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Acute HIV-1 infection is characterized by a type I interferon response, resulting in the induction of host restriction factors. HIV-1 has evolved to counteract these factors, and one such adaptation, the ability of Vpu to counteract BST2/tetherin, is associated with the evolution of simian immunodeficiency virus (SIVcpz) into pandemic group M human immunodeficiency virus type 1 (HIV-1). During transmission between individuals, very few viruses or even a single virus, the "transmitted/founder" (T/F) virus, gives rise to the new infection, but in the new host the selective pressure of the immune response yields the diverse "quasispecies" of chronic infection. Here we examine the functional characteristics of Vpu proteins encoded by T/F viruses compared to acute and chronic viruses from longitudinally sampled subjects. The studied T/F Vpu proteins showed a trend toward optimized CD4 downregulation compared to chronic Vpu proteins but did not differ substantially in their ability to downregulate BST2 or enhance virion release, although individual clones from each group were impaired in these activities. Analysis of the functionally impaired clones identified a C-terminal residue, W76, as important specifically for Vpu enhancement of virion release. Primary Vpu clones encoding a W76G polymorphism, or site-directed mutants encoding a W76G substitution, were impaired in their ability to enhance virion release, but they were not defective for BST2 surface downregulation. Conversely, the virion release function of impaired primary clones was restored by creating a G76W substitution. The identification of W76 as important for virion release enhancement that is independent of BST2 surface downregulation supports the potential to mechanistically separate these functions of Vpu.

Importance

To establish infection in a host, HIV-1 must evade the host's immune response, including the production of antiviral factors. HIV-1 encodes proteins that antagonize these defenses, including Vpu. Vpu counteracts the host protein BST2, which blocks the release of progeny viruses from the host cell. To determine the importance of Vpu activity to HIV-1 transmission, this study assessed the functionality of Vpu from viruses isolated soon after transmission ("transmitted/founder" viruses) compared to isolates from chronic infection. Although the anti-BST2 activity of Vpu proteins from the tested transmitted/founder viruses did not differ from the activity of the chronic Vpu proteins, the transmitted/founder Vpu proteins trended toward having superior activity against another host protein, CD4. Further, this study identified an amino acid near the C terminus of Vpu that is specifically important for Vpu's ability to enhance the release of progeny virus from the host cell, supporting the notion of a new mechanism for this function of Vpu.

SERINC5 is an inhibitor of retroviral infectivity that is counteracted by viral proteins, including HIV-1 Nef. Inhibition of infectivity by SERINC5 is associated with its incorporation into virions. Nef counteracts this inhibition, presumably by removing SERINC5 from sites of virion assembly at the plasma membrane. While evaluating the virion incorporation of SERINC5, we observed that a relatively high molecular weight form was preferentially present in virions. We used various glycosidases to establish that virion-associated SERINC5 is modified by N-linked, complex glycans, whereas the majority of SERINC5 in cells is of relatively low molecular weight and is modified by high-mannose glycans. Sequence alignment of SERINC family proteins led us to identify a conserved N-glycosylation site, N294, in SERINC5. We mutated this site to evaluate its effect on glycosylation, the restrictive activity of SERINC5, and the sensitivity of SERINC5 to antagonism by Nef. Our results demonstrate that N294 is the major site of N-glycosylation in SERINC5. Although N-glycosylation was required neither for restrictive activity nor for sensitivity to Nef per se, we observed a decrease in the steady-state expression of glycosylation-deficient SERINC5 (the N294A mutant) compared to the wild-type protein. Expression of this mutant was partly restored by treatment of cells with MG132 (a proteasome inhibitor) but not with bafilomycin A1 (a lysosomal inhibitor). We conclude that although not required for restrictive activity or Nef sensitivity, N-linked glycosylation is important for maintaining the steady-state expression of SERINC5 and that nonglycosylated SERINC5 is likely subjected to a quality control mechanism that induces its proteasomal degradation.IMPORTANCE SERINC5 is a member of a family of multipass transmembrane proteins that inhibit the infectivity of retroviruses, including HIV-1. These proteins are incorporated into virions and inhibit infection of target cells unless counteracted by viral antagonists such as HIV-1 Nef. The only other biological function with which these proteins have been associated is the formation of serine-containing membrane lipids. Here we show that SERINC5 is a glycosylated protein and that N-glycosylation is important for its steady-state expression. In the absence of N-glycosylation, SERINC5 is prone to proteasomal degradation. Nonetheless, N-glycosylation per se is required neither for the ability of SERINC5 to inhibit HIV-1 infectivity nor for its sensitivity to antagonism by Nef.

The restriction factor BST2 (tetherin) prevents the release of enveloped viruses from the host cell and is counteracted by HIV-1 Vpu. Vpu and BST2 interact directly via their transmembrane domains. This interaction enables Vpu to induce the surface down-regulation and the degradation of BST2, but neither of these activities fully accounts for the ability of Vpu to enhance virion release. During a study of naturally occurring Vpu proteins, we found that a tryptophan residue near the Vpu C terminus is particularly important for enhancing virion release. Vpu proteins with a W76G polymorphism degraded and down-regulated BST2 from the cell surface, yet they inefficiently stimulated virion release. Here we explore the mechanism of this anomaly. We find that Trp-76 is critical for the ability of Vpu to displace BST2 from sites of viral assembly in the plane of the plasma membrane. This effect does not appear to involve a general reorganization of the membrane microdomains associated with virion assembly, but rather is a specific effect of Vpu on BST2. Using NMR spectroscopy, we find that the cytoplasmic domain of Vpu and Trp-76 specifically interact with lipids. Moreover, paramagnetic relaxation enhancement studies show that Trp-76 inserts into the lipid. These data are consistent with a model whereby Trp-76 anchors the C terminus of the cytoplasmic tail of Vpu to the plasma membrane, enabling the movement of Vpu-bound BST2 away from viral assembly sites.

The mechanisms by which human immunodeficiency virus (HIV) circumvents and coopts cellular machinery to replicate and persist in cells are not fully understood. HIV accessory proteins play key roles in the HIV life cycle by altering host pathways that are often dependent on post-translational modifications (PTMs). Thus, the identification of HIV accessory protein host targets and their PTM status is critical to fully understand how HIV invades, avoids detection and replicates to spread infection. To date, a comprehensive characterization of HIV accessory protein host targets and modulation of their PTM status does not exist. The significant gap in knowledge regarding the identity and PTMs of HIV host targets is due, in part, to technological limitations. Here, we applied current mass spectrometry techniques to define mechanisms of viral protein action by identifying host proteins whose abundance is affected by the accessory protein Vpr and the corresponding modulation of down-stream signaling pathways, specifically those regulated by phosphorylation. By utilizing a novel, inducible HIV-1 CD4+ T-cell model system expressing either the wild type or a vpr-negative viral genome, we overcame challenges associated with synchronization and infection-levels present in other models. We report identification and abundance dynamics of over 7000 proteins and 28,000 phospho-peptides. Consistent with Vpr's ability to impair cell-cycle progression, we observed Vpr-mediated modulation of spindle and centromere proteins, as well as Aurora kinase A and cyclin-dependent kinase 4 (CDK4). Unexpectedly, we observed evidence of Vpr-mediated modulation of the activity of serine/arginine-rich protein-specific kinases (SRPKs), suggesting a possible role for Vpr in the regulation of RNA splicing. This study presents a new experimental system and provides a data-resource that lays the foundation for validating host proteins and phosphorylation-pathways affected by HIV-1 and its accessory protein Vpr.

The cellular protein bone marrow stromal antigen-2 (BST-2)/tetherin acts against a variety of enveloped viruses by restricting their release from the plasma membrane. The HIV-1 accessory protein Vpu counteracts BST-2 by downregulating it from the cell surface and displacing it from virion assembly sites. Previous comparisons of Vpus from transmitted/founder viruses and between viruses isolated during acute and chronic infection led to the identification of a tryptophan at position 76 in Vpu (W76) as a key determinant for the displacement of BST-2 from virion assembly sites. Although present in Vpus from clades B, D, and G, W76 is absent from Vpus from clades A, C, and H. Mutagenesis of the C-terminal region of Vpu from two clade C viruses led to the identification of a conserved LL sequence that is functionally analogous to W76 of clade B. Alanine substitution of these leucines partially impaired virion release. This impairment was even greater when the mutations were combined with mutations of the Vpu β-TrCP binding site, resulting in Vpu proteins that induced high surface levels of BST-2 and reduced the efficiency of virion release to less than that of virus lacking vpu Microscopy confirmed that these C-terminal leucines in clade C Vpu, like W76 in clade B, contribute to virion release by supporting the displacement of BST-2 from virion assembly sites. These results suggest that although encoded differently, the ability of Vpu to displace BST-2 from sites of virion assembly on the plasma membrane is evolutionarily conserved among clade B and C HIV-1 isolates.IMPORTANCE Although targeted by a variety of restriction mechanisms, HIV-1 establishes chronic infection in most cases, in part due to the counteraction of these host defenses by viral accessory proteins. Using conserved motifs, the accessory proteins exploit the cellular machinery to degrade or mistraffic host restriction factors, thereby counteracting them. The Vpu protein counteracts the virion-tethering factor BST-2 in part by displacing it from virion assembly sites along the plasma membrane, but a previously identified determinant of that activity is clade specific at the level of protein sequence and not found in the clade C viruses that dominate the pandemic. Here, we show that clade C Vpu provides this activity via a leucine-containing sequence rather than the tryptophan-containing sequence found in clade B Vpu. This difference seems likely to reflect the different evolutionary paths taken by clade B and clade C HIV-1 in human populations.

Latently infected CD4 T cells form a stable reservoir of HIV that leads to life-long viral persistence; the mechanisms involved in establishment of this latency are not well understood. Three scenarios have been proposed: 1) an activated, proliferating cell becomes infected and reverts back to a resting state; 2) an activated cell becomes infected during its return to resting; or 3) infection is established directly in a resting cell. The aim of this study was, therefore, to investigate the relationship between T cell activation and proliferation and the establishment of HIV latency. Isolated primary CD4 cells were infected at different time points before or after TCR-induced stimulation. Cell proliferation within acutely infected cultures was tracked using CFSE viable dye over 14 days; and cell subsets that underwent varying degrees of proliferation were isolated at end of culture by flow cytometric sorting. Recovered cell subpopulations were analyzed for the amount of integrated HIV DNA, and the ability to produce virus, upon a second round of cell stimulation. We show that cell cultures exposed to virus, prior to stimulus addition, contained the highest levels of integrated and replication-competent provirus after returning to quiescence; whereas, cells infected during the height of cell proliferation retained the least. Cells that did not divide or exhibited limited division, following virus exposure and stimulation contained greater amounts of integrated and inducible HIV than did cells that had divided many times. Based on these results, co-culture experiments were conducted to demonstrate that latent infection could be established directly in non-dividing cells via cell-to-cell transmission from autologous productively infected cells. Together, the findings from our studies implicate the likely importance of direct infection of sub-optimally activated T cells in establishment of latently infected reservoirs in vivo, especially in CD4 lymphocytes that surround productive viral foci within immune tissue microenvironments.

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HIV-1 Vpu decreases the exposure of epitopes within the viral envelope glycoprotein (Env) on the surface of infected cells by downregulating both BST2 and CD4. To test the hypothesis that inhibiting Vpu activity would increase the exposure of these epitopes and sensitize infected cells to antibody-dependent cellular cytotoxicity (ADCC), we treated cells with the Nedd8 activation enzyme (NAE) inhibitor MLN4924, which inhibits the cullin1-based ubiquitin ligase complex coopted by Vpu to degrade cellular targets. Treatment of HeLa cells with MLN4924 or expression of a dominant negative mutant of cullin1 inhibited the Vpu-mediated downregulation of CD4 but not the downregulation of BST2. NAE inhibition also increased the surface exposure of CD4-induced epitopes within Env on HEK293 cells containing an inducible HIV genome, on infected CEM T cells, and on infected primary T cells. In contrast, the Vpu-mediated downregulation of BST2 was substantially inhibited by MLN4924 only when T cells were treated with alpha interferon (IFN-α) to induce high levels of BST2 expression. As reported previously, the absence of vpu or nef and even more so the combined absence of these two genes sensitized infected cells to ADCC. However, NAE inhibition affected ADCC minimally. Paradoxically, even in infected, IFN-treated cells in which NAE inhibition substantially rescued the surface level of BST2, the surface level of Env detected with an antibody recognizing a CD4-independent epitope (2G12) was minimally increased. Mutation of the C-terminal Vpu residue W76, which supports the ability of Vpu to stimulate virion release by displacing BST2 from assembly sites on the plasma membrane by a cullin1-independent mechanism, increased the exposure of Env detected by 2G12 on infected T cells. Thus, inhibiting the displacement function of Vpu together with its ability to degrade CD4 and BST2 may be required to sensitize infected cells to ADCC.

Importance

Pathogenic viruses encode gene products that enable evasion of host immune surveillance mechanisms. One such mechanism is antibody-dependent cellular cytotoxicity (ADCC), whereby host antibodies bind envelope glycoproteins of the virus that are inserted into the cellular membrane and direct the destruction of infected cells. Targeting pharmacologically the activity of HIV-1 Vpu, which contributes to evasion of ADCC, could potentially sensitize infected cells to this immune surveillance mechanism, an outcome that would have therapeutic implications with respect to the goal of curing HIV-1 infection. The Nedd8 activation enzyme inhibitor MLN4924 blocks the activity of the host ubiquitin ligase that Vpu coopts to direct the degradation of CD4 and BST2. We observed that while MLN4924 partially reverses the activity of Vpu and could become part of a therapeutic approach by virtue of CD4-induced epitope exposure, sufficient Vpu activity as an antagonist of BST2 persists despite this drug to allow escape from ADCC.

Quantification of cell-associated replication-competent HIV, in blood samples from patients with undetectable plasma viremia, requires specialized culture conditions that include exogenous pan T cell stimulation. Different research groups have used several stimuli for this purpose; however, the relative efficacies of these T cell stimuli to induce productive HIV replication from latently infected cells ex vivo have not been systematically evaluated. To this end, we compared four commonly used T cell stimuli: 1) irradiated allogeneic cells plus phytohaemagglutinin (PHA); 2) PHA alone; 3) phorbol myristate acetate plus Ionomycin; and 4) immobilized αCD3 plus αCD28 antibodies. End-point dilutions of patient CD4 T cells were performed, using virion RNA production to quantify HIV induction. Our results demonstrated that these activation approaches were not equivalent and that antibody cross-linking of CD3 and CD28 membrane receptors was the most effective means to activate HIV replication from a resting cell state, closely followed by stimulation with irradiated allogeneic cells plus PHA.

As systems biology approaches to virology have become more tractable, highly studied viruses such as HIV can now be analyzed in new unbiased ways, including spatial proteomics. We employed here a differential centrifugation protocol to fractionate Jurkat T cells for proteomic analysis by mass spectrometry; these cells contain inducible HIV-1 genomes, enabling us to look for changes in the spatial proteome induced by viral gene expression. Using these proteomics data, we evaluated the merits of several reported machine learning pipelines for classification of the spatial proteome and identification of protein translocations. From these analyses, we found that classifier performance in this system was organelle dependent, with Bayesian t-augmented Gaussian mixture modeling outperforming support vector machine learning for mitochondrial and endoplasmic reticulum proteins but underperforming on cytosolic, nuclear, and plasma membrane proteins by QSep analysis. We also observed a generally higher performance for protein translocation identification using a Bayesian model, Bayesian analysis of differential localization experiments, on row-normalized data. Comparative Bayesian analysis of differential localization experiment analysis of cells induced to express the WT viral genome versus cells induced to express a genome unable to express the accessory protein Nef identified known Nef-dependent interactors such as T-cell receptor signaling components and coatomer complex. Finally, we found that support vector machine classification showed higher consistency and was less sensitive to HIV-dependent noise. These findings illustrate important considerations for studies of the spatial proteome following viral infection or viral gene expression and provide a reference for future studies of HIV-gene-dropout viruses.