Cytidine deaminase and HIV-1 replication

描述（由申请人提供）：G到A的超突变是灵长类慢病毒以及其他逆转录病毒在体内复制和细胞培养过程中的特征之一。然而，这一过程的分子机制仍有待阐明。最近，我们已经证明了CEM15/APOBEC3G是一种内源性的人类免疫缺陷病毒1型（HIV-1）复制抑制剂，是一种胞苷脱氨酶，能够在新合成的病毒DNA中诱导G到a的高突变。这种作用可被HIV- 1病毒粒子感染因子（Vif）抵消。我们认为这种病毒DNA突变可能诱导“致死性高突变”或新生病毒逆转录本的不稳定性，这可能解释了avif表型。重要的是，在复制病毒基因组中，CEM15/ apobec3g介导的“非致死性高突变”的积累可能会导致灵长类慢病毒群体的遗传变异。为了进一步研究CEM15/APOBEC3G诱导高突变的分子机制，我们将：(1)。进一步探讨CEM15诱导DNA脱氨的机制。特别是，我们将确定CEM15的底物特异性，通过检查CEM15与病毒蛋白之间可能的相互作用以及检查CEM15与细胞蛋白之间可能的相互作用来分析所谓的“编辑体”的组成。今后将进一步研究CEM15与HIV- 1病毒体结合的机制。（2）. 研究UDG在新生逆转录物不稳定性产生中的可能作用。病毒DNA负链上的碱基位点可以被apurinic/apyrimidinic(AP)-核酸内切酶切割的假说将被检验。（3）. 进一步确定细胞培养中病毒传代过程中发生超突变的分子机制。将使用RNA干扰（RNAi）技术来抵消非允许细胞中的CEM15。探讨CEM15介导的高突变对耐药突变体产生的可能影响。（4）. 研究CEM15基因表达的调控。特别是，cem15的启动子将被确定并进行活性研究。(5)。考察AID、APOBEC1、APOBEC2、APOBEC3A to 3F等胞苷脱氨酶在缺乏v/f的情况下是否能抑制HIV-1等逆转录病毒的复制，诱导新合成的病毒DNA发生高突变。我们相信这些替代但互补的方法将丰富我们对这种抗病毒防御系统的认识。这些项目的研究结果将有助于找到一种对抗HIV-1复制的新策略。

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.