Regulation Of HIV Replication By Host Factors

该项目旨在研究宿主和病毒因素对HIV复制的调控所涉及的细胞和分子途径。我们先前已经证明，HIV在体外外周血白细胞中的复制水平反映了许多宿主因素的诱导和抑制作用的复杂平衡，其中一些因素以HIV毒株特异性的方式发挥作用。我们的研究已经扩展到集中于了解淋巴组织(LT)微环境关于HIV在CD4+T细胞亚群中复制的潜在独特特性。使用纯化因子的实验结果表明，LT相关因子，如趋化因子和细胞外基质，可能刺激HIV生命周期事件，包括进入和前病毒整合。我们使用体外培养模型扩展了这些研究，以研究人类LT(扁桃体)中不同CD4+T细胞亚群的生产性HIV感染。我们发现，在体外，HIV感染完整的LT片段会导致激活和静止的[缺乏经典T细胞激活或增殖标志的细胞]CD4+T细胞产生病毒。总体而言，大多数产生HIV蛋白的CD4+T细胞被发现是记忆细胞。在感染静息和幼稚的CD4+T细胞方面，X4株HIV比R5株更有效。静息的CD4+T细胞支持HIV产生的能力取决于组织微环境的成分；在单独暴露于HIV的扁桃体静息的CD4+T细胞中不会产生病毒。研究发现，有效的免疫抑制剂霉酚酸可以抑制激活的但不是静止的CD4+T细胞产生HIV；而炎性细胞因子拮抗剂/抗体似乎有效地抑制静止的CD4+T细胞产生病毒。HIV包膜蛋白(Gp160)通过与表面CD4和趋化因子HIV共受体相互作用而诱导多种细胞活动，包括诱导CD4+外周血淋巴细胞表达HIV。我们研究了gp160介导的信号是否足以诱导长期静息的CD4+T病毒库中的HIV表达以及细胞的激活/增殖或凋亡。HIV gp160可通过从感染者体内分离的静止的CD4+T细胞诱导感染性HIV，这种作用与细胞活化标志物的表达、细胞周期进展或细胞凋亡无关。这些数据表明，潜伏感染HIV的静息CD4+T细胞库可能是感染性HIV的来源之一，同时保持其长期的静息T细胞表型。最后，趋化因子受体CCR7在HIV发病机制中的相关性正在研究中。CCR7是一种趋化因子受体，在将细胞归巢到LT的T细胞区和建立适当的免疫反应方面发挥重要作用。我们发现，在病毒型(每毫升50份HIV RNA)感染者的CD4+和CD8+记忆中，CCR7+细胞以及CD8+幼稚T细胞室中CCR7+细胞的频率都降低了。此外，在停止有效的HAART后最终经历病毒反弹的个体中，CD8+T细胞上CCR7+表达的减少先于血浆病毒血症和许多其他免疫激活标志的出现。这些数据表明，CCR7是与低/不可检测的艾滋病毒复制相关的早期免疫激活的敏感标志。最后，我们发现CCR7+的记忆CD4+T细胞中CCR5的表达频率比CCR7-亚群低5-20倍，这与CCR7+亚群对体外感染R5而不是X4 HIV毒株的易感性显著降低有关。

This project was designed to investigate the cellular and molecular pathways involved in the regulation of HIv replication by host and viral factors. We had previously demonstrated that the level of HIV replication in peripheral blood white cells in vitro reflects a complex balance of inductive and suppressive effects of numerous host factors, some of which exert their effects in an HIV strain-specific manner. Our studies have been expanded to focus on understanding the potentially unique properties of the lymphoid tissue (LT) microenvironment in regard to HIV replication in CD4+ T cell subsets. Results from experiments employing purified factors suggest that LT-associated factors, such as the chemokines and extracellular matrixes, may stimulate HIV life cycle events, including entry and proviral integration. We have expanded these investigations using an in vitro culture model to investigate productive HIV infection of various CD4+ T cell subsets in human LT (tonsil) . We have found in vitro HIV infection of intact LT fragments results in viral production from both activated and resting [cells lacking markers of classic T cell activation or proliferation] CD4+ T cells. Overall, the majority of CD4+ T cells producing HIV protein were found to be memory cells. X4 HIV strains were more effective than R5 HIV strains in productively infecting both resting and naive CD4+ T cells. The ability of resting CD4+ T cells to support HIV production was dependent on components of the tissue microenvironment; viral production did not occur in isolated HIV- exposed resting tonsillar CD4+ T cells. The potent immunosuppressive agent, mycophenoloic acid, was found to inhibit HIV production from activated, but not resting, CD4+ T cells; whereas inflammatory cytokine antagonists/antibodies appeared to effectively suppress viral production from resting CD4+ T cells. HIV envelope protein (gp160) has been demonstrated to induce a variety of cellular activities through interactions with surface CD4 and chemokine HIV co-receptors, including the induction of HIV expression from CD4+ PB T cells. We investigated whether gp160-mediated signaling was sufficient to induce HIV expression from the long-lived resting CD4+ T viral reservoir and cellular activation/proliferation or apoptosis. HIV gp160 was found to induce infectious HIV from resting CD4+ T cells isolated from infected individuals and this effect was not associated with expression of cellular activation markers, progression through the cell cycle or apoptosis. These data suggest that the latently HIV-infected resting CD4+ T cell reservoir may serve as a source of infectious HIV while maintaining its long-lived resting T cell phenotype. Finally, the relevance of CCR7, a chemokine receptor that plays an important role in homing of cells to T cell areas of LT and in mounting an appropriate immune response, in HIV pathogenesis is being investigated. We have found a reduced frequency of CCR7+ cells in both CD4+ and CD8+ memory and in CD8+ naive T cell compartments in viremic (> 50 copies of HIV RNA per ml) HIV-infected individuals. In addition, decreases in CCR7+ expression on CD8+ T cells precede the appearance of plasma viremia and many other markers of immune activation in individuals who ultimately experience viral rebound following cessation of effective HAART. These data demonstrate that CCR7 is a sensitive marker for early immune activation associated with low/undetectable HIV replication. Finally, we have found a 5-20 fold lower frequency of CCR5 expression in the CCR7+ versus the CCR7-subset of memory CD4+ T cells and this is associated with a considerable reduction in susceptibility of the CCR7+ subset to in vitro infection with R5, but not X4, HIV strains.