Regulation of HIV replication by proteasome degradation of Vif

本研究的目的是阐明蛋白酶体降解辅助蛋白Vif对HIV-1复制的调控。我们试图揭示(1)降解的机理(2)降解幅度的调节机制(3)降解对病毒复制的生物学意义，得到了以下结果：(1)通过脉冲/追赶实验对Vif的蛋白酶体降解进行了生化分析。已知的VIF有两种形式，胞液和细胞骨架。细胞骨架Vif比可溶性胞质Vif更稳定，并随时间积累。VIF的降解特性与细胞类型无关，在非允许细胞和允许细胞中均可观察到。对4种HIV-1辅助蛋白进行了稳定性比较分析。VIF在其短暂的半衰期和降解方面是独一无二的。我们通过免疫沉淀/Western blotting系统监测VIF泛素化。此外，免疫沉淀法观察到Vif与cullin的相互作用，提示cullin可能作为泛素连接酶发挥作用。(2)对一系列VIF缺失突变体的鉴定表明，预测对β链结构的形成起重要作用的氨基酸(氨基酸编号63-70和86-)是维持VIF正常表达水平和病毒感染性的关键。(3)与野生型相比，16种赖氨酸均被转化为精氨酸的Vif突变体表达水平极高，且不能赋予病毒复制能力。然而，我们发现这种功能的缺失是由于22^和26^赖氨酸对病毒感染性的关键作用造成的，这表明精氨酸突变体不能用于阐明降解对病毒复制的生物学意义。

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.