Chen M Y, Maldarelli F, Karczewski M K, Willey R L, Strebel K
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
J Virol. 1993 Jul;67(7):3877-84. doi: 10.1128/JVI.67.7.3877-3884.1993.
CD4 is an integral membrane glycoprotein which functions as the human immunodeficiency virus (HIV) receptor for infection of human host cells. We have recently demonstrated that Vpu, an HIV type 1 (HIV-1) encoded integral membrane phosphoprotein, induces rapid degradation of CD4 in the endoplasmic reticulum. In this report, we describe an in vitro model system that allowed us to define important parameters for Vpu-dependent CD4 degradation. The rate of CD4 decay in rabbit reticulocyte lysate was approximately one-third of that observed previously in tissue culture experiments in the presence of Vpu (40 versus 12 min) and required no other HIV-1 encoded proteins. Degradation was contingent on the presence of microsomal membranes in the assay and the coexpression of Vpu and CD4 in the same membrane compartment. By using the in vitro degradation assay, the effects of specific mutations in CD4, including C-terminal truncations and glycosylation mutants, were analyzed. The results of these experiments indicate that Vpu has the capacity to induce degradation of glycosylated as well as nonglycosylated membrane-associated CD4. Truncation of 13 C-terminal amino acids of CD4 did not affect the ability of Vpu to induce its degradation. However, the removal of 32 amino acids from the C-terminus of CD4 completely abolished sensitivity to Vpu. This suggests that Vpu targets specific sequences in the cytoplasmic domain of CD4 to induce its degradation. We also analyzed the effects of mutations in Vpu on its biological activity in the in vitro CD4 degradation assay. The results of these experiments suggest that sequences critical for this function of Vpu are located in its hydrophilic C-terminal domain.
CD4是一种整合膜糖蛋白，作为人类免疫缺陷病毒（HIV）感染人类宿主细胞的受体发挥作用。我们最近证明，Vpu是一种由1型HIV（HIV-1）编码的整合膜磷蛋白，可在内质网中诱导CD4快速降解。在本报告中，我们描述了一个体外模型系统，该系统使我们能够确定Vpu依赖性CD4降解的重要参数。在兔网织红细胞裂解物中CD4的降解速率约为先前在存在Vpu的组织培养实验中观察到的降解速率的三分之一（40分钟对12分钟），并且不需要其他HIV-1编码蛋白。降解取决于测定中微粒体膜的存在以及Vpu和CD4在同一膜区室中的共表达。通过使用体外降解测定法，分析了CD4中特定突变的影响，包括C末端截短和糖基化突变体。这些实验结果表明，Vpu有能力诱导糖基化以及非糖基化的膜相关CD4降解。CD4的13个C末端氨基酸的截短并不影响Vpu诱导其降解的能力。然而，从CD4的C末端去除32个氨基酸完全消除了对Vpu的敏感性。这表明Vpu靶向CD4胞质结构域中的特定序列以诱导其降解。我们还在体外CD4降解测定中分析了Vpu突变对其生物学活性的影响。这些实验结果表明，对Vpu此功能至关重要的序列位于其亲水性C末端结构域。
