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

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向人类的人畜共患传播的关键成分。令人兴奋的是，APOBEC3G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HIV基因组编码的6种辅助蛋白中，有一种蛋白Vif（病毒感染因子）具有抑制APOBEC3G抗病毒活性的特殊功能。在缺乏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结合病毒RNA相关的第二种活性来抑制HIV的进一步复制。Vif最初被认为是通过泛素/蛋白体途径抑制APOBEC3G的降解，但最近的证据表明，抑制可以在不降解的情况下发生。本提案的目的是获得Vif如何能够抑制APOBEC3G功能的结构理解。这两种蛋白之间的相互作用已经知道很多年了，但是由于无法获得大量的可溶性活性蛋白，这两种蛋白的体外研究一直受到阻碍。1)本提案的工作将首先寻求生成一个系统，允许表达和纯化mg量的可溶性活性APOBEC3G。为此，将采用大肠杆菌筛选系统，其中通过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。晶体试验将作为获得结构数据的一种更快速的方法，但APOBEC3G 46 kD的大小也使核磁共振结构成为可能。3)为了对Vif和APOBEC3G之间的相互作用进行分子理解，这两个蛋白将被共结晶，因为70 kD的复合物对于核磁共振研究来说太大了。从历史上看，Vif和APOBEC3G容易与其他蛋白质发生非特异性相互作用并从溶液中沉淀。更可溶形式的APOBEC3G与Vif的共表达将促进形成可溶复合物的特异性相互作用，并最大限度地减少导致沉淀的非特异性相互作用。此外，APOBEC3GA/if复合物结合APOBEC3G进入病毒粒子（如RNA和病毒核衣壳）的重要因子的能力也将被检测。公共卫生相关性：从该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HIV在个体之间的传播以及HIV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.