Cytidine deaminase and HIV-1 replication

胞苷脱氨酶和 HIV-1 复制

• 批准号: 7033935
• 负责人: Hui Zhang
• 金额: $ 30.66万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7033935
• 关键词: RNA interference; SDS polyacrylamide gel electrophoresis; amination; aminohydrolases; clinical research; cytidine; gene expression; gene mutation; human immunodeficiency virus 1; human subject; polymerase chain reaction; protein protein interaction; tissue /cell culture; virion; virus DNA; virus protein; virus replication

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.

描述（由申请人提供）：G至A超突变是灵长类慢病毒以及其他逆转录病毒在体内和细胞培养中复制期间的特征之一。然而，这一过程的分子机制仍有待阐明。最近，我们证明了 CEM15/APOBEC3G 是人类免疫缺陷病毒 1 型 (HIV-1) 复制的内源性抑制剂，是一种胞苷脱氨酶，能够在新合成的病毒 DNA 中诱导 G 到 A 超突变。这种效应可以被 HIV-1 病毒粒子感染因子 (Vif) 抵消。我们认为，这种病毒 DNA 突变体可能会诱导“致命的超突变”或新生病毒逆转录物的不稳定，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”在复制病毒基因组中的积累可能有力地促进灵长类慢病毒群体的遗传变异。为了进一步研究CEM15/APOBEC3G引起的超突变的分子机制，我们将：（1）。进一步探讨CEM15诱导DNA脱氨作用的机制。特别是，我们将鉴定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”的成分。我们将研究 CEM15 如何融入 HIV-1 病毒粒子的机制。 （2）。研究 UDG 在新生逆转录本不稳定产生中的可能作用。病毒 DNA 负链中的脱碱基位点可以被脱嘌呤/脱嘧啶 (AP) 核酸内切酶切割的假设将得到检验。 （3）。进一步确定病毒在细胞培养物中传代过程中发生超突变的分子机制。 RNA 干扰 (RNAi) 技术将用于抵消不允许的细胞中的 CEM15。研究 CEM15 介导的超突变对耐药突变体出现的可能影响。 （4）。研究 CEM15 基因表达的调控。特别是，将鉴定CEM 15的启动子并检查其活性。 （5）。研究其他胞苷脱氨酶，如AID、APOBEC1、APOBEC2和APOBEC3A至3F等是否可以抑制HIV-1和其他逆转录病毒的复制，并在没有v/f的情况下诱导新合成的病毒DNA发生超突变。我们相信这些替代但互补的方法将丰富我们关于这种抗病毒防御系统的知识。这些项目的研究结果将导致找到一种对抗 HIV-1 复制的新策略。