Tight Junction: Gate to HCV Tropism, Therapeutics and Pathogenesis

描述（由申请人提供）：丙型肝炎病毒（HCV）通过引起急性和慢性肝炎、肝硬化以及肝细胞癌，对全球健康造成严重影响。与这个冷酷的现实相矛盾的是，没有疫苗可以预防感染，聚乙二醇化IFN-a和利巴韦林的联合治疗仅对40-80%的患者有效，并且有严重的副作用。幸运的是，随着最近开发的传染性克隆，我们终于能够在分子水平上解剖整个病毒生命周期。可以想象，在可预见的未来，随着研究步伐的加快，缓解丙型肝炎相关病理的新型抗病毒疗法和治疗方案将会出现。就这一应用而言，越来越多的证据强调了紧密结（TJ）在HCV进入中的重要性。具体来说，我们和其他人报道了紧密连接蛋白闭塞蛋白（OCLN）对于HCV包膜蛋白依赖的进入是必不可少的。证据还表明，OCLN部分解释了HCV宿主范围狭窄的原因。然而，表达所有已知人类（共）受体的小鼠细胞仍然对细胞培养的HCV （HCVcc）感染具有抗性，这表明要么需要额外的人为因素，要么小鼠细胞中存在显性抑制剂。有趣的是，我们观察到在hcvcc感染的肝癌细胞中OCLN和claudin-1均下调。随后的研究表明，在HCV感染的细胞中，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，以及对hcv相关发病机制的理解可能会发展出新的见解。我们假设针对OCLN细胞外环或OCLN衍生肽的抗体可能阻止病毒的传播（在Aim 1中进行测试）；需要额外的宿主因子来支持小鼠细胞中最佳的HCV感染，或者这些细胞中存在限制因子（将在第二阶段进行测试）；HCV进入时TJ蛋白的下调可能会改变细胞生理（在Aim 3中进行测试）。我们计划通过(i)评估抗OCLN或OCLN衍生肽抗体对HCV进入的影响来验证这些假设。（ii）确定促进或限制小鼠细胞HCV感染的宿主因子。(3)分析HCV感染对TJ完整性和肝细胞功能的影响。

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.