Rhinovirus Pathogenesis and Host Range

描述(申请人提供)：人类鼻病毒(HRV)是普通感冒的主要病原体，普通感冒是最常见的人类感染之一。它们也是与哮喘恶化有关的最常见的病原体。尽管鼻病毒在医学上具有重要意义，但由于目前还没有方便的动物模型，因此对鼻病毒感染的发病机制了解甚少。这项建议中的实验涉及鼻病毒生物学的广泛实验问题，包括细胞培养中的复制、宿主范围的分子基础、对感染的先天反应以及在小鼠中建立复制。这些目标将通过以下四个具体目标来实现。1.确定与病毒2B和3A蛋白相互作用的细胞蛋白在鼻病毒复制中的作用。我们已经证明，HRV39的3A蛋白与GCP60和Finger相互作用，这些蛋白参与囊泡运输，并与参与脂质生物合成的酶CDIPT(磷脂酰肌醇合成酶)相互作用。与2B相互作用的细胞蛋白也将被识别。我们将评估这些细胞蛋白在病毒复制、膜超微结构改变和抑制内质网到高尔基体运输中的作用，方法是降低细胞中这些蛋白的水平，干扰它们的功能，使其具有显性的阴性形式，或者产生非相互作用的改变病毒。2.确定鼻病毒IRES在病毒宿主范围内的作用。我们发现，在成年小鼠的大多数器官中，包括肺在内的HRV2的IRES不介导报告蛋白的翻译。然而，HRV2 IRES介导的翻译在新生小鼠的许多器官中发生。这一特定目的的实验旨在确定RV16、RV39和RV1a的IRES是否可以在小鼠肺细胞中发挥作用。这些细胞培养将用于确定IRES的特定结构区域是否阻止成年小鼠原代肺细胞的内部启动。还计划进行实验，以确定在培养的小鼠细胞中缺乏HRV2 IRES介导的活性是由于抑制物还是缺乏所需的蛋白质，并提出了确定这些因素的实验。3.确定模式识别分子在鼻病毒复制中的作用。这一特定目的的实验旨在确定特定模式识别分子在鼻病毒复制中的作用。我们将确定RIG-I、MDA-5、TLR3和TLR7在感知鼻病毒在培养细胞中复制中的作用。我们发现RIG-I和MDA-5在感染鼻病毒的细胞中被裂解。计划进行实验以确定这些传感器分子的裂解是否有利于病毒复制。此外，我们将确定鼻病毒是否可以在树突状细胞中复制，以及TLR3或TLR7是否在这些细胞中起到感知病毒基因组的作用。识别在鼻病毒复制中起作用的细胞蛋白可能会为普通感冒的治疗干预提供新的靶点。

DESCRIPTION (provided by applicant): Human rhinoviruses (HRVs) are major causative agents of the common cold, one of the most frequent human infections. They are also the most common pathogens associated with asthma exacerbations. Despite the medical importance of rhinoviruses, the pathogenesis of rhinovirus infection is poorly understood because a convenient animal model is not available. The experiments in this proposal address a wide range of experimental questions on the biology of rhinovirus, encompassing replication in cell culture, the molecular basis of host range, innate responses to infection, and establishing replication in mice. These goals will be accomplished through the following four specific aims. 1. Determine the role in rhinovirus replication of cell proteins that interact with the viral 2B and 3A proteins. We have shown that the 3A protein of HRV39 interacts with GCP60 and FinGERS, proteins involved in vesicle traffic, and with an enzyme involved in lipid biosynthesis, CDIPT (phosphatidylinositol synthase). Cell proteins that interact with 2B will also be identified. We will assess the role of these cell proteins in viral replication, alteration of membrane ultrastructure, and inhibition of ER-to-Golgi traffic by reducing the levels of these proteins in the cell, interfering with their function with dominant negative forms of the proteins, or by producing non-interacting altered viruses. 2. Determine the role of the rhinovirus IRES in viral host range. We have found that the IRES of HRV2 does not mediate translation of a reporter protein in most organs of adult mice, including the lung. However, HRV2 IRES-mediated translation takes place in many organs of neonatal mice. Experiments in this specific aim are designed to determine whether the IRES of RV16, RV39, and RV1A can function in cells of the mouse lung. These cell cultures will be used to determine if specific structured regions of the IRES prevent internal initiation in primary lung cells from adult mice. Experiments are also planned to determine whether the lack of HRV2 IRES-mediated activity in cultured mouse cells is due to an inhibitor or absence of a required protein, and experiments to identify such factors are proposed. 3. Determine the role of pattern recognition molecules in rhinovirus replication. Experiments in this specific aim are designed to determine the role of specific pattern recognition molecules in rhinovirus replication. We will determine the roles of RIG-I, MDA-5, TLR3, and TLR7 in sensing rhinovirus replication in cultured cells. We have found that RIG-I and MDA-5 are cleaved in cells infected with rhinoviruses. Experiments are planned to determine whether cleavage of these sensor molecules benefits viral replication. In addition, we will determine whether rhinoviruses can replicate in dendritic cells, and whether TLR3 or TLR7 plays a role in sensing the viral genome in these cells. The identification of cell proteins that play a role in rhinovirus replication may reveal new targets for therapeutic intervention of the common cold.