Cytidine deaminase and HIV-1 replication

描述(由申请人提供)：G到A的高度突变是灵长类慢病毒和其他逆转录病毒在体内和细胞培养中复制过程中的特征之一。然而，这一过程的分子机制仍有待阐明。最近，我们证明了人类免疫缺陷病毒1型(HIV-1)的内源性复制抑制因子CEM15/APOBEC3G是一种胞苷脱氨酶，能够在新合成的病毒DNA中诱导G到A的高度突变。这种作用可以被HIV-1病毒粒子传染性因子(VIF)所抵消。我们认为，该病毒DNA突变体可能会导致新生病毒逆转录本的“致命性超突变”或不稳定，这可能是Avif表型的原因。重要的是，CEM15/APOBEC3G介导的“非致死性超突变”在复制的病毒基因组中的积累可能是灵长类慢病毒种群遗传变异的重要原因。为了进一步研究CEM15/APOBEC3G诱导的高突变的分子机制，我们将：(1)。进一步探讨CEM15诱导DNA脱氨基的机制。特别是，我们将确定CEM15的底物特异性，通过检测CEM15与病毒蛋白之间可能的相互作用以及CEM15与细胞蛋白的可能相互作用来分析所谓的编辑小体的组成。CEM15如何结合到HIV-1病毒粒子中的机制将被研究。(2)。研究UDG在新生逆转录不稳定性产生中的可能作用。我们将检验病毒DNA负链上的碱基位置可以被无嘌呤/脱嘧啶(AP)-核酸内切酶切割的假设。(3)。进一步确定病毒在细胞培养中传代过程中发生高突变的分子机制。将使用RNA干扰(RNAi)技术来中和非许可细胞中的CEM15。研究CEM15介导的超突变对耐药突变产生的可能影响。(4)。研究CEM15基因表达的调控。特别是，将确定CEM 15的启动子并检测其活性。(5)。研究其他胞苷脱氨酶，如AID、APOBEC1、APOBEC2和APOBEC3A至3F等，是否能在没有v/f的情况下抑制HIV-1和其他逆转录病毒的复制，并诱导新合成的病毒DNA发生高度突变。我们相信，这些替代但互补的方法将丰富我们对这一抗病毒防御系统的认识。这些项目的研究结果将导致找到抗击艾滋病毒-1复制的新战略。

DESCRIPTION (provided by applicant): G to A hypermutation is one of the characteristics of primate lentiviruses, as well as other retroviruses, during replication in vivo and in cell culture. The molecular mechanisms of this process, however, remain to be clarified. Recently, we have demonstrated that CEM15/APOBEC3G, an endogenous inhibitor of human immunodeficiency virus type 1 (HIV-1) replication, is a cytidine deaminase and is able to induce G to A hypermutation in the newly-synthesized viral DNA. This effect can be counteracted by the HIV- 1 virion infectivity factor (Vif). We suggest that this viral DNA mutator may induce either "lethal hypermutation" or instability of the incoming nascent viral reverse transcripts, which could account for the Avifphenotype. Importantly, the accumulation of CEM15/APOBEC3G-mediated "non-lethal hypermutation" in the replicating viral genome could potently contribute to the genetic variation of primate lentiviral populations.To further investigate the molecular mechanism of hypermutationqnduced by CEM15/APOBEC3G, We will: (1). Further explore the mechanism of DNA deamination induced by CEM15. Especially, we will identify the substrate specificity of CEM15, analysis the components of the so called "editosome" by examining the possible interaction between CEM15 and viral proteins and examining the possible interaction between CEM15 and cellular proteins. The mechanism how CEM15 incorporates into HIV- 1 virions will be investigated. (2). Study the possible role of UDG in the generation of instability of nascent reverse transcripts. The hypothesis that the abasic site in the minus strand of viral DNA could be cleaved by apurinic/apyrimidinic(AP)-endonuclease will be examined. (3). Further determine the molecular mechanism of hypermutation occurring during viral passage in the cell culture. An RNA interference (RNAi) technique will be used to counteract CEM15 in the non-permissive cells. Investigate the possible effect of CEM15- mediated hypermutation upon the emergence of drug-resistant mutants. (4). Investigate the regulation of CEM15 gene expression. Especially, the promoter of CEM 15 will be identified and its activity will be examined. (5). Investigate whether other cytidine deaminases, such as AID, APOBEC1, APOBEC2, and APOBEC3A to 3F, etc, could inhibit the replication of HIV-1 and other retroviruses and induce hypermutation in the newly-synthesized viral DNA in the absence of v/f. We believe that these alternative but complementary approaches would enrich our knowledge regarding this anti-viral defense system. The research result from these projects will lead to find a novel strategy to combat HIV-1 replication.