Elucidating the Structural Bases of HIV-1-Induced CD4 Degradation

项目概要： 目前可用的抗逆转录病毒疗法在治疗艾滋病毒感染方面是有效的，但不能消除它。 如果病毒感染停止，病毒反弹通常在几周内发生。感染者必须服用抗逆转录病毒药物 在他们的一生中，因此，可能会遇到抗药性和副作用。新型抗逆转录病毒药物 非常需要能够更好地治疗甚至消除HIV感染的病毒。一个有前途但不足的- 一种优选的方法是开发破坏HIV诱导的CD 4受体下调的治疗剂， 从而释放出CD 4的显著抑制能力来对抗感染。CD 4是HIV的进入受体 并且在感染的初始阶段起着至关重要的作用，但它的存在在病毒的复制周期后期强烈 抑制病毒复制并使感染细胞对免疫清除敏感。CD 4作为免疫抑制剂的效力 这种病毒反映在HIV用来对抗CD 4的严格的、高度协调的机制上。两个病毒 Nef和Vpu蛋白参与下调来自细胞表面和来自内质网的CD 4。 分别导致CD 4在溶酶体（Nef介导的途径）或 蛋白酶体（Vpu介导的途径）。艾滋病毒在这里的努力程度可以说是无与伦比的-没有其他宿主 包括众所周知的限制因素在内的因素以这种多方面的方式被对抗。这表明 恢复受感染细胞中的CD 4水平可能对病毒有显著的破坏作用， 给主人。然而，设计或开发恢复CD 4水平的治疗方法因缺乏CD 4而受到极大阻碍 有关分子组装的高分辨率结构信息，例如，Nef和Vpu是如何 招募CD 4进入被劫持的宿主贩运和退化机制。在这个项目中，我们将解决这样的高- 解析结构，以获得这种药物发现方法所需的知识。我们的具体目标是： 1)阐明病毒Nef蛋白如何劫持网格蛋白接头蛋白AP 1以使CD 4保留 在核内体中，从而促进CD 4递送至溶酶体进行降解。 2)揭示Nef如何劫持宿主运输蛋白阿利克斯将CD 4引导到多泡体中， 溶酶体降解;研究ALIX样蛋白PTPN 23是否参与，如果是，如何参与 Nef介导的CD 4和/或其他宿主因子的降解。 3)阐明病毒Vpu蛋白如何靶向ER中新合成的CD 4以介导其多聚泛素化， 通过β-TrCP/cullin 1复合物进行结合，从而将CD 4重定向至蛋白酶体进行降解。 这项工作的成功完成应揭示新的治疗方法的设计和/或开发的机会， 能够破坏HIV诱导的CD 4降解，从而恢复受感染细胞中的CD 4以抑制HIV的治疗剂 复制甚至消除感染。

PROJECT SUMMARY: Currently available antiretroviral therapy is effective in treating HIV infection but cannot eliminate it. Once treat- ment is stopped, viral rebound typically occurs within weeks. Infected individuals must take antiretroviral drugs throughout their lives and, as a consequence, may experience drug resistance and side effects. Novel antiretro- virals that can better treat or even eliminate HIV infection are highly desired. One promising and yet underap- preciated approach is to develop therapeutics that disrupt HIV-induced downregulation of the CD4 receptor and thereby unleash CD4’s remarkable inhibitory power to combat the infection. CD4 is the entry receptor for HIV and plays a vital role in the initial stage of the infection, but its presence later in the virus’ replication cycle strongly inhibits viral replication and sensitizes infected cells to immune-clearance. The potency of CD4 as an inhibitor of the virus is reflected by the stringent, highly concerted mechanisms HIV uses to antagonize CD4. Two viral proteins, Nef and Vpu, are involved in downregulating CD4 from the cell surface and from the endoplasmic reticulum, respectively, leading to degradation of CD4 in either the lysosome (Nef-mediated pathway) or the proteasome (Vpu-mediated pathway). The level of effort here by HIV is arguably unmatched—no other host factors including the well-known restriction factors are antagonized in such a multifaceted manner. This suggests that restoring CD4 levels in infected cells may be significantly damaging to the virus and significantly beneficial to the host. Designing or developing therapeutics to restore CD4 levels, however, is greatly hindered by the lack of high-resolution structural information on the pertinent molecular assemblies, e.g., how Nef and Vpu each recruit CD4 into hijacked host trafficking and degradation machineries. In this project, we will solve such high- resolution structures to gain the knowledge necessary for this drug discovery approach. Our specific aims are: 1) Elucidate how the viral Nef protein hijacks the clathrin adaptor protein AP1 to enable the retention of CD4 in endosomes, thus facilitating the delivery of CD4 to lysosomes for degradation. 2) Reveal how Nef hijacks the host trafficking protein ALIX to channel CD4 into multivesicular bodies and lysosomes for degradation; investigate whether and, if so how, the ALIX-like protein PTPN23 participates in Nef-mediated degradation of CD4 and/or other host factors. 3) Elucidate how the viral Vpu protein targets newly synthesized CD4 in the ER to mediate its polyubiquiti- nation via the β-TrCP/cullin1 complex, thereby redirecting CD4 to the proteasome for degradation. Successful completion of this work should reveal opportunities for the design and/or development of novel ther- apeutics capable of disrupting HIV-induced CD4 degradation, thus restoring CD4 in infected cells to inhibit HIV replication or even eliminate the infection.