Waheed Abdul A, MacDonald Scott, Khan Maisha, Mounts Megan, Swiderski Maya, Xu Yue, Ye Yihong, Freed Eric O
Virus-Cell Interaction Section, HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201, USA.
Laboratory of Molecular Biology, NIDDK, Bethesda, MD 20892, USA.
Sci Rep. 2016 Apr 22;6:24934. doi: 10.1038/srep24934.
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.
HIV-1辅助蛋白Vpu通过对抗宿主限制因子束缚素（tetherin）来增强病毒释放。为了进一步了解宿主细胞蛋白在Vpu功能中的作用，我们进行了酵母双杂交筛选，并鉴定出一种先前报道的与Vpu相互作用的宿主因子，即富含谷氨酰胺的小四肽重复序列蛋白（SGTA）。虽然RNA干扰介导的SGTA缺失对束缚素水平或病毒释放效率没有显著影响，但我们观察到SGTA的过表达以不依赖Vpu和束缚素的方式抑制HIV-1释放。在有Vpu存在而非无Vpu时，SGTA的过表达导致一种23 kDa的非糖基化束缚素物种显著稳定并重新定位于胞质溶胶。免疫共沉淀研究表明，非糖基化束缚素通过形成三元SGTA/Vpu/束缚素复合物而稳定。这种非糖基化束缚素的积累是由于其降解受到抑制，且不依赖内质网相关降解（ERAD）途径。由于SGTA稳定的束缚素物种部分定位于胞质溶胶，我们提出在有Vpu存在时SGTA的过表达会阻止束缚素跨内质网膜的转运，导致非糖基化束缚素物种在胞质溶胶中积累。虽然我们的结果不支持SGTA在HIV-1复制中的生理功能，但它们表明SGTA的过表达调节束缚素的表达和稳定性，从而为SGTA在内质网转运和蛋白质降解中的功能提供了见解。
