Regulation of HIV replication by proteasome degradation of Vif

本研究的目的是阐明调节HIV-1复制的蛋白酶体降解的辅助蛋白Vif。本论文试图揭示（1）降解的机制（2）降解幅度的调控机制（3）降解对病毒复制的生物学意义，并得到以下结果。（1）采用脉冲/追赶实验（pulse/chase experiment）对Vif蛋白酶体降解进行生化分析。已知Vif有两种形式，细胞溶质和细胞骨架。发现细胞骨架Vif比可溶性胞质Vif更稳定，并且随着时间积累。Vif的降解特性与细胞类型无关，在非允许性和允许性细胞中均观察到。对四种HIV-1辅助蛋白进行了比较稳定性分析。Vif在其短半衰期和降解方面是独特的。我们用免疫沉淀/Western blotting系统监测Vif的泛素化。免疫沉淀法观察到Vif与Cullin的相互作用，提示Cullin可能是泛素连接酶。（2）对一系列vif缺失突变体的鉴定表明，对β链结构形成重要的氨基酸（63 -70和86-89氨基酸）对维持Vif的正常表达水平和病毒的感染性至关重要。（3）Vif突变体，其中所有16个赖氨酸都变成了赖氨酸，发现与野生型相比表达水平极高，并且没有赋予病毒复制能力。然而，我们发现功能的缺乏是由22^<nd>和26^<th>赖氨酸对病毒感染性的关键作用引起的，这表明精氨酸突变体对于阐明降解对病毒复制的生物学意义是无用的。

An objective of this study is to elucidate regulation of HIV-1 replication by proteasome-degradation of accessory protein Vif. We have been trying to reveal (1)mechanism of the degradation (2)regulation mechanism of magnitude of the degradation (3)biological significance of the degradation on viral replication, and gotten the following results.(1)Proteasome degradation of Vif was analyzed biochemically by pulse/chase experiments. Two forms of Vif, cytosolic and cytoskeletal, have been known. Cytoskeletal Vif was found to be more stable than soluble cytosolic Vif, and accumulate with time. The degradation characteristics of Vif were cell type-independent and observed in both non-permissive and permissive cells. Comparative stability analysis of the four HIV-1 accessory proteins were performed. Vif was unique in its short half-life and in the degradation. We monitored Vif ubiquitination by immunoprecipitation/Western blotting system. Futhermore, interaction of Vif with Cullin was observed by immunoprecipitation, suggesting the possibility that Cullin works as ubiquitin ligase. (2)Characterization of a series of vif deletion mutants showed that amino acids predicted to be important for formation of β-strand structures (amino acid nos.63-70 and 86-89) were critical for maintaining a normal expression level of Vif and for viral infectivity. (3)Vif mutant, in which all 16 lysines were changed to arginines, was found to be extremely high expression level compared with wild type, and not to confer replication ability on virus. However, we showed that the lack of function was caused by the critical role of 22^<nd> and 26^<th> lysines on viral infectivity, suggesting that arginine mutants are not useful to elucidate biological significance of the degradation on viral replication.