Vpu Directs the Degradation of the Human Immunodeficiency Virus Restriction Factor BST-2/Tetherin via a βTrCP-Dependent Mechanism

Abstract

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 ␤TrCP. To identify additional cellular ␤TrCP-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 ␤TrCP. 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 ␤TrCP in this process was confirmed by demonstrating that in the absence of ␤TrCP, 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 ␤TrCP. 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.

Ubiquitination of BST-2 Protein by HIV-1 Vpu Protein Does Not Require Lysine, Serine, or Threonine Residues within the BST-2 Cytoplasmic Domain

“…Note that in both cell lines, egress of the vpu2/6 virus displays a consistent intermediate phenotype that we and others have previously observed. Although BST-2 is not degraded in the presence of Vpu2/6, several labs including our own have shown that BST-2 binds to wild type Vpu and Vpu2/6 equally well (27,40,47). Therefore, the Vpu2/6 partial egress phenotype likely results from the transient sequestration of BST-2 within an intracellular compartment.…”

“…To efficiently deliver Vpu-expression constructs to the vast majority of the cells in our experiments, we therefore utilized adenoviral vectors. We have used these adenoviruses here and elsewhere (40) in well controlled experiments showing that Vpu efficiently down-regulates total levels of BST-2, whereas Vpu2/6 does not.…”

“…Vpu Plasmids and Recombinant Adenoviral Vectors-To avoid issues arising from poor transfection efficiencies and to ensure consistent protein expression levels, we utilized adenoviral clones expressing Vpu::GFP and Vpu2/6::GFP fusions, which we have previously described (40). For transductions, cells were seeded at 3 ϫ 10 5 cells/well in a 6-well tissue culture plate (Costar, Corning, NY).…”

Oligomerization state and supramolecular structure of the HIV‐1 Vpu protein transmembrane segment in phospholipid bilayers

“…38 Vpu interferes with tetherin by targeting tetherin for degradation, in a manner similar to that by which Vpu downregulates CD4. 39,40 Sequence variations in the TM domain of tetherin that affect sensitivity to Vpu have been identified. 41,42 Formation of Vpu/tetherin hetero-oligomers through direct interactions between their TM domains may therefore be essential for Vpu function, and these hetero-oligomers may be structurally related to Vpu homo-oligomers.…”

Tetherin antagonism by Vpu protects HIV-infected cells from antibody-dependent cell-mediated cytotoxicity

“…Tetherin antagonism by Vpu also depends in part on the recruitment of βTrCP-2, an adaptor protein for an E3 ubiquitin ligase involved in targeting tetherin for degradation (24,25,30,31). Hence, substitutions in a conserved DSGxxS motif in the cytoplasmic tail of Vpu (S52,56N) that prevent the recruitment of βTrCP-2 partially impair tetherin antagonism by preventing its degradation, but not its endosomal sequestration (25,(30)(31)(32)(33). These substitutions are also known to abrogate CD4 down-regulation, but do not affect Vpu-mediated down-modulation of NTB-A (23).…”