Elucidation of the Mechanisms of CD4 Downregulation by Vpu

阐明 Vpu 下调 CD4 的机制

• 批准号: 8736937
• 负责人: JUAN BONIFACINO
• 金额: $ 7.17万
• 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/8736937
• 关键词: Acquired Immunodeficiency Syndrome; BLR1 gene; Binding; CC chemokine receptor 4; CD4 Positive T Lymphocytes; Cells; Complex; Deubiquitination; Discrimination; Dislocations; Down-Regulation; Endoplasmic Reticulum; Ensure; Excision; Genes; Genome; Glycoproteins; Goals; HIV-1; Helper-Inducer T-Lymphocyte; Immune response; In Vitro; Infection; Integral Membrane Protein; Life Cycle Stages; Ligase; Light; Lysine; Membrane; Membrane Proteins; Molecular; Mutation; Pathogenesis; Polyubiquitination; Primate Lentiviruses; Proteins; Quality Control; Serine; Staging; Surface; System; Tail; Threonine; Transmembrane Domain; Ubiquitination; Variant; Viral; Viral Genome; Virion; Work; beta-Transducin Repeat-Containing Proteins; chemokine receptor; in vivo; macrophage; monocyte; multicatalytic endopeptidase complex; nef Protein; prevent; receptor; reconstitution; superinfection; 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 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.

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 胞质结构域同时与 CD4 胞质尾部和内质网 (ER) 中的 SCF-β-TrCP E3 泛素连接酶复合物结合，导致赖氨酸和丝氨酸/苏氨酸残基上的 CD4 泛素化，然后与 VCP-UFD1L-NPL4 脱位酶复合物相互作用，随后靶向降解 蛋白酶体。 CD4 识别 Vpu 诱导的降解涉及 CD4 和 Vpu 的胞质和跨膜结构域水平上的相互作用。跨膜结构域相互作用促进 CD4 的靶向降解以及在内质网中的保留。干扰跨膜结构域相互作用可减少 CD4 多聚泛素化以及与 ER 膜的错位，从而消除体内 CD4 的降解。 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。 去年，我们在体外系统中成功重建了 Vpu 诱导的 CD4 泛素化。同样在该系统中，CD4 和 Vpu 参与跨膜域相互作用。这些相互作用的破坏降低了 CD4 多聚泛素化的程度。出乎意料的是，单独的持续合成能力差异无法解释降解和未降解的 CD4 变体的差异命运。相反，连续去泛素化被认为是最大底物区分的先决条件。这解释了底物-连接酶相互作用的微小差异如何放大为多泛素化和净降解的较大差异。除了揭示 Vpu 诱导 CD4 降解的机制之外，这些结果还为膜蛋白质量控制过程中底物辨别的一般机制提供了概念框架。