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CD4胞浆尾巴和SCF-β-TrCP E3泛素连接酶复合体结合，导致CD4泛素化，并随后被蛋白酶体靶向降解。我们的研究揭示了这一过程的以下新方面：(I)降解涉及细胞内ER相关降解(ERAD)机制的至少一些组成部分，包括VCP-UFD1L-NPL4移位酶复合体；(Ii)CD4泛素化不仅依赖于赖氨酸，还依赖于CD4尾部的丝氨酸和苏氨酸残基；(Iii)VPU除了靶向ERAD外，还介导CD4在ER中的滞留，以及(Iv)VPU的跨膜域是ER保持和ERAD靶向CD4所必需的。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。 我们对VPU诱导的CD4下调的研究中一个令人惊讶的发现是，CD4泛素化和靶向ERAD需要丝氨酸和苏氨酸残基。为了确定这一要求有多普遍，我们检查了典型的ERAD底物，T细胞抗原受体复合体的阿尔法亚单位(TCR-α)的降解情况。TCR-α是一种I型完整的膜蛋白，当它不能组装成完整的TCR复合体时，它会变得泛素化，并靶向ERAD。值得注意的是，TCR-α的胞浆尾部只有5个氨基酸残基(即RLWSS)，其中没有一个是典型的泛素受体赖氨酸。我们发现，将TCR-α胞浆尾部的两个保守的丝氨酸残基替换为丙氨酸会降低泛素化，而额外的丝氨酸残基的放置会增强泛素化。此外，胞质丝氨酸残基被其他泛素化残基(即半胱氨酸、苏氨酸或赖氨酸)取代，使得泛素化得以发生。丝氨酸依赖的泛素化与ERAD的TCR-α靶向完全相关。我们还发现，这种泛素化是由内质网定位的泛素连接酶Hrd1介导的。这些发现表明，丝氨酸依赖的、Hrd1介导的泛素化将TCR-α靶向ERAD途径。因此，VPU不诱导病毒特异性修饰，而是利用一种内源性机制进行丝氨酸依赖的泛素化，以下调CD4。

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, causing CD4 ubiquitination and its subsequent targeting for degradation by the proteasome. Our studies revealed the following novel aspects of this process: (i) degradation involves at least some components of the cellular ER-associated degradation (ERAD) machinery, including the VCP-UFD1L-NPL4 dislocase complex; (ii) CD4 ubiquitination depends on not only lysine but also serine and threonine residues in the CD4 tail; (iii) Vpu mediates CD4 retention in the ER in addition to targeting to ERAD, and (iv) the transmembrane domain of Vpu is required for both ER retention and ERAD targeting of CD4. 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. A surprising finding of our studies on Vpu-induced CD4 downregulation was the requirement of serine and threonine residues for CD4 ubiquitination and targeting to ERAD. To determine how common this requirement is, we examined the degradation of a prototypical ERAD substrate, the alpha subunit of the T-cell antigen receptor complex (TCR-alpha). TCR-alpha is a type I integral membrane protein that becomes ubiquitinated and targeted to ERAD when it fails to assemble into the complete TCR complex. Remarkably, TCR-alpha has a cytosolic tail of only five amino acid residues (i.e., RLWSS), none of which is the canonical ubiquitin-acceptor lysine. We found that substitution of two conserved serine residues in the cytosolic tail of TCR-alpha to alanine decreased ubiquitination, whereas placement of additional serine residues enhanced it. Moreover, replacement of the cytosolic serine residues by other ubiquitinatable residues (i.e., cysteine, threonine, or lysine) allowed ubiquitination to take place. Serine-dependent ubiquitination perfectly correlated with targeting of TCR-alpha for ERAD. We also found that this ubiquitination is mediated by the ER-localized ubiquitin ligase, HRD1. These findings indicated that serine-dependent, HRD1-mediated ubiquitination targets TCR-alpha to the ERAD pathway. Thus, Vpu does not induce a viral-specific modification but exploits an endogenous machinery for serine-dependent ubiquitination in order to downregulate CD4.