Roles of NELF on HIV replication and viral latency

描述（由申请人提供）：在许多情况下，高效抗逆转录病毒疗法（HAART）已显示通过将血浆病毒水平降低至低于可检测限度而显著降低HIV-1感染的发病率和死亡率。然而，最近的研究表明，在接受HAART延长时间的患者中，仍然存在一小部分含有可诱导的、有复制能力的前病毒的静息CD 4 + T细胞。这种潜伏的HIV-1储存库为成功根除病毒制造了障碍。以前的观察表明，在潜伏感染的细胞中，HIV转录主要在延伸步骤被阻断，该步骤由宿主细胞转录因子和病毒蛋白达特正向和负向调节。我们已经证明，一个细胞蛋白复合物，负延伸因子（NELF）是招募到HIV-1启动子在病毒特异性的方式。在存在和不存在达特和正延伸因子13（P-TEF）的情况下，该复合物的RD亚基直接结合HIV-1 TAR [3]，并且通过P-TEF 3的激酶活性对RD的磷酸化消除了与TAR的这种相互作用。NELF与TAR结合导致的对生产性HIV-1转录的阻断可能表明了通过在来自HAART应答者的潜伏感染的PBMC中观察到的各种测定检测到主要短的启动子近端转录物的原因。因此，NELF可能是参与从非生产性到生产性HIV-1转录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。在这项研究中，我们将使用一种新开发的siRNA技术和一种高灵敏度的检测方法，在一个模型细胞系中，以从HIV-1感染患者收集的PBMC为威尔斯孔，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种潜在的水库。本研究的结果将增加我们对HW-1发病机制的理解，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.