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 替换，这种现象称为超突变。无法表达辅助蛋白 Vif 的 HIV-1 基因组无法在原代细胞或“非许可”细胞系中复制，但可以在“许可”细胞系中复制。 APOBEC3G 是导致非允许表型的显性作用宿主限制因子，是一种胞苷脱氨酶，在缺乏 Vif 的情况下被包装到 HIV-1 病毒颗粒中，并将负链 DNA 中的脱氧胞苷脱氨基为脱氧尿苷，导致大规模 G 至 A 超突变并终止病毒复制。 HIV-1 Vif 与 APOBEC3G 结合并诱导其在病毒产生细胞中降解，从而抑制其病毒颗粒掺入并恢复病毒复制。人类 APOBEC3G 在 Vif 存在下会被降解，但猿猴 APOBEC3G 对 Vif 具有抗性；通过突变分析，我们和其他人最近表明单个氨基酸 (D128) 负责 APOBEC3G 蛋白的物种特异性。 D128 残基直接与 Vif 相互作用，或者参与 Vif 结合时发生的构象变化。为了深入了解 APOBEC3G 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敏感胞苷脱氨测定法。使用该测定，我们证明与包装的病毒和非病毒 RNA 的相互作用足以实现 APOBEC3G 病毒粒子的掺入，并且与病毒蛋白的相互作用对于病毒粒子的掺入不是必需的。我们未来的目标是阐明 APOBEC3G 的结构和功能，识别对 APOBEC3G 介导的 HIV-1 复制抑制至关重要的其他宿主蛋白，定义 Vif-APOBEC3G 相互作用的性质，并开发干扰 Vif-APOBEC3G 相互作用的药物作为潜在的抗病毒药物。