Elucidation of the Mechanisms of CD4 Downregulation by Vpu

阐明 Vpu 下调 CD4 的机制

• 批准号: 8351260
• 负责人: JUAN BONIFACINO
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8351260
• 关键词: Acquired Immunodeficiency Syndrome; Alanine; Amino Acids; BLR1 gene; Binding; CC chemokine receptor 4; CD4 Positive T Lymphocytes; Cells; Complex; Cysteine; Down-Regulation; Endoplasmic Reticulum Degradation Pathway; Ensure; Excision; Genes; Genome; Glycoproteins; Goals; HIV-1; Helper-Inducer T-Lymphocyte; Immune response; Infection; Integral Membrane Protein; Life Cycle Stages; Lysine; Mediating; Modification; Molecular; Mutation; Primate Lentiviruses; Process; Proteins; Quality Control; Serine; Staging; Surface; T-Cell Receptor; Tail; Threonine; Transmembrane Domain; Ubiquitin; Ubiquitination; Viral; Viral Genome; Virion; Work; beta-Transducin Repeat-Containing Proteins; chemokine receptor; human ESR1 protein; macrophage; monocyte; multicatalytic endopeptidase complex; nef Protein; novel; prevent; receptor; superinfection; ubiquitin ligase; ubiquitin-protein ligase; vpu Protein

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.

灵长类免疫缺陷病毒通过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）的组合结合来靶向辅助 T 细胞和巨噬细胞/单核细胞。引人注目的是，感染会导致 CD4 快速持续下调，并在较小程度上导致趋化因子受体下调。这些病毒共受体的下调可以防止重复感染，促进病毒颗粒释放并干扰免疫反应，从而导致强烈感染的建立。 CD4 下调对于人类免疫缺陷病毒 1 (HIV-1) 的生命周期非常重要，以至于病毒基因组中编码的两种辅助蛋白 Nef 和 Vpu 专门负责这项任务。事实上，Nef 和 Vpu 对于从感染到艾滋病的进展至关重要，这一事实最好的例证是感染 HIV-1 病毒株的长期非进展者的存在，这些病毒株的编码这些蛋白质的基因带有失活突变。因此，Nef 和/或 Vpu 的药理学或生物学扰动有可能预防 HIV-1 的致病作用。然而，迄今为止，这种潜力尚未实现，主要是因为 Nef 和 Vpu 没有酶活性，并且它们的作用机制尚不清楚。 在之前的工作中，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 CD4 连接到内吞和溶酶体靶向机制，从而在感染早期从宿主细胞中有效且持续地去除 CD4。当前的项目重点研究 Vpu 在感染后期下调 CD4 的机制。 Vpu 是一种小跨膜蛋白，包含短管腔结构域、单跨膜结构域 (TMD) 和胞质结构域。 Vpu 胞质结构域同时结合 CD4 胞质尾部和 SCF-β-TrCP E3 泛素连接酶复合物，导致 CD4 泛素化及其随后被蛋白酶体靶向降解。我们的研究揭示了该过程的以下新颖方面：（i）降解至少涉及细胞内质网相关降解（ERAD）机制的某些组成部分，包括VCP-UFD1L-NPL4脱位酶复合物； (ii) CD4泛素化不仅取决于赖氨酸，还取决于CD4尾部的丝氨酸和苏氨酸残基； (iii) 除了靶向 ERAD 之外，Vpu 还介导 ER 中的 CD4 保留，并且 (iv) Vpu 的跨膜结构域是 ER 保留和 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深度抑制 CD4 对 HIV-1 生命周期的重要性。 我们对 Vpu 诱导的 CD4 下调的研究的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。为了确定这种要求有多普遍，我们检查了原型 ERAD 底物（T 细胞抗原受体复合物 (TCR-alpha) 的 α 亚基）的降解。 TCR-α 是一种 I 型整合膜蛋白，当它无法组装成完整的 TCR 复合物时，它会被泛素化并靶向 ERAD。值得注意的是，TCR-α 的胞质尾部仅包含 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 胞质尾部的两个保守丝氨酸残基替换为丙氨酸会降低泛素化，而放置额外的丝氨酸残基则会增强泛素化。此外，用其他可泛素化残基（即半胱氨酸、苏氨酸或赖氨酸）替换胞质丝氨酸残基可以实现泛素化。丝氨酸依赖性泛素化与 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，丝氨酸依赖性 HRD1 介导的泛素化将 TCR-α 靶向 ERAD 通路。因此，Vpu 不会诱导病毒特异性修饰，而是利用丝氨酸依赖性泛素化的内源机制来下调 CD4。