Allostery and Hijacking of Host Membrane Traffic by HIV-1 Accessory Proteins

HIV-1 辅助蛋白对宿主膜运输的变构和劫持

• 批准号: 10092840
• 负责人: James H Hurley
• 金额: $ 62.51万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-18 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10092840
• 关键词: Acquired Immunodeficiency Syndrome; Adaptor Protein Complex 2; Adaptor Signaling Protein; Affect; Alleles; Antiviral Agents; Automobile Driving; Basic Science; Binding Sites; Biochemistry; Biophysics; CD28 gene; CD3 Antigens; CD8B1 gene; CXCR4 gene; Cell Surface Proteins; Cell membrane; Cell surface; Cells; Clathrin; Clathrin-Coated Vesicles; Complement; Complex; Cryoelectron Microscopy; Cytosol; Detergents; Down-Regulation; Family; Funding; Goals; HIV; HIV-1; Host Defense; Human; Image; Immune system; Immunity; Infection; Integral Membrane Protein; Lipids; Location; Lysosomes; Mediating; Membrane; Membrane Lipids; Membrane Protein Traffic; Monomeric GTP-Binding Proteins; Mutation; Pathway interactions; Pattern; Phenotype; Process; Production; Proteins; Reporting; Research Proposals; Role; SIV; Seminal; Signal Transduction; Site; Sorting - Cell Movement; Structure; Tail; Viral; Viral Antigens; Virion; Virus; Virus Latency; base; design; follow-up; insight; member; prevent; protein complex; reactivation from latency; receptor; reconstitution; trans-Golgi Network; viral fitness

PROJECT SUMMARY Nef is an HIV-1 accessory protein whose function is to undermine host defenses. Long term infection with HIV strains bearing defective nef alleles leads to AIDS only over many years, suggesting Nef could be targeted as part of functional cure strategies. This basic research proposal seeks will elucidate the mechanisms of action of Nef. Nef targets include CD3, CD4, CD8, CD28, CXCR4, MHC-I, SERINC3/5, and for HIV-1 group O and SIV, BST2/tetherin. Nef substrates are downmodulated by via the clathrin-coated vesicle (CCV) pathway. Nef does not interact directly with clathrin, but rather with various members of a family of heterotetrameric adaptors known as the adaptor protein (AP) complexes, AP-1 and AP-2. The ability of the human immune system to detect and kill virally infected cells relies on proper presentation of viral antigens on the cell surface by MHC-I complexes. Nef subverts this process by promoting MHC-I complex downregulation by hijacking AP-1 and its associated small GTPase Arf1 at the TGN. MHC-I contains an incomplete version of the normal Tyr-based sorting motif. Nef complements this defective motif and converts MHC-I into a substrate for AP-1 mediated sorting to the lysosome for degradation. Structures of Nef assembled with AP-1 and the MHC-I cytosolic tail in solution suggested that Nef promotes the assembly of hexagonal lattices whose symmetry matches that of clathrin. Now, the previous solution studies will be followed up by reconstitution and structure determination of Nef, MHC-I tail, AP-1 and Arf1 in their functional setting on lipid membranes. Downmodulation of CD28 by Nefs is conserved across SIV and HIV and is mediated by AP-2. CD28 downmodulation phenotypes of Nef mutations follow a distinct pattern from other receptors, and the structural basis for this mode of CD28 is unknown. The structure of the CD28:HIV-1 Nef:AP-2 complex will be determined and leveraged to design mutations that uniquely perturb the CD28 binding site. Of the many host substrates of Nef, the most significant for viral infectivity are the multipass integral membrane proteins SERINC3 and 5. The SERINC binding site appears, on the basis of Nef phenotypes, to overlap with the site used by SIVsmm Nef to downmodulate simian tetherin, but is otherwise distinct from the known locations of CD3, CD4, and MHC-I sites. SERINCs do not share any obvious motifs with other substrates. SERINCs have been purified in monodisperse form suitable for structure determination. The cryo-EM structure of lipid- or detergent embedded SERINC3 or 5 in complex with AP-2 and HIV-1 Nef will be determined, completing a major goal in the field.

项目总结 NEF是一种HIV-1辅助蛋白，其功能是破坏宿主防御。长期感染艾滋病毒 携带有缺陷的nef等位基因的菌株只会在很多年内导致艾滋病，这表明nef可以作为靶标 是功能性治疗策略的一部分。这项基础研究计划将阐明其作用机制。 Nef.NEF目标包括CD3、CD4、CD8、CD28、CXCR4、MHC-I、SERINC3/5，以及针对HIV-1 O组和 SIV、BST2/Tetherin。NEF底物通过笼蛋白包裹的囊泡(CCV)途径被下调。NEF 不直接与笼状蛋白相互作用，而是与异源四聚体适配器家族的各种成员相互作用 称为接合蛋白(AP)复合体，AP-1和AP-2。人类免疫系统的能力 检测和杀死病毒感染的细胞依赖于MHC-I在细胞表面正确呈现病毒抗原 复合体。NEF通过劫持AP-1及其受体来促进MHC-I复合体的下调，从而颠覆这一过程 TGN上相关的小GTP酶Arf1。MHC-I包含基于TYR的正常版本的不完整版本 分类主题。NEF补充了这个缺陷基序，并将MHC-I转化为AP-1介导的底物 分选到溶酶体进行降解。与AP-1和MHC-I胞浆尾巴组装的Nef的结构 解表明，Nef促进了六方晶格的组装，这些晶格的对称性与 网状蛋白。现在，在先前的解决方案研究之后，将进行重组和结构确定 NEF、MHC-I Tail、AP-1和Arf1在脂膜上的功能设置。Nef对CD28的降调 在SIV和HIV中保守，并由AP-2介导。Nef的CD28下调表型 突变遵循与其他受体不同的模式，这种CD28模式的结构基础是 未知。将确定CD28：HIV-1 Nef：AP-2复合体的结构并利用其进行设计 独一无二地干扰CD28结合位点的突变。在Nef的众多宿主底物中，最重要的是 与病毒感染性有关的是多通道完整膜蛋白SERINC3和5。SERINC结合部位 根据Nef表型，似乎与SIVsmm Nef用来下调的位点重叠 Sian tetherin，但在其他方面与CD3、CD4和MHC-I的已知位置不同。SERINC这样做 不与其他底物有任何明显的相似之处。SERINC以单分散形式纯化 适用于结构测定。包埋SERINC3或5的脂类或洗涤剂的低温电子显微镜结构 将确定与AP-2和HIV-1 Nef的复合体，完成该领域的一个主要目标。