Mechanism of APOBEC3G-Mediated Hypermutation and Inhibit

APOBEC3G 介导的超突变和抑制机制

• 批准号: 7064431
• 负责人: VINAY K. PATHAK
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/7064431
• 关键词: Retroviridae; aminoacid; aminohydrolases; cell line; cytidine; deoxycytidine; enzyme mechanism; gene expression; gene mutation; host organism interaction; human immunodeficiency virus 1; intermolecular interaction; protein binding; protein degradation; virion; virus genetics; virus protein; virus replication

HIV-1 and other retroviruses occasionally undergo a high rate of G-to-A substitutions, a phenomenon named hypermutation. HIV-1 genomes that fail to express the accessory protein Vif cannot replicate in primary cells or "nonpermissive" cell lines but can replicate in "permissive" cell lines. APOBEC3G, the dominant-acting host restriction factor responsible for the nonpermissive phenotype, is a cytidine deaminase that is packaged into HIV-1 virions in the absence of Vif and deaminates deoxycytidines in minus-strand DNA to deoxyuridines, resulting in massive G-to-A hypermutation and abrogation of viral replication. HIV-1 Vif binds to APOBEC3G and induces its proteosomal degradation in the virus producer cells, suppressing its virion incorporation and restoring viral replication. Human APOBEC3G is degraded in the presence of Vif but simian APOBEC3G is resistant to Vif; using mutational analysis, we and others recently showed that a single amino acid (D128) is responsible for the species specificity of APOBEC3G proteins. The D128 residue either directly interacts with Vif or is involved in conformational changes that occur upon Vif binding. To gain insights into the mechanism by which APOBEC3G inhibits HIV-1 replication, we recently developed a sensitive cytidine deamination assay using scintillation proximity beads. Using this assay, we demonstrated that interactions with viral and nonviral RNAs that are packaged are sufficient for APOBEC3G virion incorporation and that interactions with viral proteins are not essential for virion incorporation. Our future goals are to elucidate the structure and function of APOBEC3G, identify other host proteins that are critical for APOBEC3G-mediated inhibition of HIV-1 replication, define the nature of the Vif-APOBEC3G interactions, and develop agents that interfere with Vif-APOBEC3G interactions as potential antiviral agents.

HIV-1和其他逆转录病毒偶尔会发生高比率的G到A置换，这种现象称为超突变。不能表达辅助蛋白Vif的HIV-1基因组不能在原代细胞或“非允许”细胞系中复制，但可以在“允许”细胞系中复制。APOBEC 3G是导致非允许表型的显性作用宿主限制性因子，是一种胞苷脱氨酶，在不存在Vif的情况下被包装到HIV-1病毒粒子中，并将负链DNA中的脱氧胞苷脱氨为脱氧尿苷，导致大量的G至A超突变和病毒复制的消除。HIV-1 Vif与APOBEC 3G结合并诱导其在病毒生产细胞中的蛋白体降解，抑制其病毒体掺入并恢复病毒复制。人APOBEC 3G在Vif存在下降解，但猿猴APOBEC 3G对Vif具有抗性;使用突变分析，我们和其他人最近表明，单个氨基酸（D128）负责APOBEC 3G蛋白的种属特异性。D128残基直接与Vif相互作用或参与Vif结合后发生的构象变化。为了深入了解APOBEC 3G抑制HIV-1复制的机制，我们最近开发了一种使用闪烁邻近珠的灵敏的胞苷脱氨基测定法。使用该试验，我们证明了与包装的病毒和非病毒RNA的相互作用足以使APOBEC 3G病毒体掺入，并且与病毒蛋白的相互作用对于病毒体掺入不是必需的。我们未来的目标是阐明APOBEC 3G的结构和功能，鉴定其他对APOBEC 3G介导的HIV-1复制抑制至关重要的宿主蛋白，定义Vif-APOBEC 3G相互作用的性质，并开发干扰Vif-APOBEC 3G相互作用的药物作为潜在的抗病毒药物。