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

描述(申请人提供)：APOBEC3G(载脂蛋白B mRNA编辑酶，催化多肽样3G)已被确定为限制人畜共患逆转录病毒向人类传播的关键成分。令人兴奋的是，APOBEC3G的抗病毒活性似乎可以延长，以限制人类免疫系统和现代医学尚未攻克的病毒的感染，如艾滋病毒。在HIV基因组编码的六种辅助蛋白中，有一种蛋白Vif(病毒感染性因子)具有抑制APOBEC3G抗病毒活性的特殊功能。在没有VIF的情况下，APOBEC3G被整合到HIV病毒粒子中，并通过病毒基因组的超突变和/或与APOBEC3G与病毒RNA结合的第二活性来抑制HIV的进一步复制。VIF主要被认为通过泛素/蛋白酶体途径靶向降解APOBEC3G来抑制APOBEC3G，但最近的证据表明，这种抑制可以在没有降解的情况下发生。这项建议的目的是为了从结构上了解Vif如何能够抑制APOBEC3G功能。这两种蛋白质之间的相互作用已经知道很多年了，但由于无法获得大量的可溶性活性蛋白质，这两种蛋白质的体外研究一直受到阻碍。1)本提案的工作将首先寻求产生一种允许表达和纯化mg量的可溶性、活性APOBEC3G的系统。为此，将采用大肠杆菌筛选系统，其中将通过GFP报告融合的荧光检测具有增强溶解性的APOBEC3G突变体。2)第二个目标是获得APOBEC3G的结构。晶体试验将被认为是一种更快速地获得结构数据的方法，但APOBEC3G的大小(46kD)也使核磁共振结构成为可能。3)为了从分子上了解Vif和APOBEC3G之间的相互作用，这两种蛋白质将被共结晶，因为70kD的复合体太大，无法进行核磁共振研究。从历史上看，Vif和APOBEC3G容易与其他蛋白质非特异性相互作用并从溶液中沉淀。与Vif共表达更易溶的APOBEC3G将鼓励形成导致可溶复合体的特定相互作用，并最大限度地减少导致沉淀的非特异性相互作用。此外，还将检测APOBEC3GA/IF复合体结合APOBEC3G与病毒粒子(如RNA和病毒核衣壳)结合的重要因素的能力。公共卫生相关性：从这项提案中获得的结构数据将作为筛选和/或设计新药的模型，以抑制VIF与APOBEC3G的相互作用，从而抑制艾滋病毒在个人中的传播以及艾滋病毒感染者向艾滋病的进展。

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.