APOBEC proteins and uracil in retrovirus restriction

描述（由申请人提供）：细胞APOBEC3F和-3G蛋白限制逆转录病毒（包括HIV-1）成功感染人类细胞的能力。这种新的先天免疫防御是由apobecc催化的逆转录病毒cDNA链中新生胞嘧啶残基的脱胺作用发生的。apobecc依赖性逆转录病毒限制至少需要四个机制步骤。首先，这些APOBEC蛋白必须避开HIV-1抗防御蛋白Vif， Vif可以介导它们的破坏。其次，APOBECs必须与病毒颗粒结合。第三，在逆转录逆转录过程中，APOBECs脱氨新生cDNA胞嘧啶。这在逆转录病毒DNA中产生尿嘧啶残基，这被细胞DNA修复途径视为损伤。因此，第四步可能既包括负链尿嘧啶作为正链腺嘌呤的复制和固定（超突变），也包括其他未知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。

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.