Elucidation of the Mechanisms of CD4 Downregulation by Vpu

灵长类免疫缺陷病毒通过将病毒包膜糖蛋白与宿主细胞表面的CD4和趋化因子受体(CCR4或CXCR5)结合来靶向辅助性T细胞和巨噬细胞/单核细胞。值得注意的是，感染会导致CD4和趋化因子受体的快速和持续下调，程度较小。这些病毒协同受体的下调可防止重叠感染，促进病毒粒子的释放，并干扰免疫反应，导致建立强大的感染。CD4下调对人类免疫缺陷病毒-1(HIV-1)的生命周期非常重要，因此病毒基因组中编码的两种辅助蛋白Nef和VPu致力于这一任务。事实上，Nef和VPU对于从感染到艾滋病的进展至关重要，这一事实从感染了编码这些蛋白质的基因发生失活突变的HIV-1毒株的长期非进展者的存在就得到了最好的证明。因此，Nef和/或VPU的药理或生物扰动有可能预防HIV-1的致病作用。然而，到目前为止，这种潜力还没有被认识到，主要是因为Nef和VPU没有酶活性，它们的作用机制还不够清楚。 在以前的工作中，我们在阐明Nef下调CD4的机制方面取得了实质性进展。我们发现，Nef将表面的CD4连接到内吞和溶酶体靶向机制，导致在感染早期有效和持续地从宿主细胞中移除CD4。目前的项目重点是VPU在感染后期下调CD4的机制。VPU是一种小分子跨膜蛋白，由短腔结构域、单跨膜结构域(TMD)和胞浆结构域组成。VPU胞浆结构域同时与内质网(ER)中的CD4胞液尾部和SCF-β-TrCP E3泛素连接酶复合体结合，导致赖氨酸和丝氨酸/苏氨酸残基上的CD4泛素化，随后与VCP-UFD1L-NPL4错位酶复合体相互作用，随后被蛋白酶体靶向降解。识别VPU引起的降解需要在胞质和跨膜结构域上的相互作用。跨膜结构域的相互作用促进了内质网中CD4的降解和滞留。对跨膜区相互作用的干扰减少了CD4的多泛素化和内质网膜的错位，从而消除了体内的CD4降解。VPU参与细胞质量控制机制的多个层面强调了确保对HIV-1生命周期的深刻抑制的重要性。 在过去的一年里，我们成功地在体外系统中重建了VPU诱导的CD4泛素化。在这个系统中，CD4和VPU参与跨膜结构域的相互作用。这些相互作用的中断降低了CD4多泛素化的程度。出乎意料的是，仅靠不同的加工能力并不能解释降解和非降解的CD4变种的不同命运。相反，持续的去泛素化被认为是最大底物辨别的先决条件。这解释了底物-连接酶相互作用的微小差异如何被放大为多泛素化和净降解的更大差异。除了揭示VPU诱导的CD4降解的机制外，这些结果还为膜蛋白质量控制中底物歧视的一般机制提供了一个概念性的框架。

Primate immunodeficiency viruses target helper T-cells and macrophages/monocytes through binding of the viral envelope glycoprotein to a combination of CD4 and a chemokine receptor (CCR4 or CXCR5) on the surface of the host cells. Strikingly, infection results in rapid and sustained downregulation of CD4 and, to a lesser extent, the chemokine receptors. Downregulation of these viral co-receptors prevents superinfection, promotes virion release and interferes with the immune response, leading to the establishment of a robust infection. CD4 downregulation is so important to the life cycle of human immunodeficiency virus-1 (HIV-1) that two accessory proteins, Nef and Vpu, encoded in the viral genome are devoted to this task. Indeed, Nef and Vpu are critical for the progression from infection to AIDS, a fact that is best illustrated by the existence of long-term non-progressors that are infected with HIV-1 strains bearing inactivating mutations in the genes encoding these proteins. Therefore, pharmacologic or biologic perturbation of Nef and/or Vpu has the potential to prevent the pathogenic effects of HIV-1. To date, however, this potential has not been realized mainly because Nef and Vpu have no enzymatic activity and their mechanisms of action are insufficiently understood. In previous work, we made substantial progress towards elucidating the mechanism of CD4 downregulation by Nef. We found that Nef connects surface CD4 to both the endocytic and lysosomal targeting machineries, leading to efficient and sustained removal of CD4 from the host cells early during infection. The current project focuses on the mechanisms by which Vpu downregulates CD4 at later stages of infection. Vpu is a small transmembrane protein comprising a short luminal domain, a single transmembrane domain (TMD) and a cytosolic domain. The Vpu cytosolic domain simultaneously binds to the CD4 cytosolic tail and the SCF-beta-TrCP E3 ubiquitin ligase complex in the endoplasmic reticulum (ER), causing CD4 ubiquitination on lysine and serine/threonine residues followed by interaction with the VCP-UFD1L-NPL4 dislocase complex and subsequent targeting for degradation by the proteasome. Recognition of CD4 for Vpu-induced degradation involves interactions at the level of both the cytosolic and transmembrane domains of CD4 and Vpu. Transmembrane domain interactions promote targeting for degradation as well as retention of CD4 in the ER. Interference with transmembrane domain interactions reduce CD4 polyubiquitination as well as dislocation from the ER membrane, thus abrogating CD4 degradation in vivo. The multiple levels at which Vpu engages the cellular quality control mechanisms underscore the importance of ensuring profound suppression of CD4 to the life cycle of HIV-1. This past year we succeeded in reconstituting Vpu-induced CD4 ubiquitination in an in vitro system. Also in this system, CD4 and Vpu engaged in transmembrane domain interactions. Disruption of these interactions decreased the extent of CD4 polyubiquitination. Unexpectedly, differential processivity alone could not explain the differential fates of degraded and nondegraded CD4 variants. Instead, continuous deubiquitination was identified as a prerequisite for maximal substrate discrimination. This explains how small differences in substrate-ligase interaction can be amplified into larger differences in polyubiquitination and net degradation. In addition to shedding light on the mechanisms of Vpu-induced CD4 degradation, these results provide a conceptual framework for the general mechanisms of substrate discrimination during membrane protein quality control.