HIV-1 Accessory Protein Vpu Internalizes Cell-surface BST-2/Tetherin through Transmembrane Interactions Leading to Lysosomes

Abstract

Bone marrow stromal antigen 2 (BST-2, also known as tetherin) is a recently identified interferon-inducible host restriction factor that can block the production of enveloped viruses by trapping virus particles at the cell surface. This antiviral effect is counteracted by the human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein U (Vpu). Here we show that HIV-1 Vpu physically interacts with BST-2 through their mutual transmembrane domains and leads to the degradation of this host factor via a lysosomal, not proteasomal, pathway. The degradation is partially controlled by a cellular protein, ␤-transducin repeat-containing protein (␤TrCP), which is known to be required for the Vpu-induced degradation of CD4. Importantly, targeting of BST-2 by Vpu occurs at the plasma membrane followed by the active internalization of this host protein by Vpu independently of constitutive endocytosis. Thus, the primary site of action of Vpu is the plasma membrane, where Vpu targets and internalizes cell-surface BST-2 through transmembrane interactions, leading to lysosomal degradation, partially in a ␤TrCP-dependent manner. Also, we propose the following configuration of BST-2 in tethering virions to the cell surface; each of the dimerized BST-2 molecules acts as a bridge between viral and cell membranes. Viral protein U (Vpu)2 is an 81-amino acid type I integral membrane phosphoprotein expressed by human immunodeficiency virus type 1 (HIV-1) (1, 2) and several simian immunodeficiency viruses (3-6). Vpu is not incorporated into virus particles (7), indicating that it acts exclusively in virus-producer cells. Indeed, Vpu is known to play two distinct roles during the later stages of infection. First, Vpu interacts with newly synthesized CD4 molecules complexed with the gp160 envelope glycoprotein precursor in the endoplasmic reticulum (8, 9) and recruits the ␤-transducin repeat-containing protein 1 (␤TrCP-1) subunit of the Skp1-Cullin1-F-box ubiquitin ligase complex (10) as well as ␤TrCP-2 (11) through its phosphoserine residues at positions 52 and 56 in the cytoplasmic (CT) domain (12,13). This event results in proteasome-mediated degradation of CD4 (10, 14, 15) allowing gp160 to resume transport toward the cell surface for virion incorporation. Second, Vpu mediates the enhancement of virion release (16 -18) in a cell type-dependent manner (e.g. HeLa cells require Vpu, whereas COS7 cells do not (19,20)), and its absence leads to the accumulation of viral particles at the cell surface (21).In contrast to the effect of Vpu on CD4 degradation, little had been known about the mechanism by which Vpu enhances the release of virions. The finding that HeLa-COS7 heterokaryons exhibited HeLa-type properties suggested that Vpu-responsive HeLa cells might harbor endogenous a restriction factor(s) that could be counteracted by this viral protein (22), as seen in Vifresponsive cells harboring the antiretroviral factor APOBEC3G counteracted by Vif (23). Neil et al. (24) showed that Vpu-deficient viral particles accumulated ...

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

“…Therefore, when BST-2 is overexpressed in transiently transfected cells, processed improperly, and trapped within the ER, the observation that proteasomal inhibitors prevent its subsequent degradation is likely to be a reflection of an ER quality control mechanism and not a Vpu-dependent process. Studies that have examined Vpu action on endogenously expressed BST-2 have instead obtained data supporting the lysosomal degradation of BST-2 (30,32,40). Our own data in this and previous manuscripts supports this same conclusion, and here we have provided further evidence for lysosomal degradation, e.g.…”

“…CD4 provides yet another example of a membrane protein targeted for ubiquitination by SCF ␤TrCP , with the notable exceptions that (a) CD4 is targeted at the ER membrane instead of the cell surface, (b) the CD4/SCF ␤TrCP interaction is mediated by Vpu, and (c) CD4 is degraded by the proteasome (10). Thus far, our data suggest that SCF ␤TrCP/Vpudependent BST-2 degradation is more similar to the mechanisms described for the IFN-␣, prolactin, erythropoietin, and human growth hormone receptors than for CD4, although there remains significant disagreement as to whether Vpu acts upon BST-2 at the cell surface or within an intracellular compartment (30,32,47,65,66,68).…”

“…Additionally, CD4 and BST-2 have each been found in complexes with Vpu and ␤TrCP, and ␤TrCP is required for the Vpu-dependent degradation of CD4 and BST-2 (28,29). However, unlike CD4, BST-2 and Vpu appear to colocalize within a perinuclear/trans-Golgi network compartment, and whereas several studies using exogenously expressed BST-2 have concluded that Vpu-dependent BST-2 degradation is proteasomal, studies examining endogenously expressed BST-2 weigh more heavily in favor of lysosomal degradation (30). These observations indicate that the mechanism of Vpu-dependent downregulation for BST-2 is similar yet distinct from that of CD4.…”

“…We therefore subjected our lentiviral BST-2::HA(K18R,K21R) clone to further mutagenesis, generating simultaneous Ala substitutions for Ser-2, Ser-4, and Thr-3. Although tyrosine residues are also hydroxylated, we did not make substitutions for the BST-2 cytoplasmic tyrosines, as these have been implicated in BST-2 endocytosis (30,52,53). The resulting clone (BST-2::HA⌬STK) was then used to create 293T-and HT1080-based cell lines stably expressing this mutant BST-2.…”

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

“…Notably, the A14L substitution does not affect CD4 down-regulation (28). 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).…”