PDF(Pubmed)
Abstract：
The HIV-1 latent reservoir is the major barrier to an HIV cure. Due to low levels or lack of transcriptional activity, HIV-1 latent proviruses in vivo are not easily detectable and cannot be targeted by either natural immune mechanisms or molecular therapies based on protein expression. To target the latent reservoir, further understanding of HIV-1 proviral transcription is required. In this study, we demonstrate a novel role for cleavage and polyadenylation specificity factor 6 (CPSF6) in HIV-1 transcription. We show that knockout of CPSF6 hinders reactivation of latent HIV-1 proviruses by PMA in primary CD4+ cells. CPSF6 knockout reduced HIV-1 transcription, concomitant with a drastic reduction in the phosphorylation levels of Pol II and CDK9. Knockout of CPSF6 led to abnormal stabilization of protein phosphatase 2A (PP2A) subunit A, which then acted to dephosphorylate CDK9, downmodulating CDK9\'s ability to phosphorylate the Pol II carboxy-terminal domain. In agreement with this mechanism, incubation with the PP2A inhibitor, LB100, restored HIV-1 transcription in the CPSF6 knockout cells. Destabilization of PP2A subunit A occurs in the ubiquitin proteasome pathway, wherein CPSF6 acts as a substrate adaptor for the ITCH ubiquitin ligase. Our observations reveal a novel role of CPSF6 in HIV-1 transcription, which appears to be independent of its known roles in cleavage and polyadenylation and the targeting of preintegration complexes to the chromatin for viral DNA integration. IMPORTANCE CPSF6 is a cellular factor that regulates cleavage and polyadenylation of mRNAs and participates in HIV-1 infection by facilitating targeting of preintegration complexes to the chromatin. Our observations reveal a second role of CPSF6 in the HIV-1 life cycle that involves regulation of viral transcription through controlling the stability of protein phosphatase 2A, which in turn regulates the phosphorylation/dephosphorylation status of critical residues in CDK9 and Pol II.
摘要：
HIV-1潜伏储库是HIV治愈的主要障碍。由于低水平或缺乏转录活性，体内HIV-1潜伏前病毒不易检测,并且不能被天然免疫机制或基于蛋白质表达的分子疗法靶向。瞄准潜在的水库，需要进一步了解HIV-1前病毒转录.在这项研究中,我们证明了切割和聚腺苷酸化特异性因子6（CPSF6）在HIV-1转录中的新作用。我们表明，敲除CPSF6会阻碍PMA在原代CD4细胞中潜伏的HIV-1前病毒的再激活。CPSF6敲除降低HIV-1转录，伴随着PolII和CDK9的磷酸化水平急剧下降。敲除CPSF6导致蛋白磷酸酶2A（PP2A）亚基A的异常稳定，然后作用于去磷酸化CDK9，下调CDK9磷酸化PolII羧基末端结构域的能力。根据这一机制，与PP2A抑制剂一起孵育，LB100在CPSF6敲除细胞中恢复了HIV-1转录。PP2A亚基A的去稳定发生在泛素蛋白酶体途径中，其中CPSF6充当ITCH泛素连接酶的底物衔接子。我们的观察揭示了CPSF6在HIV-1转录中的新作用，似乎独立于其在裂解和聚腺苷酸化以及将预整合复合物靶向染色质以进行病毒DNA整合中的已知作用。重要性CPSF6是一种细胞因子，可调节mRNA的切割和聚腺苷酸化，并通过促进预整合复合物靶向染色质参与HIV-1感染。我们的观察结果揭示了CPSF6在HIV-1生命周期中的第二个作用，涉及通过控制蛋白磷酸酶2A的稳定性来调节病毒转录，进而调节CDK9和PolII中关键残基的磷酸化/去磷酸化状态。
