APOBEC proteins and uracil in retrovirus restriction

描述(申请人提供)：细胞APOBEC3F和-3G蛋白限制包括HIV-1在内的逆转录病毒成功感染人类细胞的能力。这种新的天然免疫防御是通过APOBEC催化的复制逆转录病毒的cDNA链中新生的胞嘧啶残基的脱氨基来实现的。APOBEC依赖的逆转录病毒限制至少需要四个机械性步骤。首先，这些APOBEC蛋白必须避免HIV-1对抗防御蛋白Vif，它可以介导它们的破坏。其次，APOBEC必须整合到病毒颗粒中。第三，在逆转录病毒逆转录时，APOBECs使新生的cDNA胞嘧啶脱氨基。这会在逆转录病毒DNA中产生尿嘧啶残留物，细胞DNA修复途径将其视为损伤。因此，第四步可能涉及复制和固定负链尿嘧啶为正链腺嘌呤(超突变)，以及通过其他不明细胞酶识别和处理尿嘧啶。后一种情况很可能会阻止逆转录病毒的整合。 该提案将通过描述前三个步骤所需的关键APOBEC蛋白结构域和氨基酸残基来更好地定义这些步骤。将使用模拟微生物、生化和逆转录病毒系统的强大组合，并与体细胞遗传技术相辅相成。这些系统将进一步用于识别负责第四步的细胞DNA修复酶，可能是碱基切除修复蛋白。最后，将确定在主要的人类病毒库中抑制HIV-1感染的APOBEC蛋白的特性和相对贡献。总之，拟议的研究将导致对APOBEC依赖的逆转录病毒限制的机制有一个基本的理解。这将有助于合理设计治疗方法，通过调节细胞APOBEC防御和逆转录病毒Vif对抗防御来潜在地减弱HIV-1。未能认识到这些基本步骤的后果可能是灾难性的，因为APOBEC家族成员的错误调控/表达与癌症的发生有关。

DESCRIPTION (provided by applicant): The cellular APOBEC3F and -3G proteins restrict the ability of retroviruses, including HIV-1, to successfully infect human cells. This novel innate immune defense occurs by APOBEC-catalyzed deamination of nascent cytosine residues in the cDNA strand of a replicating retrovirus. At least four mechanistic steps are required for APOBEC-dependent retroviral restriction. First, these APOBEC proteins must avoid the HIV-1 counterdefense protein Vif, which can mediate their destruction. Second, the APOBECs must incorporate into viral particles. Third, upon retroviral reverse transcription the APOBECs deaminate nascent cDNA cytosines. This produces uracil residues within the retroviral DNA, which are regarded as lesions by cellular DNA repair pathways. Thus, the fourth step may involve both the replication and fixation of minus strand uracils as plus strand adenines (hypermutation) and also the recognition and processing of uracils by other unidentified cellular enzymes. The latter event is likely to block the retrovirus from integrating. This proposal will better define these steps by delineating the key APOBEC protein domains and amino acid residues required for the first three steps. A powerful combination of model microbial, biochemical and retroviral systems will be used and complemented with somatic cell genetic techniques. These systems will be further applied to identify cellular DNA repair enzymes, likely base excision repair proteins, responsible for the fourth step. Finally, the identity and relative contributions of the APOBEC proteins that inhibit HIV-1 infection in the primary human viral reservoirs will be defined. Together the proposed studies will result in a fundamental understanding of the mechanism of APOBEC-dependent retroviral restriction. This will assist the rational design of therapies that could potentially attenuate HIV-1 by modulating the cellular APOBEC defense and the retroviral Vif counterdefense. The consequences of failing to appreciate these fundamental steps could be catastrophic as mis-regulation/expression of APOBEC family members has been associated with carcinogenesis.