Abstract
The HIV-1 accessory protein Vpu enhances virus release by counteracting the host restriction factor tetherin. To further understand the role of host cell proteins in Vpu function, we carried out yeast two-hybrid screening and identified a previously reported Vpu-interacting host factor, small glutamine-rich tetratricopeptide repeat-containing protein (SGTA). While RNAi-mediated depletion of SGTA did not significantly affect levels of tetherin or virus release efficiency, we observed that overexpression of SGTA inhibited HIV-1 release in a Vpu- and tetherin-independent manner. Overexpression of SGTA in the presence of Vpu, but not in its absence, resulted in a marked stabilization and cytosolic relocalization of a 23-kDa, non-glycosylated tetherin species. Coimmunoprecipitation studies indicated that non-glycosylated tetherin is stabilized through the formation of a ternary SGTA/Vpu/tetherin complex. This accumulation of non-glycosylated tetherin is due to inhibition of its degradation, independent of the ER-associated degradation (ERAD) pathway. Because the SGTA-stabilized tetherin species is partially localized to the cytosol, we propose that overexpression of SGTA in the presence of Vpu blocks the translocation of tetherin across the ER membrane, resulting in cytosolic accumulation of a non-glycosylated tetherin species. Although our results do not provide support for a physiological function of SGTA in HIV-1 replication, they demonstrate that SGTA overexpression regulates tetherin expression and stability, thus providing insights into the function of SGTA in ER translocation and protein degradation.
Highlights
Human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, encodes three essential structural polyproteins (Gag, Pol and Env), two regulatory proteins (Tat and Rev), and four accessory proteins (Vif, Vpr, Vpu and Nef)[1]
While we observed that SGTA depletion did not significantly impact virus release or Vpu-mediated tetherin degradation, we demonstrated that SGTA overexpression induced a marked stabilization and cytosolic accumulation of a non-glycosylated, 23-kDa form of tetherin
While we confirmed that SGTA overexpression did interfere with HIV-1 release, several observations suggest that this phenomenon is not due to an effect on Vpu-mediated tetherin degradation: 1) SGTA overexpression inhibits the release of both Vpu(+ ) and Vpu(−) virus, 2) the effect of SGTA overexpression was seen in both tetherin-positive (HeLa) and tetherin-negative (293T) cells, and 3) the inhibition of virus release imposed by SGTA overexpression is accompanied by a defect in Pr55Gag processing to CA, which is not a phenotype associated with loss of Vpu expression in tetherin-expressing cells[8,50]
Summary
Human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, encodes three essential structural polyproteins (Gag, Pol and Env), two regulatory proteins (Tat and Rev), and four accessory proteins (Vif, Vpr, Vpu and Nef)[1]. The degradation of CD4 involves the interaction of Vpu and CD4 via their cytoplasmic domains, followed by recruitment of β -TrCP to the Vpu-CD4 complex, which leads to ubiquitylation and proteasomal degradation of CD410. Tetherin is an interferon-inducible protein that inhibits virus release by trapping mature virions on the cell surface[8,9]. It is an ~180 amino acid, type-II integral membrane protein that contains a short, N-terminal CT domain, www.nature.com/scientificreports/. Vpu-induced downregulation of tetherin cell-surface expression is associated with a ubiquitin-dependent lysosomal degradation through the ESCRT machinery that involves the recruitment of the β -TRCP E3 ubiquitin ligase (reviewed in[20,21]). Accumulating evidence suggests the involvement of SGTA in a variety of cancers (reviewed in[31,32])
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