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The HIV-1 accessory protein Vpr displays various activities that can favor viral replication such as G 2 cell cycle arrest. Vpr also modulates host gene expression, although this property is poorly characterized. Here, we investigated the effect of Vpr on L-selectin (CD62L), which crucially controls leukocytes circulation and generation of immune responses against pathogens. We report that Vpr up-regulates CD62L mRNA level when individually expressed in Jurkat T cells as well as during HIV-1 infection of primary CD4 + T cells. Vpr mutant analysis and use of inhibitors suggest that the effect of Vpr on CD62L occurs independently of G 2 arrest but requires activation of the ATR kinase. Yet, induction of CD62L expression by Vpr is contrasted by down-regulation of CD62L protein by Nef that, together with Vpu, induces a net reduction of cell-surface CD62L on HIV-1-infected cells, which may impact viral spread and evasion of immune responses., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Treatment of HIV-infected patients with IFN-α results in significant, but clinically insufficient, reductions of viremia. IFN induces the expression of several antiviral proteins including BST-2, which inhibits HIV by multiple mechanisms. The viral protein Vpu counteracts different effects of BST-2. We thus asked if Vpu proteins from IFN-treated patients displayed improved anti-BST-2 activities as compared to Vpu from baseline. Deep-sequencing analyses revealed that in five of seven patients treated by IFN-α for a concomitant HCV infection in the absence of antiretroviral drugs, the dominant Vpu sequences differed before and during treatment. In three patients, vpu alleles that emerged during treatment improved virus replication in the presence of IFN-α, and two of them conferred improved virus budding from cells expressing BST-2. Differences were observed for the ability to down-regulate CD4, while all Vpu variants potently down-modulated BST-2 from the cell surface. This report discloses relevant consequences of IFN-treatment on HIV properties., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Heterosexual HIV-1 transmission has been identified as a genetic bottleneck and a single transmitted/founder (T/F) variant with reduced sensitivity to type I interferon initiates productive infection in most cases. We hypothesized that particularly active accessory protein(s) may confer T/F viruses with a selective advantage in establishing HIV infection. Thus, we tested vpu, vif and nef alleles from six T/F and six chronic (CC) viruses in assays for 9 immune evasion activities involving the counteraction of interferon-stimulated genes and modulation of ligands known to activate innate immune cells. All functions were highly conserved with no significant differences between T/F and CC viruses, suggesting that these accessory protein functions are important throughout the course of infection., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

HIV-1 NL4-3 Vpu induces downregulation of cell surface CD155, a ligand for the DNAM-1 activating receptor of NK and CD8(+) T cells, to evade NK cell mediated immune response. Here we show that the conserved alanine residues at positions 10, 14 and 18 in the TM domain of Vpu are required for the efficient downregulation of cell surface CD155. In contrast, the CK-2 phosphorylation sites and the second α-helix in the cytoplasmic Vpu domain have no influence on the surface expression of CD155. Thus, compared to Vpu׳s effect on CD4, NTB-A and tetherin, the Vpu mediated downregulation of CD155 is an independent Vpu function. We finally show that in contrast to other lentiviral strains, only Vpu and Nef from HIV-1 M NL4-3 potently interfere with CD155 surface expression. Thus, Vpu seems to subvert NK cell responses against HIV-1 infected T cells by modulation of receptors necessary for NK cell activation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Astrocytes protect neurons but also evoke a proinflammatory response to injury and viral infections including HIV. We investigated the mechanism of HIV-1 infection in primary astrocytes, which showed minimal but productive viral infection independent of CXCR4. As with ectopic-CD4-expressing astrocytes, lysosomotropic agents led to increased HIV-1 infection in wild-type but not Rabs 5, 7, and 11-ablated astrocytes. Instead, HIV-1 infection was decreased in Rab-depleted astrocytes, corroborating viral entry by endocytosis. HIV-1 produced persistent infection in astrocytes (160 days); no evidence of latent infection was seen. Notably, one caveat is that endosomal modifiers enhanced wild-type HIV-1 infection (M- and T-tropic) in astrocytes, suggesting endocytic entry of the virus. Impeding endocytosis by inhibition of Rab 5, 7 or 11 will inhibit HIV infection in astrocytes. Although the contribution of such low-level infection in astrocytes to neurological complications is unclear, it may serve as an elusive viral reservoir in the central nervous system., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A major challenge for developing effective treatments for HIV-1 is the viruses' ability to generate new variants. Inter-strain recombination is a major contributor to this high evolutionary rate, since at least 20% of viruses are observed to be recombinant. However, the patterns of recombination vary across the viral genome. A number of factors influence recombination, including sequence identity and secondary RNA structure. In addition the recombinant genome must code for a functional virus, and expressed proteins must fold to stable and functional structures. Any intragenic recombination that disrupts internal residue contacts may therefore produce an unfolded protein. Here we find that contact maps based on protein structures predict recombination breakpoints observed in the HIV-1 pandemic. Moreover, many pairs of contacting residues that are unlikely to be disrupted by recombination are coevolving. We conclude that purifying selection arising from protein structure and intramolecular coevolutionary changes shapes the observed patterns of recombination in HIV-1., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We have undertaken a genetic strategy to map Vpu regions necessary for BST-2 antagonism and viral egress. This approach is based on our identification of an egress-defective Vpu variant encoded by an HIV-1 subtype C strain. We constructed a series of chimeric Vpu molecules made from the Vpu C variant and Vpu B from a standard laboratory strain. The TM domain from the inactive Vpu C, which contains multiple non-conserved residues, was responsible for a significant decrease in egress activity and BST-2 downregulation, confirming the functional importance of the Vpu TM domain. However, for complete inactivation, both the N-terminus and TM domain from the inactive Vpu C molecule were required, suggesting a new role for the Vpu N-terminus. In addition, determinants in the C-terminus of Vpu B that may be involved in efficient TGN accumulation were also necessary for enhanced viral egress but are missing or non-functional in Vpu C., (Copyright © 2013 Elsevier Inc. All rights reserved.)

HIV-1 Vpu induces downregulation of cell surface NTB-A to evade lysis of HIV-1 infected cells by NK cells. Here we show that Vpu affects the anterograde transport and the glycosylation pattern of NTB-A by a mechanism that is distinct from the Vpu induced downregulation of CD4 and tetherin. In the presence of Vpu, only the high mannose form of NTB-A was detectable, suggesting that Vpu prevented the formation of the mature form of NTB-A. This phenomenon is associated with the ability of Vpu to downregulate cell surface NTB-A by retention of NTB-A within the Golgi-compartment. Furthermore, the Vpu-mediated effect on NTB-A glycosylation is highly conserved among Vpu proteins derived from HIV-1 and SIV and corresponds to the level of downregulation of NTB-A. Together, these results suggest that the reduction of NTB-A from the cell surface is associated with the Vpu-mediated effect on the glycosylation pattern of newly synthesized NTB-A molecules., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Previously, we reported that simian-human immunodeficiency viruses expressing either the lab adapted subtype B (SHIV(KU-1bMC33)) or subtype C (SHIV(SCVpu)) Vpu proteins of human immunodeficiency virus type 1 (HIV-1) had different rates of CD4(+) T cell loss following inoculation into macaques. In this study, we have generated SHIVs that express either the subtype B or subtype C N-terminal (NTD) and transmembrane (TMD) domains and the opposing cytoplasmic domain (SHIV(VpuBC), SHIV(VpuCB)). In culture systems, SHIV(VpuBC) replicated faster than SHIV(VpuCB) while both proteins exhibited similar ability to down-modulate CD4 surface expression. Following inoculation into macaques, SHIV(VpuBC) resulted in rapid CD4(+) T cell loss similar to the parental SHIV(KU-1bMC33), while the rate of CD4(+) T cell loss in those inoculated with SHIV(VpuCB) was intermediate of SHIV(SCVpu) and SHIV(KU-1bMC33). These results emphasize the importance of the Vpu NTD/TMD region in the rate of CD4(+) T cell loss in the pathogenic X4 SHIV/macaque model., (Copyright © 2012 Elsevier Inc. All rights reserved.)

HIV-1 Vpu antagonizes the block to particle release mediated by CD317 (BST-2/HM1.24/Tetherin) via incompletely understood mechanisms. Vpu and CD317 partially reside in cholesterol-rich lipid rafts where HIV-1 budding preferentially occurs. Here we find that lipid raft association of ectopically expressed or endogenous CD317 was unaltered upon co-expression with Vpu or following HIV-1 infection. Similarly, Vpu's lipid raft association remained unchanged upon expression of CD317. We identify amino acids V25 and Y29 of Vpu as crucial for microdomain partitioning and single substitution of these amino acids resulted in Vpu variants with markedly reduced or undetectable lipid raft association. These mutations did not affect Vpu's subcellular distribution and binding capacity to CD317, nor its ability to downmodulate cell surface CD317 and promote HIV-1 release from CD317-positive cells. We conclude that (i) lipid raft incorporation is dispensable for Vpu-mediated CD317 antagonism and (ii) Vpu does not antagonize CD317 by extraction from lipid rafts., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Tetherin is a host cell restriction factor that acts against HIV-1 and other enveloped viruses. The antiviral activity of tetherin is antagonized by the HIV-1 protein Vpu, that downregulates tetherin from the cell surface. Here, we report the specific detection of cell surface tetherin levels in primary activated CD4(+) T-cells and in CD4(+) T-cell lines. Differences were observed regarding tetherin cell surface expression, Vpu-mediated tetherin downmodulation and promotion of virus release. However, Vpu expression in all T-cell lines resulted in a 2-fold increase in numbers of infected cells after three days. This implies a Vpu-mediated effect in ongoing infection and possibly in cell-to-cell viral spread that is independent of the extent of Vpu-mediated tetherin cell surface downmodulation. Endogenous cell surface tetherin levels in T-cell lines were also downmodulated following infection with Vpu-deleted virus, suggesting an additional Vpu-independent mechanism of tetherin cell surface downmodulation following HIV-1 infection in T-cell lines., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Based on structural information, we have analyzed the mechanism of mature HIV-1 core assembly and the contributions of structural elements to the assembly process. Through the use of several in vitro assembly assay systems, we have examined details of how capsid (CA) protein helix 1, ß-hairpin and cyclophilin loop elements impact assembly-dependent protein interactions, and we present evidence for a contribution of CA helix 6 to the mature assembly-competent conformation of CA. Additional experiments with mixtures of proteins in assembly reactions provide novel analyses of the mature core assembly mechanism. Our results support a model in which initial assembly products serve as scaffolds for further assembly by converting incoming subunits to assembly proficient conformations, while mutant subunits increase the probability of assembly termination events., (Copyright © 2011 Elsevier Inc. All rights reserved.)

While the C-terminal domain of HIV-1 Vpu is critical for CD4 degradation, the transmembrane domain (TM) mediates ion channel activity, enhances virus release and is essential for counteracting CD317/Bst-2/Tetherin. Here we analyzed whether the ion channel activity of Vpu is required to antagonize CD317-mediated restriction of virion release. We examined TM-mutants of three conserved residues: the S23A mutation, which was previously shown to abrogate ion channel function, did not affect Vpu mediated augmentation of virus release. In contrast, the A14N and A18N mutation did not affect ion channel activity of Vpu, but substantially reduced its ability to support virus release and to down-regulate CD317 from the cell surface. Altogether, our data suggest that not the ion channel activity of Vpu, but its ability to remove CD317 from the cell surface is required to augment HIV-1 release., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Recent evidence suggests that transmembrane domain (TMD) interactions are essential for HIV-1 Vpu-mediated antagonism of the restriction factor BST-2/tetherin. We made Vpu TMD mutants to study the mechanism of BST-2 antagonism. Vpu-I17A, -A18F, -W22L, and -S23L co-localized with BST-2 within endosomal membranes while effectively enhancing virion release and down-regulating surface BST-2. However, Vpu-A18H was confined to an endoplasmic reticulum (ER)-like distribution, resulting in impaired down-regulation of BST-2 and reduced virion release. Brefeldin A confined wild type Vpu to the ER, resulting in a similarly impaired phenotype, as did the addition of a C-terminal ER-retention signal to Vpu. We determined the half-life of cell-surface BST-2 to be ~8 hours, whereas Vpu mediated an ~80% reduction of surface BST-2 within 6 hours, suggesting that TMD interactions between Vpu and BST-2 occur within post-ER membranes to directly and rapidly remove BST-2 from the cell surface and relieve restricted virion release., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The Vpu protein of human immunodeficiency virus type 1 (HIV-1) is known to enhance virion release from certain cell types. To accomplish this function, Vpu interacts with the restriction factor known as bone marrow stromal cell antigen 2 (BST-2)/tetherin. In this study, we analyzed whether the Vpu protein is associated with microdomains known as lipid or membrane rafts. Our results indicate that Vpu partially partitions into detergent-resistant membrane (DRM) fractions when expressed alone or in the context of simian-human immunodeficiency virus (SHIV) infection. The ability to be partitioned into rafts was observed with both subtype B and C Vpu proteins. The use of cholesterol lowering lovastatin/M-β-cyclodextrin and co-patching experiments confirmed that Vpu can be detected in cholesterol rich regions of membranes. Finally, we present data showing that raft association-defective transmembrane mutants of Vpu have impaired enhanced virus release function, but still maintain the ability to down-regulate CD4., (Copyright © 2010. Published by Elsevier Inc.)

Vpu enhances HIV-1 release by suppressing CD317-mediated tethering of virions to the cell surface. In HeLa H1 cells, Vpu(+) infection produces significant amounts of tethered virions, although efficient virus release requires Vpu. We have analyzed membrane targeting and assembly of newly synthesized Gag in infected HeLa H1 cells by quantitative pulse-chase assays in both Vpu(+) and ΔVpu virus backgrounds and in the presence and absence of CD317. Our results show that formation of tethered virions precedes release of viruses to the culture medium in the Vpu(+) infection, and CD317 knockdown reduces tethering in both Vpu(+) and ΔVpu virus backgrounds. Significantly, our results indicate that tethered Vpu(+) viruses represent precursors for extracellular viruses, and Vpu thus appears to reverse tethering in HeLa H1 cells after budding., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Pathogenic simian-human immunodeficiency viruses (SHIV) contain HIV-1 Vpu and SIV Nef, both shown to counteract BST-2 (HM1.24; CD317; tetherin) inhibition of virus release in a species-specific manner. We show that human and pig-tailed BST-2 (ptBST-2) restrict SHIV. We found that sequential "humanization" of the transmembrane domain (TMD) of the pig-tailed BST-2 (ptBST-2) protein resulted in a fluctuation in sensitivity to HIV-1 Vpu. Our results also show that the length of the TMD in human and ptBST-2 proteins is important for BST-2 restriction and susceptibility to Vpu. Taken together, our results emphasize the importance of tertiary structure in BST-2 antagonism and suggests that the HIV-1 Vpu transmembrane domain may have additional functions in vivo unrelated to BST-2 antagonism., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The cytoplasmic tail domain (CTD) of retroviral envelope (Env) proteins has been implicated in modulating Env incorporation into viral particles. We generated a panel of murine leukemia virus (MLV) Env mutants and analyzed their ability to be recruited to human immunodeficiency virus-1 (HIV-1) assembly sites. Surprisingly, the entire CTD was dispensable for recruitment to assembly sites, but a mutation that disrupted the furin cleavage site in Env abolished recruitment. To determine if MLV Env can show selectivity for homologous assembly sites, cells were co-transfected with both HIV-1 and MLV assembly components along with each MLV Env construct and assayed for infectious particle production. MLV Env selectively formed infectious particles with the MLV components at the expense of infectious HIV-1 infectious particle production, but truncation of the CTD progressively reduced this selectivity. Collectively these data suggest that there are two separable mechanisms that govern MLV Env recruitment to viral assembly sites., (Copyright 2010 Elsevier Inc. All rights reserved.)

Functional studies of HIV-1 envelope glycoproteins (Envs) commonly include the generation of pseudoviruses, which are produced by co-transfection of rev-vpu-env cassettes with an env-deficient provirus. Here, we describe six Env constructs from transmitted/founder HIV-1 that were defective in the pseudotyping assay, although two produced infectious virions when expressed from their cognate proviruses. All of these constructs exhibited an unusual gene arrangement in which the first exon of rev (rev1) and vpu were in the same reading frame without an intervening stop codon. Disruption of the rev1-vpu fusion gene by frameshift mutation, stop codon, or abrogation of the rev initiation codon restored pseudovirion infectivity. Introduction of the fusion gene into wildtype Env cassettes severely compromised their function. The defect was not due to altered env and rev transcription or a dominant negative effect of the expressed fusion protein, but seemed to be caused by inefficient translation at the env initiation codon. Although the rev1-vpu polymorphism affects Env expression only in vitro, it can cause problems in studies requiring Env complementation, such as analyses of co-receptor usage and neutralization properties, since 3% of subtype A, 20% of subtype C and 5% of CRF01_A/E viruses encode the fusion gene. A solution is to eliminate the rev initiation codon when amplifying rev-vpu-env cassettes since this increases Env expression irrespective of the presence of the polymorphism., (Copyright 2009 Elsevier Inc. All rights reserved.)

Previously, we showed that the Vpu protein from HIV-1 subtype C is more efficiently transported to the cell surface than the well studied subtype B Vpu (Pacyniak et al., 2005) and that a SHIV expressing the subtype C Vpu exhibited a decreased rate of CD4+ T cell loss following inoculation in macaques (Hill et al., 2008). In this study, we examined the role of overlapping tyrosine-based (YXXPhi) and dileucine-based ([D/E]XXXL[L/I]) motifs in the membrane proximal region of the subtype C Vpu (EYRKLL) in Vpu intracellular transport, CD4 surface expression and virus release from the cell surface. We constructed three site-directed mutants of the subtype C vpu and fused these genes to the gene for enhanced green fluorescent protein (EGFP). The first mutation made altered the tyrosine (EARKLL; VpuSCEGFPY35A), the second altered the dileucine motif (EYRKLG; VpuSCEGFPL39G), and the third contained both amino acid substitutions (EARKLG; VpuSCEGFPYL35,39AG) in this region of the Vpu protein. The VpuSCEGFPY35A protein was transported to the cell surface similar to the unmodified VpuSCEGFP1 while VpuSCEGFPL39G was expressed at the cell surface at significantly reduced levels. The VpuSCEGFPYL35,39AG was found to have an intermediate level of cell surface expression. All three mutant Vpu proteins were analyzed for the ability to prevent cell surface expression of CD4. We found that both single mutants did not significantly effect CD4 surface expression while the double mutant (VpuSCEGFPYL35,39AG) was significantly less efficient at preventing cell surface CD4 expression. Chimeric simian human immunodeficiency viruses were constructed with these mutations in vpu (SHIVSCVpuY35A, SHIVSCVpuL39G and SHIVSCVpuYL35,39AG). Our results indicate that SHIVSCVpuL39G replicated much more efficiently and was much more cytopathic than SHIVSCVpu. In contrast, SHIVSCVpuY35A and SHIVSCVpuYL35,39AG replicated less efficiently when compared to the parental SHIVSCVpu. Taken together, these results show for the first time that the membrane proximal tyrosine-based sorting motif in the cytoplasmic domain of Vpu is essential for efficient virus release. These results also indicate that the dileucine-based sorting motif affects the intracellular trafficking of subtype C Vpu proteins, virus replication, and release.

Retroviruses require integration of their RNA genomes for both stability and productive viral replication. In HIV infection of non-dividing, resting CD4 T cells, where integration is greatly impeded, the reverse transcribed HIV DNA has limited biological activity and a short half-life. In metabolically active and proliferating T cells, unintegrated DNA rapidly diminishes with cell division. HIV also infects the non-dividing but metabolically active macrophage population. In an in vitro examination of HIV infection of macrophages, we find that unintegrated viral DNA not only has an unusual stability, but also maintains biological activity. The unintegrated linear DNA, 1-LTR, and 2-LTR circles are stable for at least 30 days. Additionally, there is persistent viral gene transcription, which is selective and skewed towards viral early genes such as nef and tat with highly diminished rev and vif. One viral early gene product Nef was measurably synthesized. We also find that independent of integration, the HIV infection process in macrophages leads to generation of numerous chemokines.

RNA helicases are a large family of proteins that are able to unwind RNA duplex and remodel the structure of RNA-protein (RNP) complexes using energy derived from hydrolysis of nucleotide triphosphates (NTPs). Every step of cellular RNA metabolism involves the activity of RNA helicases. Not surprisingly, more and more RNA helicases are reported to participate in the replication of viruses including the human immunodeficiency virus type 1 (HIV-1). Here, we provide evidence that overexpression of an RNA helicase named DHX30 enhances HIV-1 gene expression, but leads to the generation of viruses that package significantly low levels of viral RNA and exhibit severely decreased infectivity. These data reveal the complex roles of DHX30 in HIV-1 replication and implicate an inhibitory activity of DHX30 in HIV-1 RNA packaging.