Characterization of retroviral restriction factor APOBEC3H

提案摘要/摘要 先天性免疫系统利用限制因子来防止外源RNA种类的整合，例如 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， 它们的自然宿主这种类型的分子军备竞赛的一个例子是APOBEC 3限制因子之间的军备竞赛。 和HIV-1感染因子Vif。提出的APOBEC 3家族限制HIV-1的机制涉及 几个步骤。首先，APOBEC 3蛋白与宿主和病毒RNA结合， 病毒粒子在新病毒颗粒的核心内容物被释放到初始宿主细胞中后，APOBEC 3 酶活性靶向病毒ssDNA用于胞苷核苷酸的脱氨基。HIV-1抵消APOBEC 3 Vif识别其天然宿主的APOBEC 3蛋白并募集宿主泛素连接酶 机器靶向这些蛋白质的蛋白酶体降解。 典型的APOBEC 3限制因子（A3 D、A3 F和A3 G）由两个同源锌- 在细胞限制活性中具有不同作用的结合结构域。一个结构域携带酶脱氨酶 功能，而另一个结构域准备与RNA更容易地相互作用，以包装APOBEC 3 转化为新的病毒体APOBEC 3限制因子的VIF相互作用区域可以定位于 域APOBEC 3 H（A3 H）特别吸引人，因为尽管其他APOBEC 3限制因子需要 两个结构域以促进所有功能，A3 H使用一个高度多功能的结构域，因此，使其成为理想的， 最小系统研究APOBEC 3限制因子的限制。 有趣的是，在猪尾猕猴模型中， A3 H是多态性最高的限制性内切酶之一。几种多态性变体 在感染动物中鉴定出显示广泛限制活性的A3 H。这些自然变异 为在分子水平上表征A3 H的各种功能提供了创新机会， 将这些生化发现与细胞表型联系起来。 这项研究计划旨在将A3 H描述为剖析APOBEC 3限制活性的模型 proteins.这项研究将集中在人类A3 H（单倍型II）与猪的多态性变体的组合， 长尾猕猴A3 H。A3 H的以下性质将通过生物化学实验进行表征 1）A3 H 3D结构，2）胞苷脱氨活性，3）RNA结合的测定和晶体学分析 和对催化的影响; 4）Vif结合和亲和力。生化活动将与限制活动相关 在细胞中。这些实验将导致更详细地了解A3 H对HIV-1的限制作用。

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.