experiments using HIV patient antibodies, vpu deletion had a major impact on both antibody binding and ADCC. This may indicate that the lack of vpu, and likely its antagonism of tetherin, contributes to the majority of antibody binding and ADCC against HIV infected lymphocytes. 3

31 The ability of tetherin to increase surface density of Env by interfering with particle release appears to have variable effects on the cell-to-cell transmission of HIV-1. On one hand tetherin increases the amount of Env at the cell surface, which may increase cell-cell interactions and virological synapse formation, while on the other hand the infectivity of the synapse formed may be decreased (19-21). Given the variable impact of vpu on cell to cell spread, it is compelling to consider other important functions of Vpu that relate to its impact on immune functions. Here we have demonstrated that Vpu antagonism of tetherin can contribute to immune evasion from ADCC, suggesting that a central role for Vpu in vivo is the evasion of infected cell clearance. In line with this supposition, four of the five surface molecules, which now includes tetherin, that are reported to be antagonized by Vpu naturally function to modulate the efficiency of NK responses during HIV infection (i.e. Tetherin, NTB-A, CD-1d and PVR). Taken together, this suggests that the principal function of vpu in vivo is the evasion of antibody- and NK cell-mediated immune clearance mechanisms. There is strong evidence indicating that ADCC responses can provide protective immunity against HIV-1 (64-66). Understanding that tetherin and Vpu can modulate antibody dependent cellular responses allows us to consider approaches to enhance vaccines or antibody-based therapies. Because Vpu can limit the detection and killing of HIV-infected cells by ADCC, in a therapeutic setting, targeting Vpu in an ADCC-inducing vaccine strategy may increase viral 31

32 antigen presentation by reducing the capacity of Vpu to antagonize tetherin. Intriguingly, ADCC responses in LTNP/elite controllers have already been observed to be broadly targeting and, in some cases, have been shown to target epitopes in Vpu (36, 47). It would be interesting to examine if targeting Vpu in vaccines may exert immune pressure on the regions required to antagonize tetherin, thereby acting synergistically to improve the ADCC responses in these patients. In this study, we have demonstrated that tetherin can facilitate HIV-infected cell clearance through ADCC. The broader implication of these findings is that tetherin may also act on other viral infections, to aid in the detection and clearance of infected cells through antibody-mediated mechanisms. In addition to ADCC, it is important to consider that the antibody Fc region can modulate various cellular effector functions including cytokine release, phagocytosis or the release of virus inhibiting chemokines. Tetherin-mediated opsonization may also induce complement dependent cytotoxicity (CDC). In addition, recent studies have implicated tetherin as an innate immune signaling receptor that functions through NF-kB pathways (67, 68). Thus, interferon-induced tetherin surface expression is likely to function as an important immune modulator during viral infections, acting as a bridge between the innate and adaptive immune responses. 32

33 Acknowledgements We thank members of the Chen Laboratory for helpful comments and advice. This work was supported by grants from the NIH, NIAID AI7442, NIDA DP1DA , the Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease Award, and the Irma T Hirschl/Monique Weill-Caulier Trust Career Scientist Award to B.K.C. R.A.A. has been supported by the Training Program in Mechanisms of Virus-host Interactions, T32 AI7647 and also by the NIH Extramural Loan Reimbursement Grant from NIDA. Downloaded from on October 19, 218 by guest 33

34 References 1. Sedaghat AR, German J, Teslovich TM, Cofrancesco J, Jr., Jie CC, Talbot CC, Jr., Siliciano RF. 28. Chronic CD4+ T-cell activation and depletion in human immunodeficiency virus type 1 infection: type I interferon-mediated disruption of T-cell dynamics. Journal of virology 82: Hyrcza MD, Kovacs C, Loutfy M, Halpenny R, Heisler L, Yang S, Wilkins O, Ostrowski M, Der SD. 27. Distinct transcriptional profiles in ex vivo CD4+ and CD8+ T cells are established early in human immunodeficiency virus type 1 infection and are characterized by a chronic interferon response as well as extensive transcriptional changes in CD8+ T cells. Journal of virology 81: Homann S, Smith D, Little S, Richman D, Guatelli J Upregulation of BST-2/Tetherin by HIV infection in vivo. J Virol 85: Neil SJ, Zang T, Bieniasz PD. 28. Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu. Nature 451: Van Damme N, Goff D, Katsura C, Jorgenson RL, Mitchell R, Johnson MC, Stephens EB, Guatelli J. 28. The interferon-induced protein BST- 2 restricts HIV-1 release and is downregulated from the cell surface by the viral Vpu protein. Cell Host Microbe 3: Arnaud F, Black SG, Murphy L, Griffiths DJ, Neil SJ, Spencer TE, Palmarini M. 21. Interplay between ovine bone marrow stromal cell antigen 2/tetherin and endogenous retroviruses. J Virol 84: Blasius AL, Giurisato E, Cella M, Schreiber RD, Shaw AS, Colonna M. 26. Bone marrow stromal cell antigen 2 is a specific marker of type I IFN-producing cells in the naive mouse, but a promiscuous cell surface antigen following IFN stimulation. J Immunol 177: Habermann A, Krijnse-Locker J, Oberwinkler H, Eckhardt M, Homann S, Andrew A, Strebel K, Krausslich HG. 21. CD317/tetherin is enriched in the HIV-1 envelope and downregulated from the plasma membrane upon virus infection. J Virol 84: Kupzig S, Korolchuk V, Rollason R, Sugden A, Wilde A, Banting G. 23. Bst-2/HM1.24 is a raft-associated apical membrane protein with an unusual topology. Traffic 4:

35 Masuyama N, Kuronita T, Tanaka R, Muto T, Hirota Y, Takigawa A, Fujita H, Aso Y, Amano J, Tanaka Y. 29. HM1.24 is internalized from lipid rafts by clathrin-mediated endocytosis through interaction with alphaadaptin. J Biol Chem 284: Jouvenet N, Neil SJ, Zhadina M, Zang T, Kratovac Z, Lee Y, McNatt M, Hatziioannou T, Bieniasz PD. 29. Broad-spectrum inhibition of retroviral and filoviral particle release by tetherin. J Virol 83: Perez-Caballero D, Zang T, Ebrahimi A, McNatt MW, Gregory DA, Johnson MC, Bieniasz PD. 29. Tetherin inhibits HIV-1 release by directly tethering virions to cells. Cell 139: Fitzpatrick K, Skasko M, Deerinck TJ, Crum J, Ellisman MH, Guatelli J. 21. Direct restriction of virus release and incorporation of the interferon-induced protein BST-2 into HIV-1 particles. PLoS Pathog 6:e Hammonds J, Wang JJ, Yi H, Spearman P. 21. Immunoelectron microscopic evidence for Tetherin/BST2 as the physical bridge between HIV-1 virions and the plasma membrane. PLoS Pathog 6:e Le Tortorec A, Willey S, Neil SJ Antiviral inhibition of enveloped virus release by tetherin/bst-2: action and counteraction. Viruses 3: Dube M, Roy BB, Guiot-Guillain P, Binette J, Mercier J, Chiasson A, Cohen EA. 21. Antagonism of tetherin restriction of HIV-1 release by Vpu involves binding and sequestration of the restriction factor in a perinuclear compartment. PLoS Pathog 6:e Van Damme N, Guatelli J. 28. HIV-1 Vpu inhibits accumulation of the envelope glycoprotein within clathrin-coated, Gag-containing endosomes. Cell Microbiol 1: Hauser H, Lopez LA, Yang SJ, Oldenburg JE, Exline CM, Guatelli JC, Cannon PM. 21. HIV-1 Vpu and HIV-2 Env counteract BST-2/tetherin by sequestration in a perinuclear compartment. Retrovirology 7: Casartelli N, Sourisseau M, Feldmann J, Guivel-Benhassine F, Mallet A, Marcelin AG, Guatelli J, Schwartz O. 21. Tetherin restricts productive HIV-1 cell-to-cell transmission. PLoS Pathog 6:e Jolly C, Booth NJ, Neil SJ. 21. Cell-cell spread of human immunodeficiency virus type 1 overcomes tetherin/bst-2-mediated restriction in T cells. J Virol 84:

36 Kuhl BD, Sloan RD, Donahue DA, Bar-Magen T, Liang C, Wainberg MA. 21. Tetherin restricts direct cell-to-cell infection of HIV-1. Retrovirology 7: Gummuluru S, Kinsey CM, Emerman M. 2. An in vitro rapid-turnover assay for human immunodeficiency virus type 1 replication selects for cellto-cell spread of virus. Journal of virology 74: Monde K, Chukkapalli V, Ono A Assembly and replication of HIV- 1 in T cells with low levels of phosphatidylinositol-(4,5)-bisphosphate. Journal of virology 85: Klimkait T, Strebel K, Hoggan MD, Martin MA, Orenstein JM The human immunodeficiency virus type 1-specific protein vpu is required for efficient virus maturation and release. Journal of virology 64: Strebel K HIV-1 Vpu - an ion channel in search of a job. Biochimica et biophysica acta. 26. Forthal DN, Moog C. 29. Fc receptor-mediated antiviral antibodies. Current opinion in HIV and AIDS 4: Huber G, Banki Z, Lengauer S, Stoiber H Emerging role for complement in HIV infection. Current opinion in HIV and AIDS 6: Sanchez-Mejorada G, Rosales C Signal transduction by immunoglobulin Fc receptors. Journal of leukocyte biology 63: Fehniger TA, Herbein G, Yu H, Para MI, Bernstein ZP, O'Brien WA, Caligiuri MA Natural killer cells from HIV-1+ patients produce C-C chemokines and inhibit HIV-1 infection. J Immunol 161: Kottilil S, Chun TW, Moir S, Liu S, McLaughlin M, Hallahan CW, Maldarelli F, Corey L, Fauci AS. 23. Innate immunity in human immunodeficiency virus infection: effect of viremia on natural killer cell function. The Journal of infectious diseases 187: Oliva A, Kinter AL, Vaccarezza M, Rubbert A, Catanzaro A, Moir S, Monaco J, Ehler L, Mizell S, Jackson R, Li Y, Romano JW, Fauci AS Natural killer cells from human immunodeficiency virus (HIV)- infected individuals are an important source of CC-chemokines and suppress HIV-1 entry and replication in vitro. The Journal of clinical investigation 12: Robinson HL Non-neutralizing antibodies in prevention of HIV infection. Expert opinion on biological therapy 13:

37 Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, Premsri N, Namwat C, de Souza M, Adams E, Benenson M, Gurunathan S, Tartaglia J, McNeil JG, Francis DP, Stablein D, Birx DL, Chunsuttiwat S, Khamboonruang C, Thongcharoen P, Robb ML, Michael NL, Kunasol P, Kim JH, Investigators M-T. 29. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. The New England journal of medicine 361: Haynes BF, Gilbert PB, McElrath MJ, Zolla-Pazner S, Tomaras GD, Alam SM, Evans DT, Montefiori DC, Karnasuta C, Sutthent R, Liao HX, DeVico AL, Lewis GK, Williams C, Pinter A, Fong Y, Janes H, DeCamp A, Huang Y, Rao M, Billings E, Karasavvas N, Robb ML, Ngauy V, de Souza MS, Paris R, Ferrari G, Bailer RT, Soderberg KA, Andrews C, Berman PW, Frahm N, De Rosa SC, Alpert MD, Yates NL, Shen X, Koup RA, Pitisuttithum P, Kaewkungwal J, Nitayaphan S, Rerks-Ngarm S, Michael NL, Kim JH Immune-correlates analysis of an HIV-1 vaccine efficacy trial. The New England journal of medicine 366: Baum LL, Cassutt KJ, Knigge K, Khattri R, Margolick J, Rinaldo C, Kleeberger CA, Nishanian P, Henrard DR, Phair J HIV-1 gp12- specific antibody-dependent cell-mediated cytotoxicity correlates with rate of disease progression. Journal of immunology 157: Wren LH, Chung AW, Isitman G, Kelleher AD, Parsons MS, Amin J, Cooper DA, investigators Asc, Stratov I, Navis M, Kent SJ Specific antibody-dependent cellular cytotoxicity responses associated with slow progression of HIV infection. Immunology 138: Scott-Algara D, Truong LX, Versmisse P, David A, Luong TT, Nguyen NV, Theodorou I, Barre-Sinoussi F, Pancino G. 23. Cutting edge: increased NK cell activity in HIV-1-exposed but uninfected Vietnamese intravascular drug users. Journal of immunology 171: Lambotte O, Ferrari G, Moog C, Yates NL, Liao HX, Parks RJ, Hicks CB, Owzar K, Tomaras GD, Montefiori DC, Haynes BF, Delfraissy JF. 29. Heterogeneous neutralizing antibody and antibody-dependent cell cytotoxicity responses in HIV-1 elite controllers. AIDS 23: Jia M, Li D, He X, Zhao Y, Peng H, Ma P, Hong K, Liang H, Shao Y Impaired natural killer cell-induced antibody-dependent cellmediated cytotoxicity is associated with human immunodeficiency virus-1 disease progression. Clinical and experimental immunology 171: Ackerman ME, Crispin M, Yu X, Baruah K, Boesch AW, Harvey DJ, Dugast AS, Heizen EL, Ercan A, Choi I, Streeck H, Nigrovic PA, 37

38 Bailey-Kellogg C, Scanlan C, Alter G Natural variation in Fc glycosylation of HIV-specific antibodies impacts antiviral activity. The Journal of clinical investigation 123: Chen BK, Gandhi RT, Baltimore D CD4 down-modulation during infection of human T cells with human immunodeficiency virus type 1 involves independent activities of vpu, env, and nef. J Virol 7: Cohen GB, Gandhi RT, Davis DM, Mandelboim O, Chen BK, Strominger JL, Baltimore D The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells. Immunity 1: Pear WS, Nolan GP, Scott ML, Baltimore D Production of hightiter helper-free retroviruses by transient transfection. Proceedings of the National Academy of Sciences of the United States of America 9: O'Doherty U, Swiggard WJ, Malim MH. 2. Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding. Journal of virology 74: Moldt B, Schultz N, Dunlop DC, Alpert MD, Harvey JD, Evans DT, Poignard P, Hessell AJ, Burton DR A panel of IgG1 b12 variants with selectively diminished or enhanced affinity for Fcgamma receptors to define the role of effector functions in protection against HIV. Journal of virology 85: Parekh BS, Berger E, Sibley S, Cahya S, Xiao L, LaCerte MA, Vaillancourt P, Wooden S, Gately D Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay. mabs 4: Stratov I, Chung A, Kent SJ. 28. Robust NK cell-mediated human immunodeficiency virus (HIV)-specific antibody-dependent responses in HIV-infected subjects. J Virol 82: Binley JM, Wrin T, Korber B, Zwick MB, Wang M, Chappey C, Stiegler G, Kunert R, Zolla-Pazner S, Katinger H, Petropoulos CJ, Burton DR. 24. Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies. Journal of virology 78: Mehandru S, Wrin T, Galovich J, Stiegler G, Vcelar B, Hurley A, Hogan C, Vasan S, Katinger H, Petropoulos CJ, Markowitz M. 24. Neutralization profiles of newly transmitted human immunodeficiency virus 38

39 type 1 by monoclonal antibodies 2G12, 2F5, and 4E1. Journal of virology 78: Hessell AJ, Rakasz EG, Tehrani DM, Huber M, Weisgrau KL, Landucci G, Forthal DN, Koff WC, Poignard P, Watkins DI, Burton DR. 21. Broadly neutralizing monoclonal antibodies 2F5 and 4E1 directed against the human immunodeficiency virus type 1 gp41 membrane-proximal external region protect against mucosal challenge by simian-human immunodeficiency virus SHIVBa-L. Journal of virology 84: Alter G, Malenfant JM, Altfeld M. 24. CD17a as a functional marker for the identification of natural killer cell activity. Journal of immunological methods 294: Shah AH, Sowrirajan B, Davis ZB, Ward JP, Campbell EM, Planelles V, Barker E. 21. Degranulation of natural killer cells following interaction with HIV-1-infected cells is hindered by downmodulation of NTB-A by Vpu. Cell Host Microbe 8: Moll M, Andersson SK, Smed-Sorensen A, Sandberg JK. 21. Inhibition of lipid antigen presentation in dendritic cells by HIV-1 Vpu interference with CD1d recycling from endosomal compartments. Blood 116: Matusali G, Potesta M, Santoni A, Cerboni C, Doria M The human immunodeficiency virus type 1 Nef and Vpu proteins downregulate the natural killer cell-activating ligand PVR. Journal of virology 86: Vigan R, Neil SJ. 21. Determinants of tetherin antagonism in the transmembrane domain of the human immunodeficiency virus type 1 Vpu protein. J Virol 84: Banks ND, Kinsey N, Clements J, Hildreth JE. 22. Sustained antibody-dependent cell-mediated cytotoxicity (ADCC) in SIV-infected macaques correlates with delayed progression to AIDS. AIDS research and human retroviruses 18: Xiao P, Zhao J, Patterson LJ, Brocca-Cofano E, Venzon D, Kozlowski PA, Hidajat R, Demberg T, Robert-Guroff M. 21. Multiple vaccineelicited nonneutralizing antienvelope antibody activities contribute to protective efficacy by reducing both acute and chronic viremia following simian/human immunodeficiency virus SHIV89.6P challenge in rhesus macaques. Journal of virology 84: Alpert MD, Harvey JD, Lauer WA, Reeves RK, Piatak M, Jr., Carville A, Mansfield KG, Lifson JD, Li W, Desrosiers RC, Johnson RP, Evans 39

40 DT ADCC develops over time during persistent infection with liveattenuated SIV and is associated with complete protection against SIV(mac)251 challenge. PLoS pathogens 8:e Egan MA, Carruth LM, Rowell JF, Yu X, Siliciano RF The ins and outs of HIV endocytosis. Trends in cell biology 7: Checkley MA, Luttge BG, Freed EO HIV-1 envelope glycoprotein biosynthesis, trafficking, and incorporation. Journal of molecular biology 41: Walker LM, Phogat SK, Chan-Hui PY, Wagner D, Phung P, Goss JL, Wrin T, Simek MD, Fling S, Mitcham JL, Lehrman JK, Priddy FH, Olsen OA, Frey SM, Hammond PW, Protocol GPI, Kaminsky S, Zamb T, Moyle M, Koff WC, Poignard P, Burton DR. 29. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science 326: Pham TN, Lukhele S, Hajjar F, Routy JP, Cohen EA HIV Nef and Vpu protect HIV-infected CD4+ T cells from antibody-mediated cell lysis through down-modulation of CD4 and BST2. Retrovirology 11: Veillette M, Desormeaux A, Medjahed H, Gharsallah NE, Coutu M, Baalwa J, Guan Y, Lewis G, Ferrari G, Hahn BH, Haynes BF, Robinson JE, Kaufmann DE, Bonsignori M, Sodroski J, Finzi A Interaction with Cellular CD4 Exposes HIV-1 Envelope Epitopes Targeted by Antibody-Dependent Cell-Mediated Cytotoxicity. J Virol 88: Carrington M, Alter G Innate immune control of HIV. Cold Spring Harbor perspectives in medicine 2:a Fauci AS, Mavilio D, Kottilil S. 25. NK cells in HIV infection: paradigm for protection or targets for ambush. Nature reviews. Immunology 5: Jost S, Altfeld M Control of human viral infections by natural killer cells. Annual review of immunology 31: Galao RP, Le Tortorec A, Pickering S, Kueck T, Neil SJ Innate sensing of HIV-1 assembly by Tetherin induces NFkappaB-dependent proinflammatory responses. Cell Host Microbe 12: Tokarev A, Suarez M, Kwan W, Fitzpatrick K, Singh R, Guatelli J Stimulation of NF-kappaB activity by the HIV restriction factor BST2. J Virol 87:

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42 Figure Legends Figure 1. Tetherin CD4+ T cell model system. (A) Heterogeneous expression of surface tetherin on unsorted CD4 + Jurkat E6 cells (left). The Jurkat E6 cells were stained for tetherin expression (red line) and then flow sorted into Tetherin low (center) and Tetherin high (right) populations, which remained stable in culture. (B) Tetherin low and Tetherin high cells were infected with WT (blue line) or Δvpu (red line) cherry fluorescent protein-expressing HIV-1 and surface tetherin on the cherry-positive infected cells was assessed by flow cytometry. The histogram plots show the modulation of surface tetherin on the HIV-infected cells in the tetherin low (left) and tetherin high (right) cells 48 hours after HIV infection. (C) Bar graph shows the mean fluorescence intensity (MFI) of surface tetherin in HIV-infected Tetherin low and Tetherin high cells. (D) Western blot show the levels of HIV-1 protein expression in the Tetherin low and Tetherin high cells infected with WT or Δvpu HIV-1. Samples were normalized to 2% infection as indicated by cherry expression. Cellular lysates were probed with a polyclonal anti-hiv antibody sera, an anti-hiv Env and an anti-cherry antibody. (E) Tetherin low and Tetherin high cells (GFP+) were infected with WT and Δvpu HIV-1 (cherry+) for 48 hours and surface tetherin (cyan) localization was assessed by spinning disk confocal microscopy. Maximum intensity projections are displayed. Figure 2. High tetherin expression correlates with anti-hiv antibody binding on HIV-infected lymphocytes. Tetherin low and Tetherin high cells were infected with WT or Δvpu HIV-1, and the levels of surface antibody binding on HIV- 42

43 infected cells was assessed using a panel of broadly neutralizing anti-hiv antibodies or polyclonal patient sera. (A) Surface binding of anti-env neutralizing antibodies b12, 2G12, 4E1 and a polyclonal anti-hiv IgG sera were measured by flow cytometry. The percentage of HIV-infected cells binding antibody is shown in bold. (B) The mean percentage of Env-positive cells from five biological replicates. (C) The mean fluorescence of the Env-positive cells is plotted (average of five independent experiments). (D) A Fluorescent Index was calculated by multiplying the percentage of infected cells by the MFI index. The graph shows the fluorescent index of anti-hiv antibody binding to the surface of the Tetherin high and Tetherin low infected populations. The levels of antibody binding in graphs B-D were calculated from five independent staining experiments. Figure 3. Tetherin surface expression increases FcγRIIIa stimulation. (A) The diagram depicts the FcγRIIIa assay. A Jurkat E6-derived indicator cell line expresses FcγRIIIa. Upon FcγIIIRa stimulation activation of the NFAT transcription factor induces luciferase expression. Tetherin low and Tetherin high cells were infected with WT or Δvpu HIV-1 for 48 hours and normalized to 15-2% infection as indicated by cherry expression. Infected populations were then co-cultured in the presence or absence of either (B) the wt b12 antibody or (C) a LALA b12 antibody with an altered Fc region that abrogates FcγRIIIa stimulation. These populations were co-cultured at a 5:1 FcγRIIIa-expressing effector to HIVinfected target cell ratio for 16hrs. After 16 hours the luciferase activity in the 43

44 lysed cells indicates FcγRIIIa activation. The graphs represent the mean of three independent experiments conducted in duplicate. p = <.5; p = <.1; p = < Figure 4. Tetherin surface expression increases Primary NK degranulation and ADCC killing. The Tetherin low and Tetherin high cells infected with either WT or Δvpu were co-cultured with primary CD56 + CD3 - NK cells at a 1:1 infected target to NK cell ratio in the presence or absence of different concentrations of the b12 antibody. (A) Gating scheme of the assays uses Cell Tracker Violet staining to discriminate HIV-infected target cells (Violet+) from NK cells, upper left panel. To assess activation of NK cells we measured surface CD17a in CD56+ cells, bottom panels. To assess infected cell killing we measured the loss of HIV-infected cells (Cherry+) from the Violet cells, upper right. (B) NK cell activation was measured after co-culture with the HIV-infected CD4+ cells for 2 hours in the presence or absence of b12 antibody. The levels of CD17a degranulation on NK cells indicate activation. (C) Graph represents the fold change in CD17a expression on the surface of NK cells in response to the HIVinfected Tetherin low and Tetherin high cells. The fold difference was calculated by dividing the levels of CD17a degranulation in the presence of b12 by those observed in the absence of antibody. (D) A primary NK ADCC assay where NK cells were co-cultured with infected Tetherin high or Tetherin low cells for 6 hours. The levels of specific killing within each HIV-infected population were assessed by flow cytometry. Dot plots depict the levels of NK-mediated ADCC in the 44

45 absence of b12 (left panel) or in the presence of 1ug/ml of b12 (Right panel). (E) Graphs represent the percentage of NK-mediated killing of Tetherin low and Tetherin high cells infected with HIV WT or Δvpu-infected following exposure to.1, 1 and 1 ug/ml of b12 antibody. The primary NK cells used in these assays were derived from four to five different donors. Each dot on the scatter plot represents the mean of technical replicates from each donor. p = <.5; p = <.1. Figure 5. Impact of Tetherin overexpression or mutation of a residue in vpu required for tetherin downmodulation on infected-cell opsonization and FcγRIIIa signaling. (A) Tetherin low cells transfected with tetherin expression construct IRAT show higher levels of tetherin staining. Cells were stained with 5 ug/ml of anti-tetherin APC antibody. Histogram flow cytometry plots depict the level of tetherin in the Tetherin low cells that were nucleofected with WT HIV-1 (upper left), WT HIV-1 + IRAT (upper right), Δvpu HIV-1 (lower left), Δvpu HIV-1+ IRAT (lower right). (B) Dot plots show the levels of surface b12 antibody binding to the surface of Tetherin low cells nucleofected with WT HIV-1 (upper left), WT HIV-1 + IRAT (upper right), Δvpu HIV-1 (lower left), Δvpu HIV-1+ IRAT (lower right). (C) The nucleofected populations were co-cultured at a 5:1 Jur-γRIIIa effector cell to infected target cell ratio for 16hrs. After co-culture, the total samples were lysed and levels of luciferase activity measured. Graph depicts the relative light units produced by FcγIIIR-expressing cells in response to HIVinfected and b12 opsinized CD4+ populations. (D) The levels of surface NTB-A, PVR and tetherin expression along with the levels of b12 binding to Tetherin high 45

46 cells infected with WT, Δvpu or vpu-a14l HIV-1 were assessed through surface antibody staining followed by flow cytometry. Human anti-ntb-a, anti-pvr and anti-tetherin antibodies were used at 5ug/ml. (E) Graph shows the levels of FcγRIIIa stimulation in response to WT, Δvpu, or vpu-a14l HIV-infected Tetherin high cells. The data is of a representative experiment done in triplicate. p = <.5; p = <.1; p = <.1 Figure 6. The magnitude of tetherin enhanced FcγRIIIa stimulation is modulated by changes in antibody Fc regions that affect FcγRIIIa binding. Tetherin high CD4 cells were infected with WT or Δvpu HIV-1 and 48 hours later normalized to approximately 2% infection. The infected populations were then incubated in the presence of WT b12 or a b12 double (S239D/I332E) or triple (S239D/I332E/A33L) Fc mutants that confer enhanced binding and signaling through the FcγRIIIa. (A-C). Graphs depict the levels of FcγRIIIa signaling in response to Tetherin high cells infected with WT or Δvpu HIV incubated with a titration of either (A) wt b12 (B) b12 double or (C) b12 triple mutant antibodies. (D) Primary CD4 + T-cells were activated for 3 days and then infected with WT or Δvpu HIV-1. Four days after infection, the populations were normalized to 1% infection and co-cultured with the Jur-γRIIIa cells at a 5:1 effector to target ratio for 16 hours. The graph depicts a representative experiment of the levels of FcγIIIR stimulation in response to primary CD4+ lymphocytes cultured with either WT or Δvpu HIV-1. The data is of representative experiments conducted in triplicate. 46

47 Figure 7. Higher Tetherin surface expression correlates with enhanced HIV positive patient-derived IgG opsonization of HIV-infected T cells and enhanced Fc receptor stimulation. Tetherin low and Tetherin high cells were infected WT and Δvpu HIV-1, and the levels of antibody binding were quantified using purified polyclonal IgG antibodies derived from two HIV negative and three HIV positive donors. (A) Overlapping histograms show the level of donor-derived IgG surface binding as measured by flow cytometry in the Tetherin low and Tetherin high cells that were infected WT or Δvpu HIV-1. (B) The graphs depict the levels of FcγRIIIa signaling in response to the Tetherin low and Tetherin high cells infected with WT and Δvpu HIV-1 and incubated with a titration of different HIVnegative (left) and HIV-positive (right) donor-derived IgG. Figure 8. Tetherin surface expression correlates with enhanced HIV positive patient-derived IgG opsonization of HIV-infected T cells and enhanced Fc receptor stimulation in primary CD4+ T cells. (A) Primary CD4+ T cells isolated from three HIV-negative donors (D389, D88, and D73) were activated for two days and infected with WT or Δvpu HIV-1. Two days after infection, the populations were normalized to 2% infection and the level of surface IgG binding was assessed. Overlapping histograms indicate the level of surface IgG bound to primary CD4+ T cells infected with WT or Δvpu HIV-1 and incubated with a titration of different HIV-negative (left) and HIV-positive (right) donor-derived IgG. (B) Graphs illustrate the correlation between the levels of antibody binding and the levels of tetherin surface expression in WT or Δvpu HIV- 47

48 infected cells that were incubated with the HIV-positive donor-derived IgG. Spearman correlation was calculated and linear regression curve plotted showing r and p values for each graph. (C) Panels depict the levels of FcγRIIIa stimulation in primary CD4+ T cells infected with WT or Δvpu HIV-1 and incubated with a titration of HIV positive donor-derived IgG from three different donors. Asterisks mark individual donors that yielded statistically significant difference between signaling stimulated by WT (filled symbols) and vpu (filled symbols) HIV-infected cells. (D) Panels show the positive correlation between the levels of FcγRIIIa signaling and levels of tetherin surface expression on HIV-1 infected primary CD4+ T cells. WT and vpu HIV-infected cells are intermixed in the graph, showing a strong positive correlation between surface Tetherin levels and Fc receptor signaling at 1, 1 and.1 ug/ml of patient IgG. Linear regression curve is plotted with p value indicated, p <.5, p <.1. (E) Cells from three different HIV negative donor cells were treated with HIV-positive patientderived IgG from three different patients, following infection with WT or vpu HIV- 1. The enhancement of Fc receptor activation in the absence of Vpu is measured by the Wilcoxon matched pair test at each concentration of antibody tested. 48

49 A Jurkat E6 Tetherin low Tetherin high Isotype WT HIV HIV vpu B D Tetherin Tetherin low Tetherin high Isotype HIV WT HIV vpu Tetherin C E Tetherin MFI HIV WT HIV vpu Tetherin low Tetherin high Tetherin low Downloaded from gp16 gp12 p55 p41 p24 Anti-ENV Anti-HIV (Gag) AntimCherry Gag-iCherry GFP-Jurkat Tetherin NK Cells WT Tetherin high vpu on October 19, 218 by guest WT vpu

50 A 2nd Ab b12 2G E HIV IgG Tetherin low Tetherin high % of anti-hiv antibody binding B WT vpu WT vpu HIV + (Cherry+) b12 2G12 4E1 2.65% 13.76% % 13.14% % 21.78% HIV IgG C MFI of anti-hiv antibody binding 28.94% 46.72% Surface Env+ b12 2G12 4E1 HIV IgG.21% % % D fluorescent index of anti-hiv antibody binding 21.22% % % % b12 2G12 4E % HIV IgG Tetherin low WT Tetherin low Δvpu Tetherin high WT Tetherin high Δvpu

51 A Jur-γIIIRa cells α-hiv Ab B FcγIIIR activation (Relative light units) C FcγIIIR activation (Relative light units) HIV-1 infected cell Env FcgIIIR NFAT concentration WT b12 (ug/ml) luciferase Tetherin low WT Tetherin low Δvpu Tetherin high WT Tetherin high Δvpu Tetherin low WT Tetherin low Δvpu Tetherin high WT Tetherin high Δvpu concentration LALA b12 (ug/ml)

52 A SCC Cell Tracker Violet B degranulation killing CD56 + CD17a + ug/ml b12 Ab SCC 1 2 HIV + (cherry + ) CD56 + NK cells Primary NK cell degranulation 1ug/ml b12 Ab D SCC E % HIV Cherry+ Killing HIV + Targets ug/ml B12 1ug/ml B12 1% 5.6% HIV WT 1ug/ml b12 Vpu WT Vpu Tetherin low Tetherin high Downloaded from C Fold induction of CD17+ NK cells % 1.13% CD17a + Primary NK cell degranulation 8 6.1ug/ml 1ug/ml 1ug/ml concentation of WT b12 Ab Tetherin low WT Tetherin low Δvpu Tetherin high WT Tetherin high Δvpu % HIV Cherry+ Killing % HIV Cherry+ Killing ug/ml b12 WT Vpu WT Vpu Tetherin low Tetherin high.1ug/ml b12 on October 19, 218 by guest WT Vpu WT Vpu Tetherin low Tetherin high

53 A WT - Tetherin + Tetherin Isotype Tetherin D Relative Light Units 1 5 FcγIIIR stimulation WT WT + IRAT vpu vpu + IRAT B WT HIV+ vpu C fluorecent index of anti-hiv antibody binding (b12) vpu Tetherin Tetherin anti-hiv b12 binding + Tetherin 6.3% 14.96% 16.78% 32.5% Teth low WT Teth low WT + IRAT Teth low vpu Teth low vpu + IRAT E F FcγIIIR activation (Relative light units) b12 concentration (ug/ml) Surface staining in HIV+ Tetherin high cells NTB-A Tetherin Fc IIIR stimulation PVR b12 1ug/ml 1ug/ml.1ug/ml.1ug/ml concentration of b12 antibody Isotype WT Vpu Teth high WT Vpu A14L Teth high Δvpu Teth high Vpu A14L