Paul M, Mazumder S, Raja N, Jabbar M A
Department of Molecular Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, Ohio 44195, USA.
J Virol. 1998 Feb;72(2):1270-9. doi: 10.1128/JVI.72.2.1270-1279.1998.
Human immunodeficiency virus type 1 Vpu is a multifunctional phosphoprotein composed of the N-terminal transmembrane (VpuTM) and C-terminal cytoplasmic domains. Each of these domains regulates a distinct function of the protein; the transmembrane domain is critical in virus release, and phosphorylation of the cytoplasmic domain is necessary for CD4 proteolysis. We carried our experiments to identify amino acids in the VpuTM domain that are important in the process of virus-like particle (VLP) release from HeLa cells. VLPs are released from the plasma membrane of HeLa cells at constitutive levels, and Vpu expression enhanced the release of VLPs by a factor of 10 to 15. Deletion of two to five amino acids from both N- and C-terminal ends or the middle of the VpuTM domain generated mutant Vpu proteins that have lost the ability to enhance VLP release. These deletion mutants have not lost the ability to associate with the wild-type or mutant Vpu proteins and formed complexes with equal efficiency. They were also transported normally to the Golgi complex. Furthermore, a Vpu protein having the CD4 transmembrane and Vpu cytoplasmic domains was completely inactive, and Vpu proteins harboring hybrid Vpu-CD4 TM domains were also defective in the ability to enhance the release of VLPs. When tested for functional complementation in cotransfected cells, two inactive proteins were not able to reconstitute Vpu activity that enhances the release of Gag particles. Coexpression of functional CD4/Vpu hybrids or wild-type Vpu with inactive mutant CD4/Vpu proteins revealed that mutations in the VpuTM domain could dominantly interfere with Vpu activity in Gag release. Taken together, these results demonstrated that the structural integrity of the VpuTM domain is critical for Vpu activity in the release of VLPs from the plasma membrane of mammalian cells.
1型人类免疫缺陷病毒（HIV-1）的Vpu是一种多功能磷蛋白，由N端跨膜结构域（VpuTM）和C端胞质结构域组成。这些结构域各自调节该蛋白的不同功能；跨膜结构域对病毒释放至关重要，而胞质结构域的磷酸化对于CD4蛋白水解是必需的。我们进行了实验，以确定VpuTM结构域中对从HeLa细胞释放病毒样颗粒（VLP）过程至关重要的氨基酸。VLP以组成型水平从HeLa细胞的质膜释放，Vpu的表达使VLP的释放增加了10到15倍。从VpuTM结构域的N端和C端或中间缺失两到五个氨基酸，会产生丧失增强VLP释放能力的突变Vpu蛋白。这些缺失突变体没有丧失与野生型或突变型Vpu蛋白结合的能力，并且能以相同效率形成复合物。它们也能正常转运到高尔基体复合物。此外，具有CD4跨膜结构域和Vpu胞质结构域的Vpu蛋白完全无活性，带有Vpu-CD4杂交跨膜结构域的Vpu蛋白在增强VLP释放的能力方面也存在缺陷。当在共转染细胞中测试功能互补时，两种无活性蛋白无法重建增强Gag颗粒释放的Vpu活性。功能性CD4/Vpu杂交体或野生型Vpu与无活性突变型CD4/Vpu蛋白的共表达表明，VpuTM结构域中的突变可显性干扰Vpu在Gag释放中的活性。综上所述，这些结果表明，VpuTM结构域的结构完整性对于Vpu在从哺乳动物细胞质膜释放VLP中的活性至关重要。
