Dataset Information

Vpu antagonizes BST-2-mediated restriction of HIV-1 release via beta-TrCP and endo-lysosomal trafficking.

ABSTRACT: The interferon-induced transmembrane protein BST-2/CD317 (tetherin) restricts the release of diverse enveloped viruses from infected cells. The HIV-1 accessory protein Vpu antagonizes this restriction by an unknown mechanism that likely involves the down-regulation of BST-2 from the cell surface. Here, we show that the optimal removal of BST-2 from the plasma membrane by Vpu requires the cellular protein beta-TrCP, a substrate adaptor for a multi-subunit SCF E3 ubiquitin ligase complex and a known Vpu-interacting protein. beta-TrCP is also required for the optimal enhancement of virion-release by Vpu. Mutations in the DSGxxS beta-TrCP binding-motif of Vpu impair both the down-regulation of BST-2 and the enhancement of virion-release. Such mutations also confer dominant-negative activity, consistent with a model in which Vpu links BST-2 to beta-TrCP. Optimal down-regulation of BST-2 from the cell surface by Vpu also requires the endocytic clathrin adaptor AP-2, although the rate of endocytosis is not increased; these data suggest that Vpu induces post-endocytic membrane trafficking events whose net effect is the removal of BST-2 from the cell surface. In addition to its marked effect on cell-surface levels, Vpu modestly decreases the total cellular levels of BST-2. The decreases in cell-surface and intracellular BST-2 are inhibited by bafilomycin A1, an inhibitor of endosomal acidification; these data suggest that Vpu induces late endosomal targeting and partial degradation of BST-2 in lysosomes. The Vpu-mediated decrease in surface expression is associated with reduced co-localization of BST-2 and the virion protein Gag along the plasma membrane. Together, the data support a model in which Vpu co-opts the beta-TrCP/SCF E3 ubiquitin ligase complex to induce endosomal trafficking events that remove BST-2 from its site of action as a virion-tethering factor.

SUBMITTER: Mitchell RS

PROVIDER: S-EPMC2679223 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Vpu antagonizes BST-2-mediated restriction of HIV-1 release via beta-TrCP and endo-lysosomal trafficking.

Mitchell Richard S RS Katsura Chris C Skasko Mark A MA Fitzpatrick Katie K Lau David D Ruiz Autumn A Stephens Edward B EB Margottin-Goguet Florence F Benarous Richard R Guatelli John C JC

PLoS pathogens 20090529 5

The interferon-induced transmembrane protein BST-2/CD317 (tetherin) restricts the release of diverse enveloped viruses from infected cells. The HIV-1 accessory protein Vpu antagonizes this restriction by an unknown mechanism that likely involves the down-regulation of BST-2 from the cell surface. Here, we show that the optimal removal of BST-2 from the plasma membrane by Vpu requires the cellular protein beta-TrCP, a substrate adaptor for a multi-subunit SCF E3 ubiquitin ligase complex and a kno ...[more]

PMID: 19478868

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Project description:The primary roles attributed to the human immunodeficiency virus type 1 (HIV-1) Vpu protein are the degradation of the viral receptor CD4 and the enhancement of virion release. With regard to CD4 downregulation, Vpu has been shown to act as an adapter linking CD4 with the ubiquitin-proteasome machinery via interaction with the F-box protein betaTrCP. To identify additional cellular betaTrCP-dependent Vpu targets, we performed quantitative proteomics analyses using the plasma membrane fraction of HeLa cells expressing either wild-type Vpu or a Vpu mutant (S52N/S56N) that does not bind betaTrCP. One cellular protein, BST-2 (CD317), was consistently underrepresented in the membrane proteome of cells expressing wild-type Vpu compared to the proteome of cells expressing the Vpu mutant. To verify the biological relevance of this phenotype for HIV pathogenesis, we showed that in T cells infected with HIV-1, BST-2 downregulation occurred in a Vpu-dependent manner. Recently, BST-2 has been identified as the interferon-inducible cellular factor Tetherin, which restricts HIV virion release in the absence of Vpu. We address here the unresolved mechanism of Vpu-mediated BST-2 downregulation. Our data show that the presence of wild-type Vpu reduced cell surface and total steady-state BST-2 levels, whereas that of the mutant Vpu had no effect. In addition, treatment of cells with the lysosome acidification inhibitor concanamycin A, but not treatment with the proteasome inhibitor MG132, reduced BST-2 downregulation by wild-type Vpu, thereby suggesting that the presence of Vpu leads to the degradation of BST-2 via an endosome-lysosome degradation pathway. The importance of betaTrCP in this process was confirmed by demonstrating that in the absence of betaTrCP, BST-2 levels were restored despite the presence of Vpu. Taken together, these data support the hypothesis that, in similarity to its role in CD4 degradation, Vpu acts as an adapter molecule linking BST-2 to the cellular ubiquitination machinery via betaTrCP. However, in contrast to the proteasome-dependent degradation of CD4, which occurs in the endoplasmic reticulum, Vpu appears to interact with BST-2 in the trans-Golgi network or in early endosomes, leading to lysosomal degradation of BST-2. Via this action, Vpu could counter the tethering function of BST-2, resulting in enhanced HIV-1 virion release. Interestingly, although HIV-2 does not express Vpu, an isolate known to exhibit enhanced viral egress can downregulate surface BST-2 by an as-yet-unknown mechanism that does not appear to involve degradation. Understanding the molecular mechanisms of both Vpu-dependent and -independent mediated antagonism of BST-2 will be critical for therapeutic strategies that exploit this novel viral function.

Project description: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.

Project description:BST-2/CD317/tetherin is a host transmembrane protein that potently inhibits human immunodeficiency virus type 1 (HIV-1) virion release by tethering the nascent virions to the plasma membrane. Viral protein U (Vpu) is an accessory protein encoded by HIV-1 as well as by some simian immunodeficiency viruses (SIVs) infecting wild chimpanzees, gorillas, or monkeys (SIVcpz, SIVgor, or SIVgsn/SIVmon/SIVmus, respectively). HIV-1 Vpu directly binds to and downregulates human BST-2. The antagonism is highly species specific because the amino acid sequences of BST-2 are different among animal species. Here, we show that Vpu proteins from several SIVcpz, SIVgsn, SIVmon, or SIVmus isolates fail to antagonize human BST-2. Only Vpu from an SIVgsn isolate (SIVgsn-99CM71 [SIVgsn71]) was able to antagonize human BST-2 as well as BST-2 of its natural host, greater spot-nosed monkey (GSN). This SIVgsn Vpu interacted with human BST-2, downregulated cell surface human BST-2 expression, and facilitated HIV-1 virion release in the presence of human BST-2. While the unique 14AxxxxxxxW22 motif in the transmembrane domain of HIV-1NL4-3Vpu was reported to be important for antagonizing human BST-2, we show here that two AxxxxxxxW motifs (A22W30 and A25W33) exist in SIVgsn71 Vpu. Only the A22W30 motif was needed for SIVgsn71 Vpu to antagonize GSN BST-2, suggesting that the mechanism of this antagonism resembles that of HIV-1NL4-3 Vpu against human BST-2. Interestingly, SIVgsn71 Vpu requires two AxxxxxxxW (A22W30 and A25W33) motifs to antagonize human BST-2, suggesting an as-yet-undefined way that SIVgsn71 Vpu works against human BST-2. These results imply an evolutionary impact of primate BST-2 on lentiviral Vpu.IMPORTANCE Genetic alterations conferring a selective advantage in protecting from life-threating pathogens are maintained during evolution. In fact, the amino acid sequences of BST-2 differ among primate animals and their susceptibility to viral proteins is species specific, suggesting that such genetic diversity has arisen through the evolutionarily controlled balance between the host and pathogens. The M (main) group of HIV-1 is thought to be derived from SIVcpz, which utilizes Nef, but not Vpu, to antagonize chimpanzee BST-2. SIVcpz Nef is, however, unable to antagonize human BST-2, and Vpu was consequently chosen again as an antagonist against human BST-2 in the context of HIV-1. Studies on how Vpu lost and acquired this ability, together with the distinct mechanisms by which SIVgsn71 Vpu binds to and downregulates human or GSN BST-2, may help to explain the evolution of this lentiviral protein as a result of host-pathogen interactions.

Dataset Information

Vpu antagonizes BST-2-mediated restriction of HIV-1 release via beta-TrCP and endo-lysosomal trafficking.

Publications

Vpu antagonizes BST-2-mediated restriction of HIV-1 release via beta-TrCP and endo-lysosomal trafficking.

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