Deciphering the Role of CPSF6 in HIV Infection

项目摘要 裂解和多聚腺苷酸化特异性因子6（CPSF 6）是一种招募到传入病毒核心的HIV宿主因子 在生命周期的早期阶段。已知HIV衣壳（CA）和CPSF 6之间的相互作用决定了 细胞核转位的决定因素，并影响整合位点的偏好，但其影响 总体病毒感染性尚不清楚。虽然细胞质CPSF 6的过表达已经显示出限制了病毒的传播， 复制、CPSF 6的敲低或CA-CPSF 6相互作用的破坏已被报道具有广泛的生物学效应。 一系列传染性表型。最近，我们的实验室发现，敲除原代CD 4 + T细胞中的CPSF 6， 在对细胞活力影响最小的情况下显著增加HIV复制。这种复制的增加与 干扰素刺激基因的诱导减少，与先前的报告相反，该报告表明CPSF 6的作用是 保护病毒不被免疫系统识别在本提案中，我们正在测试CPSF 6 在调节对HIV感染的先天免疫反应中起着关键作用， 部分是为了规避这种反应。这一假设将在三个目标，广泛寻求理解测试 CPSF 6的缺失抑制对感染的免疫反应的机制，HIV感染的影响 对CPSF 6功能的影响，以及CPSF 6调控途径在控制感染中的潜在作用。目标1： 将检验这样的假设，即CPSF 6的缺失通过诱导免疫应答直接抑制免疫应答。 选择性聚腺苷酸化（阿帕）或间接通过允许亲环素A（CYPA）的增强募集， 保护核心免受抗病毒因子TRIM 5 β的限制。CPSF 6通常作为CFIM的成员 切割因子复合物以将多聚腺苷酸化引导至3'非翻译区（UTR）的远端位点。抑制 CFIm活性触发阿帕，APA先前已涉及先天免疫的调节， 反应，并可以解释观察到的表型。另一方面，有证据表明，CPSF 6 和CYPA竞争核心结合，CYPA结合的丧失先前已与增强的限制有关 和先天感知在目标2中，我们将检验这样的假设，即通过传入病毒核心的CPSF 6募集可以 改变整个CFIm功能并诱导阿帕。不管这是否与免疫反应的减弱有关 在上文中，已经确定其他病毒劫持阿帕途径来增强它们的复制，尽管这 在艾滋病感染期间还没有被探索过。最后，在目标3中，我们将测试假设， CPSF 6调控网络可以控制病毒的感染性和对感染的免疫反应。CPSF 6活性是 受翻译后修饰和核质穿梭调节。CPSF 6的截短突变体， 强制细胞质定位已被证明可以限制HIV感染，我们将测试我们是否可以模仿它 通过其内源调节机制的遗传和/或化学干扰而产生的效应。总之，这项工作 探索了一个新描述的表型为众所周知的艾滋病毒宿主因子，CPSF 6，并寻求提供一个更好的 了解病毒通过转录后调节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.