Vpu directs the degradation of the human immunodeficiency virus restriction factor BST-2/Tetherin via a {beta}TrCP-dependent mechanism.
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.
Douglas
Janet L
JL
Oregon Health & Science University, Vaccine and Gene Therapy Institute, Beaverton, 97006, USA. dougljan@ohsu.edu
Viswanathan
Kasinath
K
McCarroll
Matthew N
MN
Gustin
Jean K
JK
Früh
Klaus
K
Moses
Ashlee V
AV
D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D058851:GPI-Linked Proteins; D015658:HIV Infections / Q000378:metabolism* / Q000821:virology; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D054298:Human Immunodeficiency Virus Proteins / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics / Q000502:physiology*; D014771:Virion / Q000235:genetics / Q000502:physiology; D017201:Virus Shedding; D044844:beta-Transducin Repeat-Containing Proteins / Q000235:genetics / Q000378:metabolism*; D000074010:Bone Marrow Stromal Antigen 2; D000086082:Viroporin Proteins
D015703:Antigens, CD; D015704:CD4 Antigens; D058851:GPI-Linked Proteins; D054298:Human Immunodeficiency Virus Proteins; D008562:Membrane Glycoproteins; D054334:Viral Regulatory and Accessory Proteins; D044844:beta-Transducin Repeat-Containing Proteins; C094572:BST2 protein, human; D000074010:Bone Marrow Stromal Antigen 2; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins