APOBEC proteins and uracil in retrovirus restriction

描述（由申请方提供）：细胞APOBEC 3F和-3G蛋白限制逆转录病毒（包括HIV-1）成功感染人细胞的能力。这种新型的先天免疫防御通过APOBEC催化的复制型逆转录病毒cDNA链中新生胞嘧啶残基的脱氨基作用而发生。APOBEC依赖性逆转录病毒限制需要至少四个机械步骤。首先，这些APOBEC蛋白必须避开HIV-1反防御蛋白Vif，后者可以介导它们的破坏。其次，APOBECs必须整合到病毒颗粒中。第三，在逆转录病毒逆转录时，APOBEC使新生的cDNA胞嘧啶脱氨基。这在逆转录病毒DNA内产生尿嘧啶残基，其被认为是细胞DNA修复途径的损伤。因此，第四步可能涉及负链尿嘧啶作为正链腺嘌呤的复制和固定（超突变），以及其他未鉴定的细胞酶对尿嘧啶的识别和加工。后一种情况可能会阻止逆转录病毒的整合。 该提案将通过描绘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。将使用模型微生物、生物化学和逆转录病毒系统的强大组合，并辅之以体细胞遗传技术。这些系统将进一步应用于鉴定细胞DNA修复酶，可能是负责第四步的碱基切除修复蛋白。最后，APOBEC蛋白的身份和相对贡献，抑制HIV-1感染的主要人类病毒水库将被定义。总之，拟议的研究将导致对APOBEC依赖性逆转录病毒限制机制的基本理解。这将有助于合理设计可能通过调节细胞APOBEC防御和逆转录病毒Vif反防御来减弱HIV-1的疗法。未能理解这些基本步骤的后果可能是灾难性的，因为APOBEC家族成员的错误调节/表达与致癌相关。

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.