APOBEC3G/CEM15 Inhibition of Lentivirus Replication

描述（由申请方提供）：慢病毒病毒粒子感染因子（Vif）是病毒在原代细胞和部分（但非全部）转化T细胞系中生产性复制所需的辅助蛋白。由于细胞蛋白APOBEC 3G/CEM 15的表达，Vif（delta-Vif）基因缺陷的HIV-1无法在非允许细胞中复制。APOBEC 3G是RNA编辑酶胞苷脱氨酶家族的成员。更简单的病毒如鼠白血病病毒不编码Vif。然而，克隆和功能分析表明，小鼠APOBEC 3G是delta-Vif和野生型HIV-1的有效抑制剂。类似地，非洲绿色猴APOBEC 3G对野生型和delta-Vif HIV-1也有活性，但仅抑制delta-Vif，而不抑制SIVagm。这些发现表明Vif和APOBEC 3G之间存在物种特异性相互作用。该项目的目标是了解APOBEC 3G降低delta-Vif HIV-1感染性的机制，并了解Vif如何增强这种抑制作用。将构建人：AGM嵌合APOBEC 3G并用于确定决定相互作用的结构域。HIV-1将适应于在表达非同源APOBEC 3G的细胞中复制。将通过免疫共沉淀检测APOBEC 3G和Vif彼此之间以及与细胞因子之间的物理相互作用。为了鉴定APOBEC 3G的病毒编辑底物，将对在存在或不存在APOBEC 3G的情况下生长的野生型和delta-Vif病毒的纯化病毒RNA、病毒mRNA、病毒cDNA和前病毒DNA进行测序。此外，为了理解APOBEC 3G的生理作用，将构建敲除小鼠。将使用几种免疫功能测定来确定缺陷对免疫系统的影响。这些研究提供的见解将对靶向APOBEC 3G：Vif相互作用以开发新型HIV治疗药物产生影响。这些药物会干扰APOBEC 3G：Vif相互作用，而不抑制APOBEC 3G功能。

DESCRIPTION (provided by applicant): The lentivirus virion infectivity factor (Vif) is an accessory protein that is required for productive replication of the virus in primary cells and some, but not all, transformed T cell lines. HIV-1 that is genetically deficient in Vif (delta-Vif) fails to replicate in nonpermissive cells as the result of the expression of the cell protein APOBEC3G/CEM15. APOBEC3G is a member of the cytidine deaminase family of RNA editing enzymes. Simpler viruses such as murine leukemia virus do not encode Vif. Nevertheless, cloning and functional analysis showed that mouse APOBEC3G is a potent inhibitor of delta-Vif and wild-type HIV-1. Similarly, African Green monkey APOBEC3G was also active against wild-type and delta-Vif HIV-1 but only inhibited delta-Vif but not SIVagm. These findings suggest a species-specific interaction between Vif and APOBEC3G. The goals of this project are to understand the mechanism by which APOBEC3G reduces the infectivity of delta-Vif HIV-1 and to understand how Vif alleviates this inhibition. Human:AGM chimeric APOBEC3Gs will be constructed and used to determine the domain that determines the interaction. HIV-1 will be adapted to replicate in cells expressing non-homologous APOBEC3G. Physical interaction of APOBEC3G and Vif with each other and with cellular factors will be detected by coimmunoprecipitation. To identify a viral editing substrate for APOBEC3G, encapsidated viral RNA, viral mRNA, viral cDNA and proviral DNA will be sequenced from wild-type and delta-Vif virus grown in the presence or absence of APOBEC3G. In addition, to understand the role physiological role of APOBEC3G, knock-out mice will be constructed. Several immune function assays will be used to determine the effects of the deficiency on the immune system. Insights provided by these studies will have implications regarding targeting the APOBEC3G:Vif interaction for the development of novel HIV therapeutics. Such drugs would interfere with the APOBEC3G:Vif interaction without inhibiting APOBEC3G function.