Characterization of retroviral restriction factor APOBEC3H

建议书摘要/摘要 先天免疫系统利用限制因素来阻止外来rna物种的整合，如 作为病毒RNA，进入宿主细胞的基因组。作为回应，病毒调整机制以逃避限制 它们的自然寄主。这种类型的分子军备竞赛的一个例子是APOBEC3限制因子之间的军备竞赛 和HIV-1传染性因子，Vif。建议的APOBEC3家族限制HIV-1的机制包括 有几个步骤。首先，APOBEC3蛋白与宿主和病毒RNA结合，并被结合到萌芽中 病毒粒子。在新病毒颗粒的核心内容被释放到幼稚的宿主细胞APOBEC3之后 针对病毒单链DNA的酶活性用于胞苷核苷酸的脱氨基。HIV-1对抗载脂蛋白BEC3 带有Vif的蛋白质，识别其自然宿主的APOBEC3蛋白并招募宿主泛素连接酶 以这些蛋白质为靶点进行蛋白酶体降解的机制。 典型的APOBEC3限制因子(A3D、A3F和A3G)由两个同源的锌- 在细胞限制活性中具有不同作用的结合域。一个结构域携带酶脱氨酶 功能，而另一个结构域准备更容易与RNA相互作用，以包装APOBEC3 分子变成新的病毒粒子。APOBEC3限制因子的VIF相互作用区可以定位于 域。APOBEC3(A3H)特别吸引人，因为当其他APOBEC3限制因素需要 两个域，方便所有的功能，A3H使用一个，高度多功能的域，因此，使其成为理想的， 用APOBEC3限制因子研究限制的最小系统。 有趣的是，在猪尾猕猴模型中，了解感染期间适应性的实验 结果表明，A3H是最具多态的限制因子之一。几种多态变体 在感染的动物中鉴定出具有广泛的限制活性的A3H。这些自然变异 为在分子水平上表征A3H的各种功能提供了一个创新的机会 这些生化发现与细胞表型有关。 本研究方案旨在将A3H表征为剖析APOBEC3限制活性的模型 蛋白质。这项研究将集中在人类A3H型(单倍型II)与猪的多态变体- 尾猴A3H型。A3H的以下特性将通过生物化学进行实验表征 1)A3H3D结构，2)胞苷脱氨活性，3)RNA结合的测定和结晶学分析 以及对催化的影响，以及4)Vif的结合和亲和力。生化活性将与限制活性相关。 在牢房里。这些实验将使我们更详细地了解A3H对HIV-1的限制。

Proposal Summary/Abstract The innate immune system utilizes restriction factors to prevent integration of foreign RNA species, such as viral RNA, into the genome of the host cell. In response, viruses adapt mechanisms to evade restriction in their natural hosts. An example of this type of molecular arms race is that between APOBEC3 restriction factors and HIV-1 infectivity factor, Vif. The proposed mechanism of restriction of HIV-1 by the APOBEC3 family involves several steps. First, APOBEC3 proteins associate with host and viral RNAs and are incorporated into budding virions. After the core contents of the new virus particle are released into a naïve host cell, the APOBEC3 enzymatic activity targets viral ssDNA for deamination of cytidine nucleotides. HIV-1 counteracts APOBEC3 proteins with Vif, which recognizes APOBEC3 proteins of its natural host and recruits the host ubiquitin ligase machinery to target these proteins for proteasomal degradation. Typical APOBEC3 restriction factors (A3D, A3F, and A3G) are comprised of two homologous zinc- binding domains with distinct roles in cellular restriction activity. One domain carries the enzymatic deaminase function, while the other domain is poised to interact more readily with RNA for packaging of APOBEC3 molecules into new virions. Vif-interacting regions of APOBEC3 restriction factors can be localized to either domain. APOBEC3H (A3H) is particularly fascinating because, while other APOBEC3 restriction factors require two domains to facilitate all functions, A3H uses one, highly multifunctional domain, thus, making it an ideal, minimal system to study restriction by APOBEC3 restriction factors. Interestingly, experiments to understand adaptations during infection in a pig-tailed macaque model revealed that the A3H was one of the most highly polymorphic restriction factors. Several polymorphic variants of A3H displaying a wide range of restriction activity were identified in the infected animals. These natural variants provide an innovative opportunity to characterize various functions of A3H on the molecular level and relate these biochemical findings to cellular phenotypes. This research proposal aims to characterize A3H as a model to dissect restriction activities of APOBEC3 proteins. This research will focus on human A3H (haplotype II) in combination with polymorphic variants of pig- tailed macaque A3H. The following properties of A3H will be characterized experimentally through biochemical assays and crystallographic analyses of 1) A3H 3D structure, 2) cytidine deamination activity, 3) RNA binding and effect on catalysis, and 4) Vif binding and affinity. Biochemical activities will be correlated to restriction activity in cells. These experiments will lead to a more detailed understanding of HIV-1 restriction by A3H.