The contribution of unintegrated HIV-1 to latency and to models of latency

 描述（由申请人提供）：由于对当前抗病毒治疗具有抗性的病毒储库的持续存在，HIV感染仍然无法治愈。在急性/早期感染期间迅速建立潜伏储库，并且在该阶段期间，许多病毒RNA+细胞缺乏活化和增殖标志物，因此类似于静息的CD 4 + T细胞。这些发现表明，潜伏期通常是通过直接感染静息T细胞建立的。HIV-1潜伏期的体外模型经常采用静息CD 4 T细胞的直接感染，包括我们最近描述的利用IL-4使T细胞可感染而不诱导T细胞活化的潜伏期系统。我们已经报道，当HIV-1感染静止的CD 4 T细胞时，产生大量潜伏感染的细胞，这些细胞含有尚未整合和不能整合的HIV-1基因组，但这些基因组能够在感染后几天激活细胞时重新产生病毒。使用基于IL-4的潜伏系统，我们发现仅含有未整合的HIV-1的细胞占所有潜伏感染细胞的约一半。我们发现，潜在的未整合的基因组响应休克和杀死剂，但他们的反应表明表观遗传调控，从集成的前病毒不同。我们假设这种新形式的潜伏期在HIV-1潜伏期的标准体外模型中很常见。我们提出的研究将定义控制这种新形式的潜伏期的机制及其对HIV-1潜伏期体外模型的贡献。我们进一步发现，HIV-1的Vpr基因是HIV-1基因组染色质化的关键调节因子，我们将定义它执行此功能的机制。我们发现Vpr对于静息T细胞中uDNA的表达是必不可少的，并且没有Vpr，这些基因组对刺激无反应并显示改变的染色质甲基化。我们假设Vpr抵消细胞防御入侵寄生DNA安装转录抑制染色质修饰。该项目将为HIV-1潜伏期的建立和调节提供新的见解。该项目还将提供有关重要的体外潜伏期模型的组成和功能的必要信息，这些模型必须在新型抗病毒疗法的开发中得到考虑。

 DESCRIPTION (provided by applicant): HIV infection remains incurable owing to the persistence of viral reservoirs that are resistant to current antiviral therapies. The latent reseroir is rapidly established during acute/early infection, and during this phase many viral RNA+ cells lack activation and proliferation markers and thus resemble resting CD4+ T cells. These findings suggest that latency is often established by direct infection of resting T cells. In vitro models o HIV-1 latency frequently employ direct infection of resting CD4 T cells, including our recently described latency system utilizing IL-4 to render T cells infectible without inducing T cell activation. We have reported that when HIV-1 infects resting CD4 T cells, large numbers of latently infected cells are generated which contain HIV-1 genomes that have not integrated and cannot integrate, yet these genomes are capable of de novo virus production when the cell is activated several days after infection. Using an IL-4-based latency system, we find that cells containing only unintegrated HIV-1 constitute about one half of all latently infected cells. We fin that latent unintegrated genomes respond to shock and kill agents but that their responses indicate epigenetic regulation that differs from integrated proviruses. We hypothesize that this new form of latency is a common occurrence in standard in vitro models of HIV-1 latency. The studies we propose will define the mechanisms controlling this new form of latency and its contribution to in vitro models of HIV-1 latency. We further find that the Vpr gene of HIV-1 is a key regulator the chromatization of HIV-1 genomes, and we will define the mechanisms by which it performs this function. We find that Vpr is essential for expression from uDNA in resting T cells, and that without Vpr, these genomes are unresponsive to stimulation and display altered chromatin methylation. We hypothesize that Vpr counteracts cellular defenses against invading parasitic DNA that install transcriptionally repressive chromatin modifications. This project will provide new insights into the establishment and regulation of HIV-1 latency. This project will also provide necessary information on the constitution and functioning of important in vitro models of latency that must be accounted for in the development of novel anti-viral therapies.