Roles of NELF on HIV replication and viral latency

NELF 对 HIV 复制和病毒潜伏期的作用

• 批准号: 6843899
• 负责人: Koh Fujinaga
• 金额: $ 22.42万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6843899
• 关键词: HIV infections; RNA interference; RNase protection assay; cell line; chromatin immunoprecipitation; clinical research; genetic promoter element; genetic transcription; helper T lymphocyte; human immunodeficiency virus 1; human subject; latent virus infection; nuclear proteins; phosphorylation; polymerase chain reaction; protein binding; small interfering RNA; tissue /cell culture; transcription factor; virus genetics; virus replication

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.

描述（由申请人提供）：高效抗逆转录病毒疗法（HAART）已被证明可以在许多情况下通过将血浆病毒水平降低至低于可检测限度来显着降低HIV-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的患者中，仍然存在一小部分含有可诱导的、具有复制能力的原病毒的静息CD4+T细胞。这种潜在的 HIV-1 储存库为成功根除该病毒造成了障碍。先前的观察表明，在潜伏感染的细胞中，HIV 转录主要在延伸步骤被阻断，该步骤受到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，细胞蛋白复合物负延伸因子（NELF）以病毒特异性方式被招募到 HIV-1 启动子上。在存在和不存在 Tat 和正延伸因子 13 (P-TEF[3) 的情况下，该复合物的 RD 亚基直接与 HIV-1 TAR 结合，并且 P-TEFI 3 的激酶活性对 RD 的磷酸化消除了这种相互作用与焦油。由 NELF 与 TAR 结合引起的对有效 HIV-1 转录的阻断可能表明通过在源自 HAART 应答者的潜伏感染 PBMC 中观察到的各种测定法检测到主要是短的启动子近端转录物的原因。因此，NELF 可能是 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 技术和模型细胞系中的高灵敏度测定以及从 HIV-1 感染患者收集的 PBMC 来定义 NELF 在 Tat 依赖性和 Tat 独立转录中的精确作用。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，希望开发出潜在的治疗方法来减少或消除这种潜伏病毒库。这项研究获得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。