Dataset Information

?-TrCP is dispensable for Vpu's ability to overcome the CD317/Tetherin-imposed restriction to HIV-1 release.

ABSTRACT:

Background

The cellular transmembrane protein CD317/BST-2/HM1.24/Tetherin restricts HIV-1 infection by physically tethering mature virions to the surface of infected cells. HIV-1 counteracts this restriction by expressing the accessory protein Vpu, yet the mechanism of this antagonism is incompletely understood. ?-TrCP is the substrate recognition domain of an E3 ubiquitin ligase complex that interacts with the di-serine motif S52/S56 in the cytoplasmic tail of Vpu to target the CD4 receptor for proteasomal degradation. Recently, it has been suggested that ?-TrCP is also critically involved in Vpu's ability to overcome the CD317-mediated virion release block.

Results

To test this model, we analyzed the consequences of several experimental strategies to interfere with the Vpu-?-TrCP protein-protein interaction. Under these conditions, we studied effects of Vpu on expression and localization of CD317 and CD4, as well as on its ability to promote HIV-1 release. Our results demonstrate a strict requirement for Vpu's di-serine motif for degradation of CD4 and also CD317, reduction of cell surface exposure of CD317, and HIV-1 release enhancement. We further show a critical role of ?-TrCP2, but not of the structurally related ?-TrCP1 isoform, for Vpu-mediated degradation of both receptors. Most importantly, Vpu remained active in downregulating CD317 from the cell surface and in overcoming the HIV-1 release restriction in ?-TrCP-depleted cells.

Conclusions

These results demonstrate that ?-TrCP is not strictly required for Vpu's ability to counteract the CD317-imposed virion release block and support the relevance of cell surface down-modulation of the restriction factor as a central mechanism of Vpu antagonism. Moreover, we propose the existence of a critical, yet to be identified cellular factor that interacts with Vpu via its di-serine motif to alter the trafficking of the restriction factor.

SUBMITTER: Tervo HM

PROVIDER: S-EPMC3049139 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

β-TrCP is dispensable for Vpu's ability to overcome the CD317/Tetherin-imposed restriction to HIV-1 release.

Tervo Hanna-Mari HM Homann Stefanie S Ambiel Ina I Fritz Joëlle V JV Fackler Oliver T OT Keppler Oliver T OT

Retrovirology 20110210

<h4>Background</h4>The cellular transmembrane protein CD317/BST-2/HM1.24/Tetherin restricts HIV-1 infection by physically tethering mature virions to the surface of infected cells. HIV-1 counteracts this restriction by expressing the accessory protein Vpu, yet the mechanism of this antagonism is incompletely understood. β-TrCP is the substrate recognition domain of an E3 ubiquitin ligase complex that interacts with the di-serine motif S52/S56 in the cytoplasmic tail of Vpu to target the CD4 rece ...[more]

PMID: 21310048

Similar Datasets

Project description:UnlabelledThe intrinsic immunity factor CD317 (BST-2/HM1.24/tetherin) imposes a barrier to HIV-1 release at the cell surface that can be overcome by the viral protein Vpu. Expression of Vpu results in a reduction of CD317 surface levels; however, the mechanism of this Vpu activity and its contribution to the virological antagonism are incompletely understood. Here, we characterized the influence of Vpu on major CD317 trafficking pathways using quantitative antibody-based endocytosis and recycling assays as well as a microinjection/microscopy-based kinetic de novo expression approach. We report that HIV-1 Vpu inhibited both the anterograde transport of newly synthesized CD317 and the recycling of CD317 to the cell surface, while the kinetics of CD317 endocytosis remained unaffected. Vpu trapped trafficking CD317 molecules at the trans-Golgi network, where the two molecules colocalized. The subversion of both CD317 transport pathways was dependent on the highly conserved diserine S52/S56 motif of Vpu; however, it did not require recruitment of the diserine motif interactor and substrate adaptor of the SCF-E3 ubiquitin ligase complex, ?-TrCP. Treatment of cells with the malaria drug primaquine resulted in a CD317 trafficking defect that mirrored that induced by Vpu. Importantly, primaquine could functionally replace Vpu as a CD317 antagonist and rescue HIV-1 particle release.ImportanceHIV efficiently replicates in the human host and induces the life-threatening immunodeficiency AIDS. Mammalian genomes encode proteins such as CD317 that can inhibit viral replication at the cellular level. As a countermeasure, HIV has evolved genes like vpu that can antagonize these intrinsic immunity factors. Investigating the mechanism by which Vpu overcomes the virion release restriction imposed by CD317, we find that Vpu subverts recycling and anterograde trafficking pathways of CD317, resulting in surface levels of the restriction factor insufficient to block HIV-1 spread. This describes a novel mechanism of immune evasion by HIV.

Project description:The Vpu accessory protein promotes HIV-1 release by counteracting Tetherin/BST-2, an interferon-regulated restriction factor, which retains virions at the cell-surface. Recent reports proposed beta-TrCP-dependent proteasomal and/or endo-lysosomal degradation of Tetherin as potential mechanisms by which Vpu could down-regulate Tetherin cell-surface expression and antagonize this restriction. In all of these studies, Tetherin degradation did not, however, entirely account for Vpu anti-Tetherin activity. Here, we show that Vpu can promote HIV-1 release without detectably affecting Tetherin steady-state levels or turnover, suggesting that Tetherin degradation may not be necessary and/or sufficient for Vpu anti-Tetherin activity. Even though Vpu did not enhance Tetherin internalization from the plasma membrane (PM), it did significantly slow-down the overall transport of the protein towards the cell-surface. Accordingly, Vpu expression caused a specific removal of cell-surface Tetherin and a re-localization of the residual pool of Tetherin in a perinuclear compartment that co-stained with the TGN marker TGN46 and Vpu itself. This re-localization of Tetherin was also observed with a Vpu mutant unable to recruit beta-TrCP, suggesting that this activity is taking place independently from beta-TrCP-mediated trafficking and/or degradation processes. We also show that Vpu co-immunoprecipitates with Tetherin and that this interaction involves the transmembrane domains of the two proteins. Importantly, this association was found to be critical for reducing cell-surface Tetherin expression, re-localizing the restriction factor in the TGN and promoting HIV-1 release. Overall, our results suggest that association of Vpu to Tetherin affects the outward trafficking and/or recycling of the restriction factor from the TGN and as a result promotes its sequestration away from the PM where productive HIV-1 assembly takes place. This mechanism of antagonism that results in TGN trapping is likely to be augmented by beta-TrCP-dependent degradation, underlining the need for complementary and perhaps synergistic strategies to effectively counteract the powerful restrictive effects of human Tetherin.

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.