HIV-1 Tat & Cell Cycle Regulatory factors in CNS Cooperative Interaction

通常发现中枢神经系统（CNS）功能障碍与HIV-1感染有关，但艾滋病相关中枢神经系统疾病的致病机制尚不清楚。在HIV-1感染个体中，大量不同的神经病理结果影响中枢神经系统中所有细胞类型，这些细胞的感染受到限制，这表明HIV-1相关的神经发病机制涉及复杂的调控途径，引发病毒和细胞因素的直接和间接影响。高滴度的HIV-1主要存在于大脑的小胶质细胞和巨噬细胞中，怀疑这些细胞通过直接释放病毒和间接分泌影响其他中枢神经系统细胞的病毒和细胞因子在诱导疾病中发挥重要作用。此外，星形胶质细胞受到了特别的关注，因为病毒基因组已经在这种细胞类型和细胞培养系统中被反复检测到，尽管它们在较小程度上支持HIV-1复制。几个实验室的研究表明，受感染细胞分泌的hiv调节蛋白、Tat和/或Tat诱导的细胞因子和免疫调节剂可以影响邻近的未感染细胞并改变重要基因的表达。为了支持这一概念，我们最近的工作已经证明Tat能够通过延长细胞周期的G1期和在G1/S边界阻滞细胞来解除对星形细胞周期细胞增殖的调节。研究表明，Tat可以改变几个关键细胞周期调节蛋白的程序化表达，包括细胞周期蛋白及其相关激酶。这些改变可能会影响下游靶蛋白（包括pRb）的磷酸化状态，通过维持E2F-1与低磷酸化pRb的复合物，使E2F-1失能。E2F-1是一种转录因子，其活性对s期进入至关重要。另一方面，我们已经证明，不含pRb的活性E2F-1能够利用跨越HIV-1 kB激活基序的序列抑制HIV-1 LTR的转录。因此，我们利用跨越HIV-1 kB激活基序的序列转录HIV-1 LTR。因此，我们的假设是HIV-1 Tat及其应答细胞因子，即TNFalpha和TGFbeta，对细胞周期调节装置有影响，解除了对正常细胞功能重要的级联事件的调节。这反过来又影响病毒基因的表达和复制，因为抑制HIV-1基因转录的E2F-1通过与其细胞伴侣pRb的络合而保持隔离。在这项提议中，我们打算研究Tat和Tat诱导的细胞因子，包括TNFalpha和tgfβ -1，与人类初级小胶质细胞和星形胶质细胞的细胞周期调节蛋白之间的相互作用：(i)在细胞周期的不同阶段用HIV-1 Tat和相关细胞因子处理细胞，并检查对细胞周期控制重要的关键参数；（ii）表达E2F-1，同时研究NFkappaB激活前后HIV-1病毒基因组的表达和复制，并评估E2F-1和NFkappaB在这些细胞中与HIV-1 LTR上的共同基序以及彼此之间的体内关联。此外，与神经病理学和组织培养核心（核心A）合作，将研究病毒和相关细胞蛋白在临床标本中的表达和相互作用。

Central nervous system (CNS) dysfunction is often found in association with HIV-1 infection, yet the pathogenic mechanisms of AIDS-related CNS disorders are poorly understood. Massive and diverse neuropathological findings in HIV-1 infected individuals that affect all cell types in the CNS with restricted infection of these cells suggests that HIV-1 associated neuropathogenesis involves complex regulatory pathways that elicit direct and indirect effects of both viral and cellular factors. A high titers of HIV-1 are found predominantly in microglial cells and macrophages of the brain, it is suspected that these cells play an important role in inducing disease both directly by releasing virus, and indirectly by secreting viral and cellular factors that have an impact upon other CNS cells. Also, astroglial cells received special attention as the viral genome has been repeatedly detected in this cell type and in cell- culture system they support, albeit to a lesser extent, HIV-1 replication. Work from several laboratories has inducted that the secretion of the HIV-regulatory protein, Tat, and/or Tat-induced cytokines and immunomodulators by infected cells could affect neighboring uninfected cells and alter the expression of important genes. In support of this concept, our recent work has demonstrated that Tat has the ability to deregulate astrocyctic cell proliferation by elongating the G1 phase of the cell cycle and arresting cells at the G1/S boundary. Studies have revealed that Tat can alter the programmed expression of several key cell cycle regulatory proteins, including cyclins and their associated kinases. These alterations, which may affect the phosphorylation status of their downstream target protein, including pRb, can incapacitate E2F-1, a transcription factor whose activity is essential for S-phase entry, by maintaining E2F-1 in complex with hypophosphorylated pRb. On the other hand, we have demonstrated that active E2F-1, free from pRb, has the ability to suppress transcription of the HIV-1 LTR by utilizing a sequence spanning the HIV-1 kB activation motif. Thus, our transcription of the HIV-1 LTR by utilizing a sequence spanning the HIV-1 kB activation motif. Thus, our hypothesis is that HIV-1 Tat and its responsive cytokines, i.e. TNFalpha and TGFbeta, which have an impact upon the cell cycle regulatory apparatus, deregulate the cascade of events which are important for normal cell function. This, in turn, affects viral gene expression and replication since E2F-1, which suppresses HIV-1 gene transcription, remains sequestered by complexation with its cellular partner, pRb. In this proposal, we intend to study the interplay between Tat and Tat-induced cytokines, including TNFalpha and TGFbeta-1, with cell cycle regulatory proteins from human primary microglia and astrocytes by: (i) treating cells with HIV-1 Tat and the relevant cytokines at various stages of the cell cycle and examining key parameters which are important for control of the cell cycle, and (ii) expressing E2F-1 while investigating expression and replication of the HIV-1 viral genome before and after activation of NFkappaB and assess the in vivo association of E2F-1 and NFkappaB with their common motif on the HIV-1 LTR and with each other in these cells. Furthermore, in collaboration with the Neuropathology and Tissue Culture Core (Core A), expression and interaction on viral and relevant cellular proteins in clinical specimens will be investigated.