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

中枢神经系统(CNS)功能障碍常与HIV-1感染相关，但艾滋病相关CNS功能障碍的致病机制尚不清楚。在HIV-1感染者中，大量和多样化的神经病理学结果影响到中枢神经系统中所有类型的细胞，这些细胞受到限制性感染，表明HIV-1相关的神经发病机制涉及复杂的调控途径，引起病毒和细胞因子的直接和间接影响。高滴度的HIV-1主要存在于脑内的小胶质细胞和巨噬细胞中，推测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而在致病过程中发挥重要作用。此外，星形胶质细胞受到了特别的关注，因为在这种细胞类型和细胞培养系统中反复检测到病毒基因组，它们支持HIV-1复制，尽管程度较小。几个实验室的研究表明，感染细胞分泌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，我们最近的工作表明，TAT有能力通过延长细胞周期的G1期并将细胞滞留在G1/S边界来解除对星形细胞增殖的调控。研究表明，TAT可以改变几种关键的细胞周期调节蛋白的程序性表达，包括细胞周期蛋白及其相关的激酶。这些改变可能影响其下游靶蛋白(包括pRb)的磷酸化状态，通过保持E2F-1与低磷酸化的pRb形成复合体，使E2F-1失活，E2F-1是进入S期所必需的转录因子。另一方面，我们已经证明了不含pRB的活性E2F-1通过利用跨越HIV-1kB激活基序的序列来抑制HIV-1LTR的转录。因此，我们通过利用一个跨越HIV-1kB激活基序的序列来转录HIV-1LTR。因此，我们的假设是，HIV-1Tat及其反应性细胞因子，即对细胞周期调节装置产生影响的TNFpha和TGFbeta，解除了对正常细胞功能至关重要的一系列事件的调节。这反过来会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通过与其细胞伙伴pRb的络合而保持隔离状态。在这项研究中，我们打算研究TAT和TAT诱导的细胞因子，包括TNFpha和TGFbeta-1，与人原代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，方法是：(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，并检测对控制细胞周期至关重要的关键参数；(Ii)在研究NFkappaB激活前后HIV-1病毒基因组的表达和复制的同时，表达E2F-1，并评估E2F-1和NFkappaB与它们在HIV-1 LTR上的共同基序以及在这些细胞中彼此之间的关系。此外，将与神经病理学和组织培养核心(核心A)合作，研究临床标本中病毒和相关细胞蛋白的表达和相互作用。

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.