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人类逆转录病毒的协同因子的作用和发病机制。在表面和细胞内的病毒-细胞相互作用可以改变正常细胞过程的调节控制的微妙平衡，例如导致致病后果的细胞因子产生和/或作用。像细胞一样，包膜病毒在其细胞表面上含有一组复杂的蛋白质、碳水化合物和脂质。最近的证据表明，病毒和细胞的分子决定簇之间的多重相互作用是有效的病毒融合和进入所必需的。其中一些用于HIV进入的附着因子可以是糖胺聚糖和鞘糖脂。我们已经表明，至少有两种不同的鞘糖脂在原代T细胞可以用于HIV感染。由于病毒进入抑制剂已成为重要的抗HIV化合物，我们最近报道了肽T（来自V2-茎附近的八肽gp 120序列），是一种有效的抗病毒进入抑制剂，用于早期传代患者R5或R5/X4分离株，以及实验室R5菌株如BaL。肽T通过抑制进入来阻断感染，主要是通过干扰gp 120与R5的结合来通过R5受体。我们正在确定肽T是否破坏CD 4/gp 120复合物的形成，gp 120与R5或其他附着因子的结合，或通过多种相互作用。HIV和宿主蛋白质之间的细胞内相互作用可以导致抑制病毒产生或激活免疫细胞的潜伏HIV储库。我们以前已经确定了两种机制，激活潜伏的HIV前病毒的表达，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（DNMT 1）的活性导致细胞和病毒基因甲基化的细胞能力增加。干扰素-γ（IFN-γ）是一种在HIV感染期间甲基化的细胞基因。这种高甲基化导致IFN表达降低、1型免疫应答降低和病毒传播增加。HIV前病毒整合对于刺激这些甲基化是不必要的。在长期HAART治疗的96名患者中，约有49名患者产生IFN-γ的能力大大降低，这种能力可以通过IFN-γ启动子的低甲基化来恢复。亚硫酸氢盐基因组测序表明，IFN γ启动子在HAART治疗期间可以保持高甲基化。与艾滋病毒不同，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，这些蛋白对于有效结合和进入至关重要。使用可溶形式的包膜的人逆转录病毒HTLV-Ⅰ，我们发现，细胞表面蛋白的表达的病毒进入的关键是在免疫激活过程中增加。此外，我们鉴定了原始CD 4 + T细胞作为第一种不具有可检测的HTLV-I结合蛋白的人类细胞类型。这种HTLV-I SU结合蛋白可能在正常免疫功能中起作用，因为HTLV-I SU是混合白细胞反应的有效抑制剂。除了遗传和生物化学方法来确定参与病毒结合的包膜蛋白残基外，我们还确定了在HTLV进入中发挥关键作用的细胞表面分子，无论是作为初级受体还是作为附着因子。我们最近已经确定，胆固醇消耗的膜上显着减少进入，和大多数逆转录病毒，聚阳离子抑制HTLV Env的结合和进入表明，HTLV-1具有新的进入因子或新的结构复合物。

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.