Roles of NELF on HIV replication and viral latency

描述（由申请人提供）：在许多情况下，高活性抗逆转录病毒疗法（HAART）通过将血浆病毒水平降低到可检测限度以下，已被证明可显著降低艾滋病毒- 1感染的发病率和死亡率。然而，最近的研究表明，在长期接受HAART治疗的患者中，仍有一小部分静止的CD4+ T细胞含有可诱导的、具有复制能力的前病毒。这种潜伏的HIV-1病毒库为成功根除这种病毒制造了障碍。先前的观察表明，在潜伏感染的细胞中，HIV转录主要在延伸阶段被阻断，这一阶段受宿主细胞转录因子和病毒蛋白Tat的正向和负向调节。我们已经证明了一种细胞蛋白复合物，负延伸因子（NELF）以病毒特异性的方式被募集到HIV-1启动子上。无论Tat和正延伸因子13（P-TEF）是否存在，该复合物的RD亚基都直接与hiv - 1 TAR结合[3]，并且通过P-TEFI 3的激酶活性使RD磷酸化，从而消除了与TAR的相互作用。NELF与TAR结合导致HIV-1转录受阻，这可能是在HAART应答者衍生的潜伏感染pbmc中检测到主要是短的启动子近端转录的原因。因此，NELF可能是参与HIV-1转录从非生产性向生产性转变的关键参与者之一。我们假设NELF是导致潜伏感染细胞中HIV转录水平低的一个因素。在这项拟议的研究中，我们将使用新开发的siRNA技术和在模型细胞系以及从HIV-1感染患者收集的pbmc中进行的高灵敏度测定来确定NELF在tat依赖性和tat非依赖性转录中的确切作用。此外，我们将尝试通过调节NELF活性来激活潜伏感染细胞中的病毒转录，希望开发潜在的治疗方法来减少或消除这个潜伏库。本研究结果将增加我们对HW-1发病机制的认识，并可能提出新的治疗方向。

DESCRIPTION (provided by applicant): Highly active antiretroviral therapy (HAART) has been shown to significantly lower morbidity and mortality rates from HIV-l infection by reducing levels of plasma virus to below detectable limits in many instances. However, recent studies indicate that there remains a small population of resting CD4+ T cells containing inducible, replication competent provirus in patients receiving HAART for extended periods of time. This latent HIV-1 reservoir creates an obstacle for the successful eradication of the virus. Previous observations suggest that in latently infected cells HIV transcription is blocked mainly at the elongation step, which is regulated positively and negatively by host cellular transcription factors and the viral protein Tat. We have demonstrated that a cellular protein complex, negative elongation factor (NELF) is recruited to the HIV-1 promoter in a virus specific manner. The RD subunit of this complex binds directly to HIV-l TAR in the presence and absence of Tat and the positive elongation factor 13(P-TEF[3), and phosphorylation of RD by the kinase activity of P-TEFI 3 abolishes this interaction with TAR. The block to productive HIV-1 transcription caused by the binding of NELF to TAR may indicate a reason for the detection of predominantly short, promoter proximal transcripts by various assays observed in the latently infected PBMCs derived from HAART responders. Therefore NELF may be one of the key players involved in the transition from nonproductive to productive HIV-1 transcription. We hypothesize that NELF is a factor contributing to the low level of HIV transcription in latently infected cells. In this proposed study, we will define the precise roles of NELF in Tat-dependent and Tat-independent transcription using a newly developed siRNA technology and a highly sensitive assay in a model cell line, as wells as PBMCs collected from HIV-1 infected patients. Moreover, we will attempt to activate viral transcription in latently infected cells by regulating NELF activity in hopes of developing potential treatments to reduce or abolish this latent reservoir. The results obtained from this study will increase our understanding of HW-1 pathogenesis and may suggest new therapeutic directions.