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Vif is a lentiviral accessory protein that counteracts the antiviral activity of cellular APOBEC3 (A3) cytidine deaminases in infected cells. The exact contribution of each member of the A3 family for the restriction of HIV-2 is still unclear. Thus, the aim of this work was to identify the A3s with anti-HIV-2 activity and compare their restriction potential for HIV-2 and HIV-1. We found that A3G is a strong restriction factor of both types of viruses and A3C restricts neither HIV-1 nor HIV-2. Importantly, A3B exhibited potent antiviral activity against HIV-2, but its effect was negligible against HIV-1. Whereas A3B is packaged with similar efficiency into both viruses in the absence of Vif, HIV-2 and HIV-1 differ in their sensitivity to A3B. HIV-2 Vif targets A3B by reducing its cellular levels and inhibiting its packaging into virions, whereas HIV-1 Vif did not evolve to antagonize A3B. Our observations support the hypothesis that during wild-type HIV-1 and HIV-2 infections, both viruses are able to replicate in host cells expressing A3B but using different mechanisms, probably resulting from a Vif functional adaptation over evolutionary time. Our findings provide new insights into the differences between Vif protein and their cellular partners in the two human viruses. Of note, A3B is highly expressed in some cancer cells and may cause deamination-induced mutations in these cancers. Thus, A3B may represent an important therapeutic target. As such, the ability of HIV-2 Vif to induce A3B degradation could be an effective tool for cancer therapy. IMPORTANCE Primate lentiviruses encode a series of accessory genes that facilitate virus adaptation to its host. Among those, the vif-encoded protein functions primarily by targeting the APOBEC3 (A3) family of cytidine deaminases. All lentiviral Vif proteins have the ability to antagonize A3G; however, antagonizing other members of the A3 family is variable. Here, we report that HIV-2 Vif, unlike HIV-1 Vif, can induce degradation of A3B. Consequently, HIV-2 Vif but not HIV-1 Vif can inhibit the packaging of A3B. Interestingly, while A3B is packaged efficiently into the core of both HIV-1 and HIV-2 virions in the absence of Vif, it only affects the infectivity of HIV-2 particles. Thus, HIV-1 and HIV-2 have evolved two distinct mechanisms to antagonize the antiviral activity of A3B. Aside from its antiviral activity, A3B has been associated with mutations in some cancers. Degradation of A3B by HIV-2 Vif may be useful for cancer therapies.

Tetherin/BST-2 is an antiviral protein that blocks the release of enveloped viral particles by linking them to the membrane of producing cells. At first, BST-2 genes were described only in humans and other mammals. Recent work identified BST-2 orthologs in nonmammalian vertebrates, including birds. Here, we identify the BST-2 sequence in domestic chicken (Gallus gallus) for the first time and demonstrate its activity against avian sarcoma and leukosis virus (ASLV). We generated a BST-2 knockout in chicken cells and showed that BST-2 is a major determinant of an interferon-induced block of ASLV release. Ectopic expression of chicken BST-2 blocks the release of ASLV in chicken cells and of human immunodeficiency virus type 1 (HIV-1) in human cells. Using metabolic labeling and pulse-chase analysis of HIV-1 Gag proteins, we verified that chicken BST-2 blocks the virus at the release stage. Furthermore, we describe BST-2 orthologs in multiple avian species from 12 avian orders. Previously, some of these species were reported to lack BST-2, highlighting the difficulty of identifying sequences of this extremely variable gene. We analyzed BST-2 genes in the avian orders Galliformes and Passeriformes and showed that they evolve under positive selection. This indicates that avian BST-2 is involved in host-virus evolutionary arms races and suggests that BST-2 antagonists exist in some avian viruses. In summary, we show that chicken BST-2 has the potential to act as a restriction factor against ASLV. Characterizing the interaction of avian BST-2 with avian viruses is important in understanding innate antiviral defenses in birds. IMPORTANCE Birds are important hosts of viruses that have the potential to cause zoonotic infections in humans. However, only a few antiviral genes (called viral restriction factors) have been described in birds, mostly because birds lack counterparts of highly studied mammalian restriction factors. Tetherin/BST-2 is a restriction factor, originally described in humans, that blocks the release of newly formed virus particles from infected cells. Recent work identified BST-2 in nonmammalian vertebrate species, including birds. Here, we report the BST-2 sequence in domestic chicken and describe its antiviral activity against a prototypical avian retrovirus, avian sarcoma and leukosis virus (ASLV). We also identify BST-2 genes in multiple avian species and show that they evolve rapidly in birds, which is an important indication of their relevance for antiviral defense. Analysis of avian BST-2 genes will shed light on defense mechanisms against avian viral pathogens.

SUMMARY Human mannose receptor 1 (hMRC1) is expressed on the surface of most tissue macrophages, dendritic cells, and select lymphatic or liver endothelial cells. HMRC1 contributes to the binding of HIV-1 to monocyte-derived macrophages (MDMs) and is involved in the endocytic uptake of HIV-1 into these cells. Here, we identify hMRC1 as an antiviral factor that inhibits virus release through a bone marrow stromal antigen 2 (BST-2)-like mechanism. Virions produced in the presence of hMRC1 accumulated in clusters at the cell surface but were fully infectious. HIV-1 counteracted the effect by transcriptional silencing of hMRC1. The effect of hMRC1 was not virus isolate specific. Surprisingly, deletion of the Env protein, which is known to interact with hMRC1, did not relieve the hMRC1 antiviral activity, suggesting the involvement of additional cellular factor(s) in the process. Our data reveal an antiviral mechanism that is active in primary human macrophages and is counteracted by HIV-1 through downregulation of hMRC1., In Brief hMRC1 is a surface receptor in macrophages that binds glycoproteins and contributes to innate immunity. Sukegawa et al. report that hMRC1 inhibits detachment of mature and infectious HIV-1 virions from the surface of infected cells. The hMRC1-imposed restriction of virus release is similar to, but independent of, the BST-2/tetherin-imposed restriction.

APOBEC3G (A3G) is a cytidine deaminase with potent antiviral activity that is antagonized by Vif. A3G is expressed in a cell type-specific manner and some semi-permissive cells, including A3.01, express A3G but fail to block replication of Vif-null HIV-1. Here we explored the semi-permissive nature of A3.01 cells and found it to be defined exclusively by the levels of A3G. Indeed, minor changes in A3G levels rendered A3.01 cells either fully permissive or non-permissive for Vif-null HIV-1. Our data indicate that A3.01 cells express sub-lethal levels of catalytically active A3G that affects Vif-null HIV-1 at the proviral level but does not completely block virus replication due to purifying selection. Attempts to use the selective pressure exerted by such sub-lethal levels of A3G to select for APOBEC-resistant Vif-null virus capable of replicating in H9 cells failed despite passaging virus for five months, demonstrating that Vif is a critical viral accessory protein.

Macrophages are one of the first and also a major site of filovirus replication and, in addition, are a source of multiple cytokines, presumed to play a critical role in the pathogenesis of the viral infection. Some of these cytokines are known to induce macrophage phenotypic changes in vitro, but how macrophage polarization may affect the cell susceptibility to filovirus entry remains largely unstudied. We generated different macrophage subsets using cytokine pre-treatment and subsequently tested their ability to fuse with beta-lactamase containing virus-like particles (VLP), pseudotyped with the surface glycoprotein of Ebola virus (EBOV) or the glycoproteins of other clinically relevant filovirus species. We found that pre-incubation of primary human monocyte-derived macrophages (MDM) with interleukin-10 (IL-10) significantly enhanced filovirus entry into cells obtained from multiple healthy donors, and the IL-10 effect was preserved in the presence of pro-inflammatory cytokines found to be elevated during EBOV disease. In contrast, fusion of IL-10-treated macrophages with influenza hemagglutinin/neuraminidase pseudotyped VLPs was unchanged or slightly reduced. Importantly, our in vitro data showing enhanced virus entry are consistent with the correlation established between elevated serum IL-10 and increased mortality in filovirus infected patients and also reveal a novel mechanism that may account for the IL-10-mediated increase in filovirus pathogenicity.

SAMHD1 is a cellular dNTPase that restricts lentiviral infection presumably by lowering cellular dNTP levels to below a critical threshold required for reverse transcription; however, lowering cellular dNTP levels may not be the sole mechanism of restriction. In particular, an exonuclease activity of SAMHD1 was reported to contribute to virus restriction. We further investigated the requirements for SAMHD1 restriction activity in both differentiated U937 and cycling HeLa cells. Using hydroxyurea treatment to lower baseline dNTP levels in HeLa cells, we were able to document efficient SAMHD1 restriction without significant further reduction in dNTP levels by SAMHD1. These results argue against a requirement for additional myeloid-specific host factors for SAMHD1 function but further support the notion that SAMHD1 possesses an additional dNTP-independent function contributing to lentiviral restriction. However, our own experiments failed to associate this presumed additional SAMHD1 antiviral activity with a reported nuclease activity.

Vpr is a lentiviral accessory protein that is expressed late during the infection cycle and is packaged in significant quantities into virus particles through a specific interaction with the P6 domain of the viral Gag precursor. Characterization of the physiologically relevant function(s) of Vpr has been hampered by the fact that in many cell lines, deletion of Vpr does not significantly affect viral fitness. However, Vpr is critical for virus replication in primary macrophages and for viral pathogenesis in vivo. It is generally accepted that Vpr does not have a specific enzymatic activity but functions as a molecular adapter to modulate viral or cellular processes for the benefit of the virus. Indeed, many Vpr interacting factors have been described by now, and the goal of this review is to summarize our current knowledge of cellular proteins targeted by Vpr.

Background Gene knockouts are a common tool used to study gene function in various organisms. However, designing gene knockouts is complicated in viruses, which frequently contain sequences that code for multiple overlapping genes. Designing mutants that can be traced by the creation of new or elimination of existing restriction sites further compounds the difficulty in experimental design of knockouts of overlapping genes. While software is available to rapidly identify restriction sites in a given nucleotide sequence, no existing software addresses experimental design of mutations involving multiple overlapping amino acid sequences in generating gene knockouts. Results Pyviko performed well on a test set of over 240,000 gene pairs collected from viral genomes deposited in the National Center for Biotechnology Information Nucleotide database, identifying a point mutation which added a premature stop codon within the first 20 codons of the target gene in 93.2% of all tested gene-overprinted gene pairs. This shows that Pyviko can be used successfully in a wide variety of contexts to facilitate the molecular cloning and study of viral overprinted genes. Conclusions Pyviko is an extensible and intuitive Python tool for designing knockouts of overlapping genes. Freely available as both a Python package and a web-based interface (http://louiejtaylor.github.io/pyViKO/), Pyviko simplifies the experimental design of gene knockouts in complex viruses with overlapping genes. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0920-3) contains supplementary material, which is available to authorized users.

BST-2/CD317/HM1.24/tetherin is a B-cell antigen overexpressed on the surface of myeloma cell lines and on neoplastic plasma cells of patients with multiple myeloma. Antibodies to BST-2 are in clinical trial for the treatment of multiple myeloma and are considered for the treatment of solid tumors with high BST-2 antigen levels. Functionally, BST-2 restricts the secretion of retroviruses, including human immunodeficiency virus type 1, as well as members of the herpesvirus, filovirus, and arenavirus families, presumably by tethering nascent virions to the cell surface. Here we report that BST-2 antibody treatment facilitates virus release from BST-2 + cells by interfering with the tethering activity of BST-2. BST-2 antibodies were unable to release already tethered virions and were most effective when added early during virus production. BST-2 antibody treatment did not affect BST-2 dimerization and did not reduce the cell surface expression of BST-2. Interestingly, BST-2 antibody treatment reduced the nonspecific shedding of BST-2 and limited the encapsidation of BST-2 into virions. Finally, flotation analyses indicate that BST-2 antibodies affect the distribution of BST-2 within membrane rafts. Our data suggest that BST-2 antibody treatment may enhance virus release by inducing a redistribution of BST-2 at the cell surface, thus preventing it from accumulating at the sites of virus budding.

Viruses face a variety of obstacles when infecting a new host. The past few years have brought exciting new insights into the function of restriction factors, which form part of the host's innate immune system. One of the most recently identified restriction factors is bone marrow stromal antigen 2 (BST-2)/tetherin. BST-2 is an interferon-inducible gene whose expression dramatically reduces the release of viruses from infected cells. This effect of BST-2 is not specific to human immunodeficiency virus but affects a broad range of enveloped viruses. Since the identification of BST-2 as a restriction factor in 2008, much progress has been made in understanding the molecular properties and functional characteristics of this host factor. The goal of this review was to provide an update on our current understanding of the role of BST-2 in regulating virus release and to discuss its role in controlling virus spread during productive infection with special emphasis on human immunodeficiency virus-1.

HIV-1 Vif counteracts the antiviral activity of APOBEC3G by inhibiting its encapsidation into virions. Here, we compared the relative sensitivity to Vif of APOBEC3G in stable HeLa cells containing APOBEC3G (HeLa-A3G cells) versus that of newly synthesized APOBEC3G. We observed that newly synthesized APOBEC3G was more sensitive to degradation than preexisting APOBEC3G. Nevertheless, preexisting and transiently expressed APOBEC3G were packaged with similar efficiencies into vif -deficient human immunodeficiency virus type 1 (HIV-1) virions, and Vif inhibited the encapsidation of both forms of APOBEC3G into HIV particles equally well. Our results suggest that HIV-1 Vif preferentially induces degradation of newly synthesized APOBEC3G but indiscriminately inhibits encapsidation of “old” and “new” APOBEC3G.

The activation of IRF-3 during the early stages of viral infection is critical for the initiation of the antiviral response; however the activation of IRF-3 in HIV-1 infected cells has not yet been characterized. We demonstrate that the early steps of HIV-1 infection do not lead to the activation and nuclear translocation of IRF-3; instead, the relative levels of IRF-3 protein are decreased due to the ubiquitin-associated proteosome degradation. Addressing the molecular mechanism of this effect we show that the degradation is independent of HIV-1 replication and that virion-associated accessory proteins Vif and Vpr can independently degrade IRF-3. The null mutation of these two genes reduced the capacity of the HIV-1 virus to down modulate IRF-3 levels. The degradation was associated with Vif- and Vpr-mediated ubiquitination of IRF-3 and was independent of the activation of IRF-3. N-terminal lysine residues were shown to play a critical role in the Vif- and Vpr-mediated degradation of IRF-3. These data implicate Vif and Vpr in the disruption of the initial antiviral response and point to the need of HIV-1 to circumvent the antiviral response during the very early phase of replication.

Human immunodeficiency virus type 1 (HIV-1) Vif counteracts the antiviral activity of the human cytidine deaminase APOBEC3G (APO3G) by inhibiting its incorporation into virions. This has been attributed to the Vif-induced degradation of APO3G by cytoplasmic proteasomes. We recently demonstrated that although APO3G has a natural tendency to form RNA-dependent homo-multimers, multimerization was not essential for encapsidation into HIV-1 virions or antiviral activity. We now demonstrate that a multimerization-defective APO3G variant (APO3G C97A) is able to assemble into RNase-sensitive high-molecular-mass (HMM) complexes, suggesting that homo-multimerization of APO3G and assembly into HMM complexes are unrelated RNA-dependent processes. Interestingly, APO3G C97A was highly resistant to Vif-induced degradation even though the two proteins were found to interact in coimmunoprecipitation experiments and exhibited partial colocalization in transfected HeLa cells. Surprisingly, encapsidation and antiviral activity of APO3G C97A were both inhibited by Vif despite resistance to degradation. These results demonstrate that targeting of APO3G to proteasome degradation and interference with viral encapsidation are distinct functional properties of Vif.

Vif is a primate lentiviral accessory protein that is crucial for viral infectivity. Vif counteracts the antiviral activity of host deaminases such as APOBEC3G and APOBEC3F. We now report a novel function of African green monkey simian immunodeficiency virus (SIVagm) Vif that promotes replication of SIVagm in human cells lacking detectable deaminase activity. We found that cyclophilin A (CypA) was excluded from wild-type SIV particles but was efficiently packaged into vif -deficient SIVagm virions. The presence of CypA in vif -defective SIVagm was correlated with reduced viral replication. Infection of CypA knockout Jurkat cells or treatment of Jurkat cells with cyclosporine A eliminated the Vif-sensitive inhibition and resulted in replication profiles that were similar for wild-type and vif -deficient SIVagm. Importantly, the inhibitory effect of CypA was restricted to virus-producing cells and was TRIM5α independent. The abilities of SIVagm Vif to inhibit encapsidation of CypA and to increase viral infectivity were shared by rhesus macaque SIV Vif and thus seem to be general properties of SIV Vif proteins. Exclusion of CypA from SIVagm particles was not associated with intracellular degradation, suggesting a mode of Vif action distinct from that proposed for APOBEC3G. This is the first report of a novel vif -sensitive antiviral activity of human CypA that may limit zoonotic transmission of SIV and the first demonstration of CypA encapsidation into a virus other than human immunodeficiency virus type 1.

Current antiretroviral therapy (ART) is not sufficient to completely suppress disease progression in the CNS, as indicated by the rising incidence of HIV-1-associated neurocognitive disorders (HAND) among infected individuals on ART. It is not clear why some HIV-1-infected patients develop HAND, despite effective repression of viral replication in the circulation. SIV-infected nonhuman primate models are widely used to dissect the mechanisms of viral pathogenesis in the CNS. Here, we identified 4 amino acid substitutions in the cytoplasmic tail of viral envelope glycoprotein gp41 of the neurovirulent virus SIVsm804E that enhance replication in macrophages and associate with enhanced antagonism of the host restriction factor BM stromal cell antigen 2 (BST-2). Rhesus macaques were inoculated with a variant of the parental virus SIVsmE543-3 that had been engineered to contain the 4 amino acid substitutions present in gp41 of SIVsm804E. Compared with WT virus-infected controls, animals infected with mutant virus exhibited higher viral load in cerebrospinal fluid. Together, these results are consistent with a potential role for BST-2 in the CNS microenvironment and suggest that BST-2 antagonists may serve as a possible target for countermeasures against HAND.

HIV accessory genes are expressed throughout the viral life cycle and regulate wide-ranging aspects of virus replication including viral infectivity (Vif and Nef), viral gene expression (Vpr) and progeny virion production (Vpu). While in many cases the molecular basis of accessory protein function is not fully understood, a consensus is emerging that these viral products are generally devoid of enzymatic activity and instead act as multifunctional adapters, subverting normal cellular processes to serve the needs of the virus. This review focuses on presenting our current knowledge of the HIV-1-specific Vpu protein and its essential role in regulating viral particle release, viral load and expression of the CD4 receptor.

For nearly 20 years, the principal biological function of the HIV-2/SIV Vpx gene has been thought to be required for optimal virus replication in myeloid cells. Mechanistically, this Vpx activity was recently reported to involve the degradation of Sterile Alpha Motif and HD domain-containing protein 1 (SAMHD1) in this cell lineage. Here we show that when macaques were inoculated with either the T cell tropic SIVmac239 or the macrophage tropic SIVmac316 carrying a Vpx point mutation that abrogates the recruitment of DCAF1 and the ensuing degradation of endogenous SAMHD1 in cultured CD4+ T cells, virus acquisition, progeny virion production in memory CD4+ T cells during acute infection, and the maintenance of set-point viremia were greatly attenuated. Revertant viruses emerging in two animals exhibited an augmented replication phenotype in memory CD4+ T lymphocytes both in vitro and in vivo, which was associated with reduced levels of endogenous SAMHD1. These results indicate that a critical role of Vpx in vivo is to promote the degradation of SAMHD1 in memory CD4+ T lymphocytes, thereby generating high levels of plasma viremia and the induction of immunodeficiency., Author Summary Primate lentiviruses, such as HIV and its SIV simian relative, encode accessory proteins that suppress cellular restriction factors interfering with efficient replication. One of these, designated Vpx, is produced in infected cells by HIV-2 and some SIV strains, which cause endemic infections in African monkeys. The primary function of Vpx has long been thought to facilitate infectivity in dendritic cells and macrophage by degrading the Sterile Alpha Motif and HD domain-containing protein 1 (SAMHD1), which restricts virus replication in these cells. Using SIVmac carrying a mutated Vpx gene with a single amino acid change that prevents it from binding to DCAF1 and subsequently mediating the degradation of SAMHD1, we show that virus infection of CD4+ T lymphocytes is markedly compromised both in vitro and in vivo. The SIV Vpx mutant is severely attenuated in establishing new infections in inoculated rhesus monkeys, in producing high levels of virus progeny, in degrading SAMHD1 in memory CD4+ T cell in infected animals, and in inducing symptomatic disease. Thus, although once considered to be only critical for optimal replication in macrophage based on earlier studies performed with cultured cells, the SIV Vpx protein is functionally important in vivo for establishing the primary infection in rhesus macaques, sustaining high levels of virus replication in CD4+ T lymphocytes, and promoting the onset of symptomatic immunodeficiency.

One of the hallmarks of human immunodeficiency virus type I (HIV-1) infection is the rapid removal of the viral receptor CD4 from the cell surface. This remarkably efficient receptor interference requires the activity of three separate viral proteins: Env, Vpu, and Nef. We have investigated whether this unusually tight interference on cell surface CD4 expression had a more essential function during the viral life cycle than simply preventing superinfection. We now report that the removal of cell surface CD4 is required for optimal virus production by HIV-1. Indeed, maintenance of CD4 surface expression in infected cells lead to a 3–5-fold decrease in viral particle production. This effect was not due to the formation of intracellular complexes between CD4 and the gp160 viral envelope precursor but instead required the presence of CD4 at the cell surface and was specifically mediated by CD4 but not closely related plasma membrane receptors. The finding that CD4 had no significant effect on particle release by a Vpu-deficient variant indicates that CD4 acts by inhibiting the particle release-promoting activity of Vpu. Co-immunoprecipitation experiments further showed that CD4 and Vpu physically interact at the cell surface, suggesting that CD4 might inhibit Vpu activity by disrupting its oligomeric structure.

We have recently shown that the envelope glycoprotein of the ROD10 isolate of human immunodeficiency virus type 2 (HIV-2) has the ability to positively regulate HIV-2 viral particle release. The activity provided by the ROD10 Env was remarkably similar to that of the HIV-1 Vpu protein, thus raising the possibility that the two proteins act in a related fashion. We now show that the ROD10 Env can functionally replace Vpu to enhance the rate of HIV-1 particle release. When provided in trans, both Vpu and the ROD10 Env restored wild-type levels of particle release in a Vpu-deficient mutant of the NL4-3 molecular clone with indistinguishable efficiencies. This effect was independent of the presence of the HIV-1 envelope protein. The ROD10 Env also enhanced HIV-1 particle release in the context of HIV-2 chimeric viruses containing the HIV-1 gag-pol, indicating a lack of need for additional HIV-1 products in this process. In addition, we show for the first time that HIV-1 Vpu, as well as ROD10 Env, has the ability to enhance simian immunodeficiency virus (SIV) particle release. The effects of Vpu and ROD10 Env on SIV particle release were indistinguishable and were observed in the context of full-length SIVmac239 and simian-human immunodeficiency virus chimeras. These results further demonstrate that ROD10 Env can functionally complement Vpu with respect to virus release. In contrast, we found no evidence of a destabilizing activity of ROD10 Env on the CD4 molecule. HIV-1 and HIV-2 thus appear to have evolved genetically distinct but functionally similar strategies to resolve the common problem of efficient release of progeny virus from infected cells.

APOBEC3G (A3G) is a host cytidine deaminase that serves as a potent intrinsic inhibitor of retroviral replication. A3G is packaged into human immunodeficiency virus type 1 virions and deaminates deoxycytidine to deoxyuridine on nascent minus-strand retroviral cDNA, leading to hyper-deoxyguanine-to-deoxyadenine mutations on positive-strand cDNA and inhibition of viral replication. The antiviral activity of A3G is suppressed by Vif, a lentiviral accessory protein that prevents encapsidation of A3G. In this study, we identified dominant negative mutants of Vif that interfered with the ability of wild-type Vif to inhibit the encapsidation and antiviral activity of A3G. These mutants were nonfunctional due to mutations in the highly conserved HCCH and/or SOCS box motifs, which are required for assembly of a functional Cul5-E3 ubiquitin ligase complex. Similarly, mutation or deletion of a PPLP motif, which was previously reported to be important for Vif dimerization, induced a dominant negative phenotype. Expression of dominant negative Vif counteracted the Vif-induced reduction of intracellular A3G levels, presumably by preventing Vif-induced A3G degradation. Consequently, dominant negative Vif interfered with wild-type Vif's ability to exclude A3G from viral particles and reduced viral infectivity despite the presence of wild-type Vif. The identification of dominant negative mutants of Vif presents exciting possibilities for the design of novel antiviral strategies.

CD317/Bst-2/tetherin is a host factor that restricts the release of human immunodeficiency virus type 1 (HIV-1) by trapping virions at the plasma membrane of certain producer cells. It is antagonized by the HIV-1 accessory protein Vpu. Previous light microscopy studies localized CD317 to the plasma membrane and the endosomal compartment and showed Vpu induced downregulation. In the present study, we performed quantitative immunoelectron microscopy of CD317 in cells producing wild-type or Vpu-defective HIV-1 and in control cells. Double-labeling experiments revealed that CD317 localizes to the plasma membrane, to early and recycling endosomes, and to the trans-Golgi network. CD317 largely relocated to endosomes upon HIV-1 infection, and this effect was partly counteracted by Vpu. Unexpectedly, CD317 was enriched in the membrane of viral buds and cell-associated and cell-free viruses compared to the respective plasma membrane, and this enrichment was independent of Vpu. These results suggest that the tethering activity of CD317 critically depends on its density at the cell surface and appears to be less affected by its density in the virion membrane.

Members of the APOBEC family of cellular cytidine deaminases represent a recently identified group of proteins that provide immunity to infection by retroviruses and protect the cell from endogenous mobile retroelements. Yet, HIV-1 is largely immune to the intrinsic antiviral effects of APOBEC proteins because it encodes Vif (viral infectivity factor), an accessory protein that is critical for in vivo replication of HIV-1. In the absence of Vif, APOBEC proteins are encapsidated by budding virus particles and either cause extensive cytidine to uridine editing of negative sense single-stranded DNA during reverse transcription or restrict virus replication through deaminase-independent mechanisms. Thus, the primary function of Vif is to prevent encapsidation of APOBEC proteins into viral particles. This is in part accomplished by the ability of Vif to induce the ubiquitin-dependent degradation of some of the APOBEC proteins. However, Vif is also able to prevent encapsidation of APOBEC3G and APOBEC3F through degradation-independent mechanism(s). The goal of this review is to recapitulate current knowledge of the functional interaction of HIV-1 and its Vif protein with the APOBEC3 subfamily of proteins and to summarize our present understanding of the mechanism of APOBEC3-dependent retrovirus restriction.

APOBEC3G (APO3G) is a cellular cytidine deaminase with potent antiviral activity. Initial studies of the function of APO3G demonstrated extensive mutation of the viral genome, suggesting a model in which APO3G's antiviral activity is due to hypermutation of the viral genome. Recent studies, however, found that deaminase-defective APO3G mutants transiently expressed in virus-producing cells exhibited significant antiviral activity, suggesting that the antiviral activity of APO3G could be dissociated from its deaminase activity. To directly compare the antiviral activities of wild-type (wt) and deaminase-defective APO3G, we used two approaches: (i) we titrated wt and deaminase-defective APO3G in transient-transfection studies to achieve similar levels of virus-associated APO3G and (ii) we constructed stable cell lines and selected clones expressing comparable amounts of wt and deaminase-defective APO3G. Viruses produced under these conditions were tested for viral infectivity. The results from the two approaches were consistent and suggested that the antiviral activity of deaminase-defective APO3G was significantly lower than that of wt APO3G. We conclude that efficient inhibition of vif -defective human immunodeficiency virus type 1 requires catalytically active APO3G.

Background APOBEC3 (A3) proteins constitute a family of cytidine deaminases that provide intracellular resistance to retrovirus replication and to transposition of endogenous retroelements. A3A has significant homology to the C-terminus of A3G but has only a single cytidine deaminase active site (CDA), unlike A3G, which has a second N-terminal CDA previously found to be important for Vif sensitivity and virus encapsidation. A3A is packaged into HIV-1 virions but, unlike A3G, does not have antiviral properties. Here, we investigated the reason for the lack of A3A antiviral activity. Results Sequence alignment of A3G and A3A revealed significant homology of A3A to the C-terminal region of A3G. However, while A3G co-purified with detergent-resistant viral nucleoprotein complexes (NPC), virus-associated A3A was highly detergent-sensitive leading us to speculate that the ability to assemble into NPC may be a property conveyed by the A3G N-terminus. To test this model, we constructed an A3G-3A chimeric protein, in which the N-terminal half of A3G was fused to A3A. Interestingly, the A3G-3A chimera was packaged into HIV-1 particles and, unlike A3A, associated with the viral NPC. Furthermore, the A3G-3A chimera displayed strong antiviral activity against HIV-1 and was sensitive to inhibition by HIV-1 Vif. Conclusion Our results suggest that the A3G N-terminal domain carries determinants important for targeting the protein to viral NPCs. Transfer of this domain to A3A results in A3A targeting to viral NPCs and confers antiviral activity.

Background Efficient incorporation of the cellular cytidine deaminase APOBEC3G (APO3G) into HIV-1 virions is necessary for its antiviral activity. Even though cellular RNAs are known to be non-specifically incorporated into virus particles, we have previously found that encapsidation of APO3G into HIV-1 virions is specifically enhanced by viral genomic RNA. Intracellularly, APO3G was found to form large RNA-protein complexes involving a variety of cellular RNAs. The goal of this study was to investigate the possible contribution of host RNAs recently identified in intracellular APO3G ribonucleoprotein complexes to APO3G's encapsidation into HIV-1 virions. Results Our results show that 7SL RNA, a component of signal recognition particles, and hY1, hY3, hY4, hY5 RNAs were present in intracellular APO3G complexes and were packaged into HIV-1 particles lacking viral genomic RNA unlike APO3G, which was not packaged in significant amounts into genomic RNA-deficient particles. These results indicate that packaging of 7SL or hY RNAs is not sufficient for the packaging of APO3G into HIV-1 virions. We also tested the encapsidation of several other cellular RNAs including β-actin, GAPDH, α-tubulin, and small nuclear RNAs and determined their effect on the packaging of APO3G into nascent virions. Again, we were unable to observe any correlation between APO3G encapsidation and the packaging of any of these cellular RNAs. Conclusion The results from this study support our previous conclusion that viral genomic RNA is a critical determinant for APO3G incorporation into HIV-1 virions. While most cellular RNAs tested in this study were packaged into viruses or virus-like particles we failed to identify a correlation between APO3G encapsidation and the packaging of these cellular RNAs.

The virus infectivity factor (Vif) is a protein encoded by most primate lentiviruses. Recent evidence suggests that HIV-1 Vif reduces the intracellular levels of the host cytidine deaminase APOBEC3G (Apo3G) and inhibits its packaging into virions. These functions of Vif are thought to be species-specific. Accordingly, HIV-1 Vif can target only human Apo3G (hApo3G), whereas, African green monkey simian immunodeficiency virus (SIVagm) Vif can inhibit African green monkey but not human Apo3G. Consistent with this, we found that SIVagm Vif does not affect the stability of exogenously and endogenously expressed hApo3G and does not prevent packaging of exogenous and endogenous hApo3G into SIVagm virions. Nevertheless, SIVagm Vif supported spreading infection of SIVagm virus in the hApo3G-positive human A3.01 T cell line and rescued infectivity of viruses produced from Apo3G-expressing HeLa cells. Sequence analysis verified that SIVagm Vif inhibited the accumulation of hApo3G-induced mutations, suggesting that SIVagm Vif is indeed active in human cells. Our data suggest that SIVagm Vif can inhibit hApo3G activity without inducing its intracellular degradation or preventing its packaging into virions.

Replication of human immunodeficiency virus type 1 (HIV-1) in most primary cells and some immortalized T-cell lines depends on the activity of the viral infectivity factor (Vif). Vif has the ability to counteract a cellular inhibitor, recently identified as CEM15, that blocks infectivity of Vif-defective HIV-1 variants. CEM15 is identical to APOBEC3G and belongs to a family of proteins involved in RNA and DNA deamination. We cloned APOBEC3G from a human kidney cDNA library and confirmed that the protein acts as a potent inhibitor of HIV replication and is sensitive to the activity of Vif. We found that wild-type Vif inhibits packaging of APOBEC3G into virus particles in a dose-dependent manner. In contrast, biologically inactive variants carrying in-frame deletions in various regions of Vif or mutation of two highly conserved cysteine residues did not inhibit packaging of APOBEC3G. Interestingly, expression of APOBEC3G in the presence of wild-type Vif not only affected viral packaging but also reduced its intracellular expression level. This effect was not seen in the presence of biologically inactive Vif variants. Pulse-chase analyses did not reveal a significant difference in the stability of APOBEC3G in the presence or absence of Vif. However, in the presence of Vif, the rate of synthesis of APOBEC3G was slightly reduced. The reduction of intracellular APOBEC3G in the presence of Vif does not fully account for the Vif-induced reduction of virus-associated APOBEC3G, suggesting that Vif may function at several levels to prevent packaging of APOBEC3G into virus particles.

Packaging of the human immunodeficiency virus type 1 Vif protein into virus particles is mediated through an interaction with viral genomic RNA and results in the association of Vif with the nucleoprotein complex. Despite the specificity of this process, calculations of the amount of Vif packaged have produced vastly different results. Here, we compared the efficiency of packaging of Vif into virions derived from acutely and chronically infected H9 cells. We found that Vif was efficiently packaged into virions from acutely infected cells (60 to 100 copies per virion), while packaging into virions from chronically infected H9 cells was near the limit of detection (four to six copies of Vif per virion). Superinfection by an exogenous Vif-defective virus did not rescue packaging of endogenous Vif expressed in the chronically infected culture. In contrast, exogenous Vif expressed by superinfection of wild-type virus was readily packaged (30 to 40 copies per virion). Biochemical analyses suggest that the differences in the relative packaging efficiencies were not due to gross differences in the steady-state distribution of Vif in chronically or acutely infected cells but are likely due to differences in the relative rates of de novo synthesis of Vif. Despite its low packaging efficiency, endogenously expressed Vif was sufficient to direct the production of viruses with almost wild-type infectivity. The results from our study provide novel insights into the biochemical properties of Vif and offer an explanation for the reported differences regarding Vif packaging.

The human immunodeficiency virus type 1 (HIV-1) Vif protein is specifically packaged into virus particles through an interaction with viral genomic RNA in which it associates with the viral nucleoprotein complex. We now demonstrate for the first time that virus-associated Vif is subject to proteolytic processing by the viral protease (Pr). Pr-dependent processing of Vif was observed both in vivo and in vitro. In vivo processing of Vif was cell type independent and evident by the appearance of a 7-kDa processing product, which was restricted to cell-free virus preparations. Processing of Vif required an active viral Pr and was sensitive to Pr inhibitors such as ritonavir. The processing site in Vif was characterized both in vivo and in vitro and mapped to Ala 150 . Interestingly, the Vif processing site is located in a domain that is highly conserved among HIV-1, HIV-2, and simian immunodeficiency virus Vif isolates. Mutations at or near the processing site did not affect protein stability or packaging efficiency but had dramatic effects on Vif processing. In general, mutations that markedly increased or decreased the sensitivity of Vif to proteolytic processing severely impaired or completely abolished Vif function. In contrast, mutations at the same site that had little or no effect on processing efficiency also did not influence Vif function. None of the mutants affected the ability of the virus to replicate in permissive cell lines. Our data suggest that mutations in Vif that cause a profound change in the sensitivity to Pr-dependent processing also severely impaired Vif function, suggesting that intravirion processing of Vif is important for the production of infectious viruses.

The human immunodeficiency virus type 1 (HIV-1) Vif protein plays a critical role in the production of infectious virions. Previous studies have demonstrated the presence of small amounts of Vif in virus particles. However, Vif packaging was assumed to be nonspecific, and its functional significance has been questioned. We now report that packaging of Vif is dependent on the packaging of viral genomic RNA in both permissive and restrictive HIV-1 target cells. Mutations in the nucleocapsid zinc finger domains that abrogate packaging of viral genomic RNA abolished packaging of Vif. Additionally, an RNA packaging-defective virus exhibited significantly reduced packaging of Vif. Finally, deletion of a putative RNA-interacting domain in Vif abolished packaging of Vif into virions. Virion-associated Vif was resistant to detergent extraction and copurified with components of the viral nucleoprotein complex and functional reverse transcription complexes. Thus, Vif is specifically packaged into virions as a component of the viral nucleoprotein complex. Our data suggest that the specific association of Vif with the viral nucleoprotein complex might be functionally significant and could be a critical requirement for infectivity of viruses produced from restrictive host cells.

The human immunodeficiency virus type 1-specific Vpu protein is a small integral membrane phosphoprotein that induces degradation of the virus receptor CD4 in the endoplasmic reticulum and, independently, increases the release of progeny virions from infected cells. To address the importance of Vpu for virus replication in primary human cells such as peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM), we used three different sets of monocyte-tropic molecular clones of human immunodeficiency virus type 1: a primary isolate, AD8+, and two chimeric variants of the T-cell-tropic isolate NL4-3 carrying the env determinants of either AD8+ or SF162 monocyte-tropic primary isolates. Isogenic variants of these chimeric viruses were constructed to express either wild-type Vpu or various mutants of Vpu. The effects of these mutations in the vpu gene on virus particle secretion from infected MDM or PBMC were assessed by determination of the release of virion-associated reverse transcriptase into culture supernatants, Western blot (immunoblot) analysis of pelleted virions, and steady-state or pulse-chase metabolic labeling. Wild-type Vpu increased virus release four- to sixfold in MDM and two- to threefold in PBMC, while nonphosphorylated Vpu and a C-terminal truncation mutant of Vpu were partially active on virus release in primary cells. These results demonstrate that Vpu regulates virus release in primary lymphocyte and macrophage cultures in a similar manner and to a similar extent to those previously observed in HeLa cells or CD4+ T-cell lines. Thus, our findings provide evidence that Vpu functions in a variety of human cells, both primary cells and continuous cell lines, and mutations in Vpu affect its biological activity independent of the cell type and virus isolate used.

A deletion mutation affecting vpu was introduced into an infectious molecular clone of human immunodeficiency virus type 1, and the resultant phenotype was examined after infection of human T lymphocytes. The absence of vpu resulted in an accumulation of cell-associated viral proteins and impaired the release of progeny virions. Both electron microscopic and biochemical analyses indicated that a large proportion of the mutant particles was attached to the surface of infected cells. Significant variation in the size and shape of these progeny virions was observed. In addition, intracytoplasmic particles, some of which formed aberrant budding structures, were visualized in T cells infected with the vpu mutant. Indirect immunofluorescence analyses of cultures inoculated with wild-type virus with use of a vpu-specific antiserum demonstrated that vpu is mainly localized to a perinuclear region in the cytoplasm of virus-producing cells.

Background The human immunodeficiency virus Vif protein overcomes the inhibitory activity of the APOBEC3G cytidine deaminase by prohibiting its packaging into virions. Inhibition of APOBEC3G encapsidation is paralleled by a reduction of its intracellular level presumably caused by the Vif-induced proteasome-dependent degradation of APOBEC3G.

Defined segments of the cloned foot-and-mouth disease virus genome corresponding to all parts of the coding region were expressed in Escherichia coli as fusions to the N-terminal part of the MS2-polymerase gene under the control of the inducible lambda PL promoter. All constructs yielded large amounts of proteins, which were purified and used to raise sequence-specific antisera in rabbits. These antisera were used to identify the corresponding viral gene products in 35S-labeled extracts from foot-and-mouth disease virus gene products in the nucleotide sequence, to identify precursor-product relationships, and to detect several foot-and-mouth disease virus gene products not previously identified in vivo or in vitro.

A cDNA clone of Foot and Mouth Diseases Virus (FMDV), strain C1, has been sequenced. The limits of the structural genes were defined by comparison with the available protein data. We identified two potential translation initiation sites for the viral polyprotein separated by 84 nucleotides. We suggest that these two initiation sites could be used to express two proteins differing only at the N-terminal, P16 and P20a. This model is supported by the fact that antiserum against a bacterially synthesized polypeptide corresponding to the anterior region of the polyprotein precipitates specifically both P16 and P20a. Comparison of the C1 sequence with two other serotypes, O1K and A10 revealed variability in the major immunogenic structural protein, VP1, and also in two other capsid proteins, VP2 and VP3. P16/P20a, VP4, and the N-terminal part of the precursor of the nonstructural genes, P52, are rather conserved between the different FMDV strains.

APOBEC3G (APO3G) is a cellular cytidine deaminase with potent antiviral activity. In the case of HIV, the antiviral activity of APO3G is counteracted by the viral Vif protein. Monocyte-derived macrophages (MDM) are terminally differentiated, non-dividing cells susceptible to HIV infection. Human MDM are known to express APO3G and HIV replication in these cells is dependent on Vif. Here we analyzed the correlation between HIV-1 replication and APO3G expression in MDM. Replication of wild type HIV-1 induced a gradual 4-5-fold reduction in APO3G expression. The efficiency of APO3G downregulation correlated with the efficiency of virus replication. Interestingly, despite downregulation of APO3G, the relative infectivity of viruses rapidly declined during the course of infection and was already reduced approximately 90% prior to peak virus production. Cell-free virus preparations showed increased levels of a 41 kDa MA-CA processing intermediate. Sequence analysis around the MA-CA cleavage site and the protease and LTR regions did not reveal deaminase-induced hypermutation of the viral genome, suggesting that APO3G activity is not responsible for the incomplete Gag processing. Thus, the loss of infectivity of HIV-1 viruses produced from long-term infected primary macrophages is due to an APO3G-independent mechanism.

The envelope glycoprotein of HIV-2 ROD10 has the intriguing ability to enhance the rate of viral particle release from infected cells. However, not all HIV-2 envelope glycoproteins are active in this regard. Indeed, we have previously noted that, despite a high degree of identity with that of ROD10, the envelope protein of the ROD14 isolate was unable to enhance virus production. In this study, site-directed mutagenesis was employed to reveal that a single naturally occurring alanine-to-threonine substitution at position 598, located in the extracellular part of the TM subunit, fully accounted for the lack of activity of the ROD14 Env in HeLa and 12D7 cells. A second mutation at position 422, substituting a lysine residue in ROD10 for an arginine in ROD14, was additionally required for efficient virus release from infected H9 cells, suggesting cell-type-specific requirements for this activity. Interestingly, the ROD14 Env protein exhibited a trans-dominant negative effect on particle release by ROD10 Env, suggesting that the viral release activity of the HIV-2 ROD envelope protein may be regulated by its ability to assemble into functional oligomeric structures.

The genome of the human immunodeficiency virus (HIV) contains several open reading frames (ORFs) not present in other viruses. The 'A' gene, also known as Q2 P'3, ORF-1(4) or sor5, partially overlaps the pol gene; its protein product has a relative molecular mass of 23,000 (Mr 23K) and is present in productively infected cells. The function of this protein is unclear; mutant viruses deleted in 'A' replicate in and kill CD4+ lymphocyte lines, but the high degree of conservation of the deduced amino-acid sequence in nine different HIV isolates (80%) and the presence of analogous genes in HIV-2 and other lentiviruses suggest that the gene function is an important one. Here we describe a mutant virus deficient in the 'A' gene which produces virion particles normally; however, the particles are approximately 1,000 times less infective than wild type. Transcomplementation experiments partially restore infectivity. The mutant virus spreads efficiently when virus-producing cells are co-cultivated with CD4+ lymphocytes, however, indicating that HIV can spread from cell to cell in a mechanism that does not require the 'A' gene product and probably does not require the production of infective virus particles.

We investigated genetic recombination of the human immunodeficiency virus (HIV) in a tissue culture system. A clonal cell line expressing a single integrated HIV provirus with a termination codon affecting pol gene expression was transfected with different defective mutants derived from an infectious molecular clone of HIV. Replication-competent viral particles were recovered, passaged, and plaque purified. Restriction analyses of the proviral DNA corresponding to several of these viruses indicated that their emergence was the result of genetic recombination.

We have performed a detailed analysis of the biochemical properties of the human immunodeficiency virus (HIV) type 1 vpu gene product to elucidate its function during virus replication. Our data suggest that vpu is posttranslationally modified by phosphorylation, since a 16-kilodalton phosphoprotein can be specifically immunoprecipitated with both a serum from an HIV-positive individual (HIV-positive serum) and a vpu-specific antiserum. In contrast, our results suggest that vpu is not glycosylated, even though the protein contains a potential glycosylation site. In vitro translation studies demonstrated that vpu is cotranslationally integrated into microsomal membranes, suggesting that vpu is an integral membrane protein. While vpu was found in significant quantities in virus-producing cells, the protein could not be detected in cell-free culture fluids and is therefore most likely not viron associated. Processing of viral precursor proteins was unaffected by the absence of vpu, and no differences were detected in the protein compositions of wild-type and mutant virions. However, virus release from cultures producing vpu-defective virus was found to be delayed, resulting in the intracellular accumulation of viral proteins. Our data suggest that vpu has a function in the release of virus particles from infected cells.

A 16-kilodalton protein expressed in cells producing the human immunodeficiency virus (HIV-1) was identified as the gene product of the vpu open reading frame. When expressed in vitro, the 81-amino acid vpu protein reacted with about one-third of the serum samples from AIDS patients that were tested, indicating that the vpu open reading frame is expressed in vivo as well. Introduction of a frame-shift mutation into the vpu open reading frame did not significantly interfere with expression of the major viral proteins in a transient expression system. However, a five- to tenfold reduction in progeny virions was observed after the infection of T lymphocytes with the mutant virus. These data suggest that the vpu gene product is required for efficient virus replication and may have a role in assembly or maturation of progeny virions.