Regulation Of HIV Replication By Host Factors

该项目旨在研究宿主和病毒因子调节HIV复制的细胞和分子途径。我们以前已经证明，在体外外周血白色细胞中的HIV复制水平反映了许多宿主因子的诱导和抑制作用的复杂平衡，其中一些以HIV株特异性方式发挥其作用。我们的研究已经扩展到关注了解淋巴组织（LT）微环境在CD 4 + T细胞亚群中HIV复制方面的潜在独特特性。采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，可能会刺激HIV生命周期事件，包括进入和前病毒整合。我们已经扩大了这些调查使用体外培养模型，以调查生产HIV感染的各种CD 4 + T细胞亚群在人类LT（扁桃体）。我们已经发现，在体外HIV感染的完整LT片段的结果在病毒生产从激活和休息[细胞缺乏经典的T细胞活化或增殖的标志物]的CD 4 + T细胞。总的来说，大多数产生HIV蛋白的CD 4 + T细胞被发现是记忆细胞。X4 HIV毒株比R5 HIV毒株更有效地感染静息和初始CD 4 + T细胞。静息CD 4 + T细胞支持HIV产生的能力取决于组织微环境的组分;在分离的暴露于HIV的静息扁桃体CD 4 + T细胞中不发生病毒产生。发现有效的免疫抑制剂霉酚酸抑制活化的但不抑制静息的CD 4 + T细胞的HIV产生;而炎性细胞因子拮抗剂/抗体似乎有效抑制静息的CD 4 + T细胞的病毒产生。HIV包膜蛋白（gp 160）已被证明通过与表面CD 4和趋化因子HIV共受体的相互作用诱导多种细胞活性，包括诱导来自CD 4 + PB T细胞的HIV表达。我们研究了gp 160介导的信号传导是否足以诱导HIV从长寿命的静息CD 4 + T病毒库表达和细胞活化/增殖或凋亡。发现HIV gp 160从分离自受感染个体的静息CD 4 + T细胞诱导感染性HIV，并且该效应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，潜伏性HIV感染的静息CD 4 + T细胞库可能作为感染性HIV的来源，同时保持其长寿命的静息T细胞表型。 最后，正在研究CCR 7在HIV发病机制中的相关性，CCR 7是一种趋化因子受体，在细胞归巢至LT的T细胞区域和建立适当的免疫应答中起重要作用。我们已经发现，在病毒血症（> 50个HIV RNA拷贝/ml）的HIV感染个体中，在CD 4+和CD 8+记忆中以及在CD 8+初始T细胞区室中，CCR 7+细胞的频率降低。此外，在停止有效HAART后最终经历病毒反弹的个体中，CD 8 + T细胞上CCR 7+表达的降低先于血浆病毒血症和许多其他免疫活化标志物的出现。这些数据表明，CCR 7是与低/不可检测的HIV复制相关的早期免疫激活的敏感标志物。最后，我们发现记忆性CD 4 + T细胞的CCR 7+亚群中CCR 5表达的频率比CCR 7-亚群低5-20倍，这与CCR 7+亚群对R5而不是X4 HIV株体外感染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.