Deciphering the Role of CPSF6 in HIV Infection

项目总结 裂解和多聚腺苷化特异性因子6(CPSF6)是一种募集到新的病毒核心的HIV宿主因子 在生命周期的早期阶段。已知HIV衣壳蛋白(CA)和CPSF6之间的相互作用决定了 核移位的细胞决定因素和影响整合位点偏好，但其影响 总体上，病毒的传染性尚不清楚。而细胞质CPSF6的过度表达已被证明限制了病毒 据报道，CPSF6的复制、敲除或CA-CPSF6相互作用的中断具有广泛的 传染性表型的范围。最近，我们的实验室发现，在原代CD4T细胞中，CPSF6的敲除 极大地增加了艾滋病毒的复制，而对细胞活力的影响最小。复制数量的增加与 与先前报道的CPSF6作用相反，干扰素刺激基因的诱导减少 保护病毒免受免疫识别。在这项提案中，我们正在检验CPSF6的总体假设 在调节对艾滋病毒感染的先天免疫反应方面起着关键作用，该病毒招募CPSF6 在一定程度上是为了绕过这种回应。这一假设将在三个广泛寻求理解的目标中得到检验 CPSF6缺失抑制感染免疫反应的机制--HIV感染的影响 CPSF6的功能，以及CPSF6调控通路在控制感染中的潜在作用。在目标1中，我们 将检验CPSF6缺失直接通过诱导 选择性多聚腺苷化(APA)或间接地允许亲环素A(CyPA)增强募集到 保护核心免受抗病毒因子TRIM5的限制。CPSF6通常作为CFIm的成员 切割因子复合体将多聚腺苷作用定向到3‘非翻译区(UTR)的远端。抑制 CFIm活动触发APA，此前已有研究表明APA参与调节先天免疫 反应，并可以解释观察到的表型。或者，有证据表明CPSF6 而CyPA竞争核心结合，而CyPA结合的丧失此前已与增强限制联系在一起 与生俱来的感觉。在目标2中，我们将测试由传入的病毒核心重新招募CPSF6可以 改变整体CFIm功能，诱发APA。不管这是否与抑制免疫反应有关 如上所述，众所周知，其他病毒劫持APA途径以增强其复制，尽管这 在艾滋病毒感染期间还没有被研究过。最后，在目标3中，我们将测试假设 CPSF6调控网络可以控制病毒的传染性和对感染的免疫反应。CPSF6活动是 受翻译后修饰和核质穿梭调控。CPSF6的截断突变体 强制细胞质定位已被证明可以限制艾滋病毒感染，我们将测试是否可以模仿这一点 基因和/或化学扰动对其内源调节机制的影响。总而言之，这项工作 探索了一种新描述的HIV宿主因子CPSF6的表型，并试图提供一种更好的 通过转录后调节来理解病毒对先天免疫反应的操纵。

PROJECT SUMMARY Cleavage and polyadenylation specificity factor 6 (CPSF6) is an HIV host factor recruited to incoming viral cores during the early stage of the lifecycle. The interaction between HIV capsid (CA) and CPSF6 is known to dictate the cellular determinants of nuclear translocation and influence integration site preference, but its impact on overall viral infectivity is unclear. While overexpression of cytoplasmic CPSF6 has been shown to restrict viral replication, knock-down of CPSF6 or disruption of the CA-CPSF6 interaction has been reported to have a broad range of infectivity phenotypes. Recently, our lab found that knock-out of CPSF6 in primary CD4+ T cells dramatically increases HIV replication with minimal impact on cell viability. This increase in replication correlates with decreased induction of interferon-stimulated genes, contrary to prior reports that suggest CPSF6 acts to shield the virus from immune recognition. In this proposal, we are testing the overall hypothesis that CPSF6 plays a critical role in regulating the innate immune response to HIV infection and that the virus recruits CPSF6 in part to circumvent this response. This hypothesis will be tested in three aims that broadly seek to understand the mechanism by which loss of CPSF6 dampens the immune response to infection, the impact of HIV infection on CPSF6 function, and the potential role of CPSF6 regulatory pathways in controlling infection. In Aim 1, we will test the hypothesis that loss of CPSF6 acts to dampen the immune response directly by induction of alternative polyadenylation (APA) or indirectly by allowing enhanced recruitment of Cyclophilin A (CYPA) to protect the core from restriction by the antiviral factor TRIM5. CPSF6 normally acts as a member of the CFIm cleavage factor complex to direct polyadenylation to distal sites of the 3' untranslated region (UTR). Inhibition of CFIm activity triggers APA, which has been previously implicated in the regulation of the innate immune response, and could explain the observed phenotype. Alternatively, there is evidence to suggest that CPSF6 and CYPA compete for core binding and loss of CYPA binding has been previously linked to enhanced restriction and innate sensing. In Aim 2, we will test the hypothesis that CPSF6 recruitment by incoming viral cores can alter overall CFIm function and induce APA. Regardless if this is linked to dampening of the immune response above, it is well established that other viruses hijack the APA pathway to enhance their replication, though this hasn't been explored during HIV infection. Finally, in Aim 3, we will test the hypothesis that perturbation of the CPSF6 regulatory network can control viral infectivity and the immune response to infection. CPSF6 activity is regulated by post-translational modification and nuclear-cytoplasmic shuttling. Truncation mutants of CPSF6 that force cytoplasmic localization have been shown to restrict HIV infection, and we will test if we can mimic that effect by genetic and/or chemical perturbation of its endogenous regulatory mechanisms. Altogether, this work explores a newly described phenotype for a well-known HIV host factor, CPSF6, and seeks to provide a better understanding of viral manipulation of the innate immune response by post-transcriptional regulation.