Mechanism of APOBEC3G-Mediated Hypermutation and Inhibit

HIV-1和其他逆转录病毒偶尔会经历高速率的G-to-A取代，这种现象被称为超突变。不能表达辅助蛋白Vif的HIV-1基因组不能在原代细胞或“非受纳”细胞系中复制，但可以在“受纳”细胞系中复制。APOBEC3G是导致非允许表型的主要宿主限制因子，它是一种胞苷脱氨酶，在缺乏Vif的情况下被包装到HIV-1病毒粒子中，并将负链DNA中的脱氧胞苷脱胺为脱氧尿苷，导致大量G-to-A高突变和病毒复制的终止。HIV-1 Vif与APOBEC3G结合并诱导其在病毒产生细胞内的蛋白体降解，抑制其病毒粒子合并并恢复病毒复制。人类APOBEC3G在Vif存在下降解，但猿猴APOBEC3G对Vif有抗性；通过突变分析，我们和其他人最近发现一个氨基酸（D128）负责APOBEC3G蛋白的物种特异性。D128残基要么直接与Vif相互作用，要么参与Vif结合后发生的构象变化。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接近珠的敏感胞苷脱胺测定方法。通过该实验，我们证明了与包装的病毒和非病毒rna的相互作用足以整合APOBEC3G病毒粒子，而与病毒蛋白的相互作用对于病毒粒子的整合并不是必需的。我们未来的目标是阐明APOBEC3G的结构和功能，鉴定其他对APOBEC3G介导的HIV-1复制抑制至关重要的宿主蛋白，确定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。

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.