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

描述（由申请方提供）：APOBEC 3G（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向人类传播的关键组分。令人兴奋的是，APOBEC 3G的抗病毒活性似乎可以扩展到限制人类免疫系统和现代医学尚未征服的病毒（如HIV）的感染。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）执行抑制APOBEC 3G抗病毒活性的特定功能。在不存在Vif的情况下，APOBEC 3G被掺入HIV病毒体中，并通过病毒基因组的超突变和/或与APOBEC 3G结合病毒RNA相关的第二活性来抑制HIV的进一步复制。Vif主要被认为通过靶向APOBEC 3G以经由泛素/蛋白体途径降解而抑制APOBEC 3G，但最近的证据表明，抑制可以在没有降解的情况下发生。该提案的目的是获得Vif如何能够抑制APOBEC 3G功能的结构理解。这两种蛋白质之间的相互作用已经知道了很多年，但这两种蛋白质的体外研究一直受到无法获得大量可溶性活性蛋白质的阻碍。1)该提案的工作将首先寻求产生允许表达和纯化mg量的可溶性活性APOBEC 3G的系统。要做到这一点，E。将采用大肠杆菌筛选系统，其中具有增强的溶解性的APOBEC 3G突变体将通过GFP报告融合物的荧光检测。2)第二个目标是获得APOBEC 3G的结构。晶体试验将被视为获得结构数据的更快速方法，但APOBEC 3G的大小（46 kD）也使NMR结构成为可能。3)为了确定Vif和APOBEC 3G之间相互作用的分子理解，这两种蛋白质将共结晶，因为复合物的大小（70 kD）对于NMR研究来说太大了。从历史上看，Vif和APOBEC 3G倾向于与其他蛋白质非特异性相互作用并从溶液中沉淀。更可溶形式的APOBEC 3G与Vif的共表达将促进导致可溶性复合物的特异性相互作用的形成，并使导致沉淀的非特异性相互作用最小化。此外，将检查APOBEC 3GA/IF复合物结合用于将APOBEC 3G掺入病毒体的重要因子（例如RNA和病毒核衣壳）的能力。公共卫生相关性：从该提议中获得的结构数据将用作筛选和/或设计新药的模型，以抑制Vif与APOBEC 3G的相互作用，从而抑制HIV在个体中的传播以及HIV感染个体向AIDS的进展。

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.