Tight Junction: Gate to HCV Tropism, Therapeutics and Pathogenesis

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)通过引起急性和慢性肝炎、肝硬变以及肝细胞癌，对全球健康造成严重损害。与这一冷酷的现实相矛盾的是，目前还没有疫苗可以预防感染，聚乙二醇化干扰素-a和利巴韦林联合治疗只对40%-80%的患者有效，而且有严重的副作用。幸运的是，有了最近开发的感染性克隆，我们终于能够在分子水平上剖析病毒的整个生命周期。可以想象，鉴于研究步伐的加快，缓解丙型肝炎病毒相关病理的新型抗病毒疗法和治疗方案将在可预见的未来变得可用。就这项应用而言，越来越多的证据突显了紧密连接(TJ)在丙型肝炎病毒进入中的重要性。具体地说，我们和其他人报道了紧密连接蛋白阻断素(OCLN)是丙型肝炎病毒包膜蛋白依赖的进入必不可少的。证据还表明，OCLN部分解释了丙型肝炎病毒宿主范围狭窄的原因。然而，表达所有已知的人类(辅助)受体的小鼠细胞仍然对细胞培养生长的丙型肝炎病毒(HCVcc)感染具有抵抗力，这表明要么需要额外的人类因素，要么小鼠细胞中存在明显的抑制物。有趣的是，我们观察到OCLN和claudin-1在HCVcc感染的肝癌细胞中都下调了。随后的研究表明，在感染丙型肝炎病毒的细胞中，OCLN被越来越多地切割。根据这些发现，我们相信新的治疗方法、新的感染系统和对了解丙型肝炎相关发病机制的新见解可能会被开发出来。我们假设，针对OCLN胞外环或OCLN衍生多肽的抗体可能阻止病毒的传播(在目标1中进行测试)；需要额外的宿主因素来支持在小鼠细胞中的最佳丙型肝炎病毒感染或这些细胞中存在限制因素(在目标2中进行测试)；在丙型肝炎病毒进入过程中TJ蛋白的下调可能改变细胞生理(在目标3中进行测试)。我们计划通过(I)评估针对OCLN或OCLN衍生多肽的抗体对丙型肝炎病毒进入的影响来检验这些假说。(Ii)确定促进或限制小鼠细胞感染丙型肝炎病毒的宿主因素。(3)分析丙型肝炎病毒感染对TJ完整性和肝细胞功能的影响。 公共卫生相关性：丙型肝炎病毒感染对美国公共卫生构成严重威胁，目前还没有疫苗可用。这项拟议研究的完成将有助于开发新的治疗方法、小动物模型，并有助于我们了解丙型肝炎病毒相关的发病机制。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) exacts a heavy toll on global health by causing acute and chronic hepatitis, cirrhosis, as well as hepatocellular carcinoma. Paradoxical to this cold reality, there is no vaccine available to prevent the infection, and combination therapy with pegylated IFN-a and ribavirin is only effective in 40-80% of patients and has severe side effects. Fortunately, with the recently developed infectious clones, we are finally able to dissect the entire viral life cycle at the molecular level. It is conceivable that novel antiviral therapies and treatment regimens relieving HCV associated pathologies will become available within foreseeable future given the accelerated pace of research. As far as what this application concerns, accumulating evidence has highlighted the importance of tight junction (TJ) in HCV entry. Specifically, we and others reported that the tight junction protein occludin (OCLN) is indispensible for HCV envelope protein-dependent entry. Evidence also indicated that OCLN partially accounts for the narrow host range of HCV. However, murine cells expressing all known human (co-)receptors remained resistant to cell culture grown HCV (HCVcc) infection, suggesting either additional human factors are required or dominant inhibitors exist in murine cells. Interestingly, we observed that both OCLN and claudin-1 were downregulated in HCVcc-infected hepatoma cells. Subsequent study suggested that OCLN is increasingly cleaved in HCV infected cells. In light of these findings, we believe novel therapeutics, novel infection systems, and novel insights in understanding HCV-associated pathogenesis may be developed. We hypothesize that antibody against OCLN extracellular loops or OCLN-derived peptide may block the spreading of virus (to be tested in Aim 1); additional host factors are required to support optimal HCV infection in murine cells or restriction factors exist in these cells (to be tested in Aim 2); downregulation of TJ proteins during HCV entry may alter cell physiology (to be tested in Aim 3). We plan to test the hypotheses by (i) assessing the effect of antibodies against OCLN or OCLN-derived peptides on HCV entry. (ii) identifying host factors promoting or restricting HCV infection of murine cells. (iii) analyzing the impact of HCV infection on TJ integrity and hepatocyte functions. PUBLIC HEALTH RELEVANCE: HCV infection poses a serious threat to US public health and there is currently no vaccine available. Completion of the proposed research will shed lights to the development of novel therapeutics, small animal models, and to our understanding of HCV-associated pathogenesis.