Pathogenic Effects of Human Retroviruses on Hematopoieti

该项目的目标是更好地了解宿主细胞和其他辅助因素对人类逆转录病毒和致病机制的影响。病毒与细胞表面和细胞内的相互作用可以改变正常细胞过程调控的微妙平衡，如细胞因子的产生和/或导致致病后果的行为。与细胞一样，被包裹的病毒在其细胞表面含有一套复杂的蛋白质、碳水化合物和脂肪。最近的证据表明，病毒的分子决定因素和细胞之间的多重相互作用是有效的病毒融合和进入所必需的。其中一些使艾滋病毒进入的依附因素可能是糖基氨基聚糖和糖鞘糖脂。我们已经证明，原代T细胞上至少有两种不同的鞘糖脂可以用于HIV的感染性。由于病毒进入抑制剂已经成为重要的抗HIV化合物，我们最近报道了多肽T(源自V2-茎附近的八肽gp120序列)是一种有效的抗病毒进入抑制剂，适用于早期传代患者R5或R5/X4分离株以及实验室R5毒株，如Bal。肽T通过抑制进入而阻止感染，主要是通过干扰gp120与R5的结合而通过R5受体。我们正在确定多肽T是否干扰了CD4/gp120复合体的形成，gp120与R5的结合，或其他附着因子，或通过多种相互作用。HIV和宿主蛋白之间的细胞内相互作用可以导致抑制病毒的产生，也可以激活潜在的HIV免疫细胞储存库。我们以前已经确定了两种激活潜伏的HIV前病毒表达的机制，免疫激活和低甲基化。另一方面，急性HIV感染通过增加DNA甲基转移酶-1(DNMT1)的活性，导致细胞对细胞和病毒基因甲基化的能力增强。干扰素-γ是一种在HIV感染过程中甲基化的细胞基因。这种高甲基化导致干扰素表达减少，1型免疫反应减弱，病毒传播加快。HIV前病毒的整合不是刺激这些甲基化所必需的。在长期接受HAART治疗的96名患者中，约有49名患者产生干扰素-γ的能力大大减弱，而这种能力可以通过干扰素-γ启动子的低甲基化来恢复。亚硫酸氢盐基因组测序显示，在HAART治疗期间，干扰素γ启动子可以保持高甲基化状态。与艾滋病毒不同，人们对HTLV-I进入的要求知之甚少。我们正在确定HTLV包膜蛋白和宿主细胞蛋白之间的相互作用，这对有效结合和进入至关重要。利用人类逆转录病毒HTLV-I包膜的可溶性形式，我们发现在免疫激活过程中，对病毒进入至关重要的细胞表面蛋白的表达增加。此外，我们发现初始的CD_4~+T细胞是第一个不具有HTLV-I结合蛋白的人类细胞类型(S)。这种HTLV-ISu结合蛋白可能在正常免疫功能中发挥作用，因为HTLV-ISu是一种有效的混合白细胞反应抑制因子。除了用遗传和生物化学方法识别与病毒结合有关的包膜蛋白残基外，我们还在识别在HTLV进入过程中起关键作用的细胞表面分子，无论是作为主要受体还是作为附着因子。我们最近确定，膜上胆固醇的消耗大大减少了进入，并且与大多数逆转录病毒不同，聚阳离子抑制了HTLV Env的结合和进入，这表明HTLV-1要么有新的进入因子，要么有新的结构复合体。

The goal of this project is to better understand the effects of host cells and other co factors of human retroviral and pathogenesis. Viral-cell interactions both at the surface and intracellularly can alter the delicate balance of regulatory controls of normal cellular processes such as cytokine production and/or action leading to pathogenic consequences. Like cells, the enveloped viruses contain a complex set of protein, carbohydrates and lipids on their cell surface. Recent evidence suggests that multiple interactions between molecular determinants of viruses and cells are needed for efficient viral fusion and entry. Some of these attachment factors for HIV entry can be glycosylaminoglycans and glycosphingolipids. We have shown at least two different glycosphingolipids on primary T cells can be used for HIV infectivity. Since viral entry inhibitors have become important anti-HIV compounds, we recently reported that peptide T (derived from an octapeptide gp120 sequence near the V2-stem), is a potent anti-viral entry inhibitor for early passage patient R5 or R5/X4 isolates, as well as laboratory R5 strains like BaL. Peptide T blocks infection by inhibiting entry, primarily through R5 receptors by interfering with gp120 binding to R5. We are determining if peptide T disrupts formation of the CD4/gp120 complex, the binding of gp120 to R5, or other attachment factors or by multiple interactions. Intracellular interactions between HIV and host proteins can either lead to suppressing viral production or activating latent HIV reservoirs of immune cells. We have previously identified two mechanisms that activate expression of latent HIV provirus, immune activation and hypomethylation. On the other hand, acute HIV infection leads to an increased cellular capacity to methylate cellular and viral genes through increase activity of DNA methyltransferase-1 (DNMT1). Interferon-gamma (IFN-gamma) is one cellular gene that is methylated during HIV infection. This hypermethylation leads to a decreased IFN expression, decreased type1 immune response and increased viral spread. HIV proviral integration is not necessary for stimulating these on methyaltion. Some 49/96 patients on long term HAART therapy have greatly diminished ability to produce IFN-gamma that can be restored though hypomethylation of the IFN-gamma promoter. Bisulfite genomic sequencing has shown that the IFN gamma promoter can remain hypermethylated during HAART therapy. Unlike HIV, little is known about the requirements for HTLV-I entry. We are determining the interactions between the HTLV envelope proteins and host cell proteins that are critical for efficient binding and entry. Using a soluble form of the envelope of the human retrovirus HTLV-I, we found that expression of a cell surface protein critical for viral entry is increased during immune activation. In addition, we identified nave CD4+ T cells as the first human cell type not possessing detectable HTLV-I binding protein(s). This HTLV-I SU binding protein may play a role in normal immune function as the HTLV-I SU is a potent inhibitor of a mixed leukocyte reaction. In addition to genetic and biochemical approaches to identify residues on the envelope protein that are involved in viral binding, we are also identifying cell-surface molecules that play a critical role in HTLV entry, either as primary receptors or as attachment factors. We have recently determined that cholesterol depletion on the membrane dramatically reduces entry, and that unlike most retroviruses, polycations inhibit HTLV Env binding and entry suggesting that HTLV-1 has either novel entry factors or new structural complexes.