Identification of cellular factors that mediate HCV cell-to-cell spread

 描述(由申请人提供)：病毒进入允许细胞是确定感染的第一步，因此是常见且往往有效的抗病毒药物靶点。然而，在病毒颗粒在最初感染的细胞中复制和组装后，许多病毒，如丙型肝炎病毒，可以通过两种途径传播到其他细胞：无细胞传播和细胞间传播。由于细胞间传播可能是建立持续感染的一个因素，并与抗病毒治疗期间病毒逃逸突变株的传播有关，因此阐明病毒细胞间传播的机制(S)不仅提供了一个机会，进一步加深我们对病毒学和发病机制这一相对较少研究的方面的理解，还可能为在治疗过程中增强抗病毒耐药性屏障的全球策略提供洞察力。这与丙型肝炎病毒(丙型肝炎病毒)有关，因为尽管有效的不含干扰素的直接作用抗病毒治疗组合已可用于治疗丙型肝炎病毒，但病毒逃逸的风险尚未在不理想的依从性人群中确定，这些药物的天文数字成本使其对世界上大多数丙型肝炎病毒阳性人群来说昂贵得令人望而却步。为了解决丙型肝炎病毒特有的和更普遍的公共卫生问题，我们的长期目标包括阐明丙型肝炎病毒无细胞传播和细胞间传播的分子机制，评估细胞间传播在建立和维持慢性病毒感染中的作用，以及评估阻断病毒细胞间传播是否可以影响不同联合治疗方法的协同潜力和耐药性障碍。这一小型试点R03提案的更重点的目标是通过定向siRNA击倒筛选已知参与病毒从生产者细胞无细胞出口或无细胞进入目标细胞的询问基因，识别参与丙型肝炎病毒细胞间传播的宿主细胞因子/途径。虽然我们已经知道无细胞进入和细胞间进入具有一些相同的因素和机制(例如SRB1、CLDN1、OCLN)，但我们有初步数据确定不同所需的进入因素。更独特的是，我们还发现，无细胞传染性病毒颗粒排出所需的某些细胞因子，不是细胞间传播所必需的。因此，我们假设，虽然无细胞传播和细胞间传播之间有一些共同之处，但存在关键的机制差异。重要的是，一旦确定了这些差异，就可以利用它们作为研究工具和开发有效的抗病毒策略。因此，这项先导性研究将提供所需的基本信息，以便设计对这些关键病毒-宿主相互作用进行更深入的机制研究，并确定两种传播途径中涉及的因素是否可以作为高效的抗病毒靶标，增强病毒耐药性的屏障，这是治疗病毒感染的一个广泛的临床相关问题。

 DESCRIPTION (provided by applicant): Viral entry into permissive cells is the first step in establishing infection and is thus a common and often effective antiviral drug target. However, after replication and assembly of viral particles in the initially infected cell, many viruses suchas HCV can spread to infect additional cells by two routes: cell-free and cell-to-cell spread. Because cell-to-cell spread can be a factor in the establishment of persistent infections and has been implicated in the spread of viral escape mutants during antiviral treatment, elucidating the mechanism(s) of viral cell-to-cell spread provides an opportunity to not only further our understanding of this relatively understudied aspect of virology and pathogenesis, but also perhaps provide insight into global strategies for enhancing the barrier to antiviral resistance during treatment. This is relevant for hepatitis C virus (HCV) because while effective interferon-free direct acting antiviral therapeutic combinations are becoming available for the treatment of HCV, the risk of viral escape has not been determined in less than ideal compliance populations and the astronomical cost of these drugs makes them prohibitively expensive for the majority of the world's HCV-positive populations. To address both this HCV-specific and more general public health issue, our long term goals include elucidating the molecular mechanisms of cell-free and cell-to-cell HCV spread, assessing the role of cell-to-cell spread in the establishment and maintenance of chronic viral infections, and assessing how blocking viral cell- to-cell spread, or not, can impact the synergy potential and barrier to resistance of different combination therapy approaches. The more focused objective of this small pilot R03 proposal is to identify the host cell factors/pathways involved in HCV cell-to-cell spread through directed siRNA knockdown screening interrogating genes already known to be involved in cell-free viral egress from the producer cell or cell-free entry into a target cells. While we already know that cell-free and cell-to-cell entry share some of the same factors and mechanisms (e.g. SRB1, CLDN1, OCLN), we have preliminary data identifying differentially required entry factors. More uniquely, we have also found that certain cellular factors required for the egress of cell-free infectious virus particles, are not required for the cell-to-cell spread. Hence, we hypothesize tha while there are some commonalities between cell-free and cell-to-cell spread, that there key mechanistic differences. Importantly, once identified, these differences can be exploited both as research tools and to develop effective antiviral strategies. As such, this pilot study will provid the fundamental information needed to enable the design of more in-depth mechanistic studies of these critical virus-host interactions and to determine whether factors involved in both routes of spread can serve as highly effective antiviral targets that enhance the barrier to viral resistance, a broad clinically relevant issue for the treatment of viral infections.