Structural Determination of Full-length APOBEC3G and the APOBEC3G/Vif Complex

全长 APOBEC3G 和 APOBEC3G/Vif 复合物的结构测定

• 批准号: 7913046
• 负责人: Troy Christopher Krzysiak
• 金额: $ 5.05万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-06 至 2011-08-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7913046
• 关键词: Acquired Immunodeficiency Syndrome; Ammonium; Antiviral Agents; Apolipoproteins; Baculoviruses; Binding; Biochemical; Biological Assay; Biological Factors; Calorimetry; Cells; Communities; Complex; Cytidine Deaminase; Data; Deamination; Engineering; Enzymes; Escherichia coli; Fluorescence; Future; Generations; Genome; Genomics; Goals; HIV; HIV Infections; Human; Human Genome; Immune system; Immunity; In Vitro; Individual; Infection; Ions; Knowledge; Lead; Length; Libraries; Link; Messenger RNA; Methods; Modeling; Modern Medicine; Molecular; Molecular Conformation; Monitor; Mutagenesis; Mutate; Mutation; Names; Nucleocapsid; Oligonucleotides; Pathway interactions; Pharmaceutical Preparations; Precipitation; Preclinical Drug Evaluation; Property; Proteins; RNA; Rabies; Reagent; Reporter; Research Personnel; Retroelements; Retroviridae; Sampling; Screening procedure; Site-Directed Mutagenesis; Solubility; Solutions; Structure; Subfamily lentivirinae; Surface; Surface Plasmon Resonance; System; Therapeutic; Titrations; Ubiquitin; Variant; Viral; Viral Genome; Virion; Virus; Virus Diseases; Work; X-Ray Crystallography; apolipoprotein B mRNA editing enzyme; base; design; homologous recombination; milligram; model design; multicatalytic endopeptidase complex; mutant; polypeptide; prevent; protein degradation; public health relevance; rapid technique; research study; structural biology; success; transmission process; vif Gene Products; viral RNA

DESCRIPTION (provided by applicant): APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G) has been identified as a key component in restricting the zoonotic transmission of retroviruses to humans. Excitingly, it appears that the antiviral activity of APOBEC3G could be extended to restrict the infection of viruses, such as HIV, that the human immune system and modern medicine have yet to conquer. Of the six accessory proteins encoded by the HIV genome, one protein, Vif (viral infectivity factor), performs the specific function of inhibiting APOBEC3G antiviral activity. In the absence of Vif, APOBEC3G is incorporated into HIV virions, and inhibits further HIV replication through hypermutation of the viral genome and/or a second activity linked to APOBEC3G binding to viral RNA. Vif is primarily believed to inhibit APOBEC3G by targeting it for degradation via the ubiquitin/proteosome pathway, but recent evidence suggests that inhibition can occur without degradation. The purpose of this proposal is to obtain a structural understanding of how Vif is able to inhibit APOBEC3G function. The interaction between these two proteins has been know for many years but in vitro studies of both proteins have been hampered by the inability to acquire large amounts of soluble, active protein. 1) Work from this proposal will first seek to generate a system allowing for expression and purification of mg amounts of soluble, active APOBEC3G. To do this, an E. coli screening system will be employed in which APOBEC3G mutants with enhanced solubility will be detected through the fluorescence of a GFP reporter fusion. 2) The second goal is to obtain the structure of APOBEC3G. Crystal trials will be conduted as a more rapid method to obtain structural data, but the size of APOBEC3G, 46 kD, makes an NMR structure also possible. 3) To detevelope a molecular understanding of the interaction between Vif and APOBEC3G, the two proteins will be cocrystalized as the size of the complex, 70 kD, is too large for NMR studies. Historically, Vif and APOBEC3G have been prone to nonspecifically interact with other proteins and precipitate from solution. Coexpression of a more soluble form of APOBEC3G with Vif will encourage formation of specific interactions leading to soluble complexes and and minimize the nonspecific interactions that lead to precipitation. Also, the ability of APOBEC3GA/if complexes to bind important factors for the incorporation of APOBEC3G into virions, such as RNA and viral nucleocapsid, will be examined. Public Health Relevance: The structural data obtained from this proposal will serve as a model for the screening and/or design of new drugs to inhibit the interaction of Vif with APOBEC3G and therefore inhibit HIV spread among individuals as well as progression of HIV infected individuals to AIDS.

描述（由申请人提供）：apobec3g（载脂蛋白B mRNA编辑酶，催化多肽样3G）已被确定为限制逆转录病毒对人的人畜共动性传播的关键成分。令人兴奋的是，似乎可以扩展APOBEC3G的抗病毒活性以限制诸如HIV之类的病毒感染，即人类免疫系统和现代医学尚未征服。在由HIV基因组编码的六种辅助蛋白中，一种蛋白质，VIF（病毒感染因子）执行了抑制APOBEC3G抗病毒活性的特定功能。在没有VIF的情况下，将APOBEC3G掺入HIV病毒体中，并通过病毒基因组的过度突击和/或第二种与APOBEC3G与病毒RNA结合的活动抑制进一步的HIV复制。 VIF主要据信通过通过泛素/蛋白质体途径靶向降解来抑制APOBEC3G，但最近的证据表明，抑制可能不会出现而不会降解。该建议的目的是获得对VIF如何抑制APOBEC3G功能的结构理解。这两种蛋白质之间的相互作用已经知道多年了，但是对两种蛋白质的体外研究都无法因获得大量可溶性活性蛋白而受到阻碍。 1）该提案的工作将首先寻求生成一个系统，以表达和纯化Mg量的可溶性Aptive Apobec3g。为此，将采用大肠杆菌筛选系统，其中将通过GFP报告基因融合的荧光检测具有增强溶解度的APOBEC3G突变体。 2）第二个目标是获得apobec3g的结构。晶体试验将被指定为获得结构数据的更快的方法，但是Apobec3g（46 kd）的大小也使NMR结构也成为可能。 3）为了确定对VIF与Apobec3g之间相互作用的分子理解，将两种蛋白质共结合为NMR研究，将两种蛋白共结合为70 kD的尺寸。从历史上看，VIF和APOBEC3G易于非特异性与其他蛋白质相互作用，并从溶液中沉淀出来。 apobec3g与VIF的更易溶形式的共表达将鼓励形成特定相互作用，从而导致可溶性复合物，并最大程度地减少导致沉淀的非特异性相互作用。同样，将检查apobec3ga/IF复合物结合重要因素以将apobec3g掺入病毒体中的重要因素（例如RNA和病毒核蛋白酶）。公共卫生相关性：从该提案获得的结构数据将作为筛查和/或设计新药的模型，以抑制VIF与APOBEC3G的相互作用，从而抑制个体之间的HIV传播以及HIV感染的个体向AIDS的进展。