APOBEC3G/CEM15 Inhibition of Lentivirus Replication

描述(申请人提供)：慢病毒病毒感染性因子(Vif)是一种辅助蛋白，是病毒在原代细胞和一些(但不是全部)转化的T细胞系中高效复制所必需的。由于细胞蛋白APOBEC3G/CEM15的表达，Vif(Delta-Vif)基因缺陷的HIV-1未能在非允许细胞中复制。APOBEC3G是核糖核酸编辑酶胞苷脱氨酶家族的成员。更简单的病毒，如小鼠白血病病毒，不编码Vif。然而，克隆和功能分析表明，小鼠APOBEC3G对Delta-Vif和野生型HIV-1具有很强的抑制作用。同样，非洲绿猴APOBEC3G对野生型和Delta-Vif HIV-1也有活性，但只抑制Delta-Vif，而不抑制SIVagm。这些发现表明Vif和APOBEC3G之间存在物种特异性的相互作用。该项目的目标是了解APOBEC3G降低Delta-Vif HIV-1传染性的机制，并了解Vif如何缓解这种抑制。人：AGM嵌合APOBEC3G将被构建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APOBEC3G的细胞中复制。APOBEC3G和VIF之间的物理相互作用以及与细胞因子的相互作用将通过免疫共沉淀进行检测。为了确定APOBEC3G的病毒编辑底物，对APOBEC3G存在和不存在的情况下生长的野生型和Delta-Vif病毒的病毒RNA、病毒mRNA、病毒cDNA和前病毒DNA进行了测序。此外，为了了解APOBEC3G的生理作用，将构建基因敲除小鼠。将使用几种免疫功能分析来确定缺陷对免疫系统的影响。这些研究提供的见解将对以APOBEC3G：VIF相互作用为目标开发新的艾滋病毒疗法产生影响。这类药物会干扰APOBEC3G：VIF相互作用，而不会抑制APOBEC3G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.