APOBEC proteins and uracil in retrovirus restriction

逆转录病毒限制中的 APOBEC 蛋白和尿嘧啶

• 批准号: 7846580
• 负责人: Reuben S Harris
• 金额: $ 8.7万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-05 至 2010-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7846580
• 关键词: Acquired Immunodeficiency Syndrome; Address; Adenine; Amino Acids; Anti-Retroviral Agents; Attenuated; Base Excision Repairs; Biochemical; Biological Assay; Biological Models; Bypass; Cells; Chimera organism; Cleaved cell; Complement; Complementary DNA; Cytosine; DNA; DNA Repair Enzymes; DNA Repair Pathway; Data; Deaminase; Deamination; Dimerization; Electrophoretic Mobility Shift Assay; Enzymes; Equilibrium; Essential Amino Acids; Event; Failure; Family member; Gagging; Genetic Techniques; Genome; HIV-1; Human; Immune; Infection; Intervention; Invaded; Knowledge; Lead; Lesion; Maps; Mediating; Methods; Modeling; Mutagenesis; Mutation; Nucleic Acids; Oligonucleotides; Outcome; Patients; Pharmacologic Substance; Physiological; Process; Protein Region; Proteins; Publishing; Regulation; Relative (related person); Research; Research Personnel; Retroviridae; Reverse Transcription; Role; Site; Site-Directed Mutagenesis; Somatic Cell Genetics; Southern Blotting; Subfamily lentivirinae; System; Tertiary Protein Structure; Testing; Therapeutic; Uracil; Variant; Viral; Virion; Yeasts; base; carcinogenesis; design; domain mapping; in vivo; microbial; novel; particle; programs; repair enzyme; retroviral-mediated; sample fixation; small hairpin RNA; therapy design; uracil-DNA glycosylase; vif Gene Products; virus host interaction

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.

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。这种新颖的先天免疫防御是通过 APOBEC 催化复制逆转录病毒 cDNA 链中新生胞嘧啶残基的脱氨基作用而发生的。 APOBEC 依赖性逆转录病毒限制至少需要四个机械步骤。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，APOBEC 必须融入病毒颗粒中。第三，逆转录病毒逆转录后，APOBEC 使新生 cDNA 胞嘧啶脱氨。这会在逆转录病毒 DNA 中产生尿嘧啶残基，这被视为细胞 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。后一个事件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。将使用模型微生物、生化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识别细胞 DNA 修复酶，可能是负责第四步的碱基切除修复蛋白。最后，将定义抑制人类主要病毒库中 HIV-1 感染的 APOBEC 蛋白的特性和相对贡献。所提出的研究将共同​​促进对 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 家族成员的错误调节/表达与致癌有关。