Role of PARP-1 in HIV-1 latent infection

 描述(由申请人提供)：我们的长期目标是通过寻找消除潜伏期的策略来为根除艾滋病毒感染做出贡献。HIV-1感染通过以下方式逃脱根除 建立一个潜在的储集层。因此，细胞因子的鉴定牵涉到 潜伏期的建立或维持是这一应用的重点，可以揭示抑制HIV-1感染的新的治疗靶点。我们最近发现了PARP-1在沉默人类CD4+T细胞中HIV-1基因表达方面的新作用。我们的数据表明，PARP-1基因敲除(KD)的CD4+T细胞对HIV-1复制的容许性是对照细胞的90倍。此外，在KD细胞中重新表达PARP-1大大降低了它们对HIV-1的敏感性。此外，一种针对PARP-1锌指结构域的小分子，而不是与该酶活性部位结合的抑制剂，也将CD4+T细胞中的病毒复制增加60倍。重要的是，HIV-1DNA整合或HIV-1生命周期的生产阶段不受PARP-1缺乏或药物干预的影响，表明PARP-1在整合后步骤影响HIV-1复制，更有可能是在基因表达水平。值得注意的是，PARP-1的作用需要CD4/CXCR4介导的病毒进入；即PARP-1不影响VSV-G假型病毒的感染。基于这些结果，我们设想PARP-1负向调节HIV Env诱导的CD4/CXCR4信号，以这种方式限制特定转录因子的可获得性，从而有利于潜伏期的建立。多项发现支持我们的中心假设：(1)HIV Env诱导的CD4/辅受体信号转导增加了促进病毒复制的细胞因子(即NF-ATS、AP-1和NF-kB)的表达。(2)PARP-1负性调节T细胞活化诱导的多个基因的转录，部分是通过抑制NF-AT或NF-kB依赖的转录。(3)HIV-1基因的表达在很大程度上受特定细胞转录因子的可用性和潜伏期的影响 在缺乏转录因子的细胞中受到青睐。我们很好地开展了拟议的研究，因为我们在表征HIV复制中细胞辅因子的分子机制方面拥有丰富的经验，特别是在PARP-1中。与HIV-1潜伏期和T细胞信号领域的专家建立的合作将补充并非常有效地与我们自己在这些领域的专业知识协同。在本研究的结论中，我们 希望已经确定了PARP-1在HIV潜伏期中的作用，并更好地确定了其作用机制。拟议的工作很重要，因为可以发现根除艾滋病毒感染的新治疗目标。

 DESCRIPTION (provided by applicant): Our long-term goal is to contribute to the eradication of HIV infection by finding strategies to eliminate latency. HIV-1 infection escapes eradication by establishing a latent reservoir. Consequently, identification of cellular factors implicated in the establishment or maintenance of latency, which is the focus of this application, could reveal new therapeutic targets for suppression of HIV-1 infection. We have recently discovered a novel role of PARP-1 in silencing HIV-1 gene expression in human CD4+ T cells. Our data indicated that PARP-1 knockdown (KD) CD4+ T cells are up to 90-fold more permissive to HIV-1 replication than control cells. Furthermore, re-expression of PARP-1 in the KD cells substantially diminished their susceptibility to HIV-1. In addition, a small molecule that targets the zinc finge domains of PARP-1, but not inhibitors binding to the active site of this enzyme, also increased viral replication in CD4+ T cells by 60-folds. Importantly, HIV-1 DNA integration or the production phase of the HIV-1 life cycle were not affected by PARP-1 deficiency or pharmacological interference, indicating that PARP-1 affects HIV-1 replication at a post-integration step, more likely at the level of gene expression. Notably, the effect of PARP-1 required CD4/CXCR4-mediated viral entry; i.e. PARP-1 did not affect infection by VSV-G pseudotyped viruses. Based on these results, we envision that PARP-1 negatively regulates HIV Env-induced CD4/CXCR4 signaling, limiting in this manner the availability of specific transcription factors and consequently favoring the establishment of latency. Multiple findings support our central hypothesis: (1) HIV Env-induced CD4/co-receptor signaling increases expression of cellular factors (i.e. NF-ATs, AP-1, and NF-kB) that promote viral replication. (2) PARP-1 negatively regulates the transcription of multiple genes induced by T cell activation, in part through inhibition of NF-AT- or NF-kB-dependent transcription. (3) HIV-1 gene expression is largely influenced by the availability of specific cellular transcription factors and latency is favored in transcription factor-deprived cells. We are well positioned to carry out the proposed studies because of our extensive experience in the characterization of the molecular mechanisms of cellular cofactors in HIV replication, and in particular in PARP-1. Collaborations established with experts in HIV-1 latency and T cell signaling fields will complement and very efficiently synergize with our own expertise in these fields. At the conclusion of this research we expect to have defined the role of PARP-1 in HIV latency and better identified its mechanism of action. The work proposed is important because new therapeutic targets to eradicate HIV infection could be discovered.