Regulation of HIV transcription by Tat and P-TEPb

描述（由申请人提供）：本提案的直接目标是阐明达特和P-TEFb在HIV-1潜伏期和再活化中的作用，并研究细胞因子HIC和颗粒蛋白对这些蛋白质的调节。P-TEFb（细胞周期蛋白T1/CDK 9）是一种细胞转录延伸因子，其与病毒蛋白Tal合作以实现HIV基因组的进行性转录。我们将研究HIV感染的控制自然发生的变化达特序列来自HIV患者的记忆T细胞，和P-TEFb和病毒生产的影响，伴随着潜在感染细胞的激活。HIC和颗粒蛋白与达特和细胞周期蛋白T1的新的相互作用将被表征以了解它们如何调节HIV基因表达。这些研究的长期意义在于控制HIV宿主和开发干扰HIV-1感染的化合物。我们的具体目标是：1.检验部分活性达特突变体与病毒潜伏期相关的假设。对潜伏感染细胞系和临床样本的研究表明，受损的达特反式激活活性可能有助于潜伏期的建立。我们的研究已经表明，中度衰弱的达特突变体的反式激活活性优先由诱导P-TEFb的有丝分裂原刺激。为了探索达特衰减在潜伏期中的作用，我们将（i）检查HAART患者静息CD 4 + T细胞活化后出现的病毒中存在的达特序列，（ii）研究感染含有部分活性达特变体的病毒的细胞，以及（iii）对变体进行生物化学表征。2.探讨P-TEFb活性与潜伏期再激活的关系。虽然HIV可以通过诱导P-TEFb的治疗重新激活，但P-TEFb在Tat相关潜伏期中的作用仍有待确定。使用目的1中产生的潜伏感染的细胞系，我们将检查暴露于已知激活T细胞和巨噬细胞的细胞因子和药理学试剂后病毒产生和P-TEFb诱导之间的相关性。我们将使用RNA干扰和抑制剂来直接确定P-TEFb是否涉及。3.表征细胞因子HIC和颗粒蛋白对达特和细胞周期蛋白T1的调节。P-TEFb支持达特反式激活的能力可由与细胞周期蛋白T1和达特相互作用的细胞蛋白正向或负向调节。我们已经分离出两种这样的蛋白质，即HIC（人含I-mfa结构域的蛋白质）和颗粒蛋白，它们结合达特和细胞周期蛋白T1并调节HIV-1启动子的反式激活。我们将探讨HIC和颗粒蛋白调节HIV启动子反式激活的能力，并确定这些蛋白质与细胞周期蛋白T1和达特结合所需的序列。

DESCRIPTION (provided by applicant): The immediate objectives of this proposal are to shed light on the role of Tat and P-TEFb in HIV-1 latency and reactivation, and to investigate the regulation of these proteins by the cellular factors HIC and granulin. P-TEFb (cyclin T1/CDK9) is a cellular transcription elongation factor that cooperates with the viral protein Tal to attain processive transcription of the HIV genome. We will study the control of HIV infection by naturally occurring variations in Tat sequences derived from HIV patients' memory T cells, and the effects on P-TEFb and virus production that accompany the activation of latently infected cells. The novel interactions of HIC and granulin with Tat and cyclin T1 will be characterized to understand how they modulate HIV gene expression. Long-term implications of these investigations lie in the control of HIV reservoirs and the development of compounds to interfere with HIV-1 infection. Our specific aims are to: 1. Test the hypothesis that partially active Tat mutants are associated with viral latency. Work with latently infected cell lines and clinical samples suggests that impaired Tat transactivation activity may contribute to the establishment of latency. Our studies have shown that the transactivation activity of moderately debilitated Tat mutants is preferentially stimulated by a mitogen that induces P-TEFb. To explore the role of Tat attenuation in latency, we will (i) examine the Tat sequences present in viruses that emerge after activation of resting CD4+ T cells from patients on HAART, (ii) study cells infected with viruses containing partially active Tat variants, and (iii) characterize the variants biochemically. 2. Explore the relationship between P-TEFb activity and the reactivation from latency. Although HIV can be reactivated by treatments that induce P-TEFb, the role of P-TEFb in Tat-related latency remains to be established. Using the latently infected cell lines generated in aim 1, we will examine the correlation between viral production and P-TEFb induction following exposure to cytokines and pharmacological agents that are known to activate T cells and macrophages. We will use RNA interference and inhibitors to determine directly whether P-TEFb is implicated. 3. Characterize the regulation of Tat and cyclin T1 by the cellular factors HIC and granulin. The ability of P-TEFb to support Tat transactivation can be positively or negatively modulated by cellular proteins that interact with cyclin T1 and Tat. We have isolated two such proteins, namely HIC (human I-mfa domain containing protein) and granulin, which bind to both Tat and cyclin T1 and regulate transactivation of the HIV-1 promoter. We will explore the ability of HIC and granulin to regulate transactivation of the HIV promoter and define the sequences of these proteins necessary for binding to cyclin T1 and Tat.