Rhinovirus Pathogenesis and Host Range

人鼻病毒（Human rhinoviruses，HRV）是普通感冒的主要病原体， 人类感染。它们也是与哮喘急性发作相关的最常见病原体。 尽管鼻病毒在医学上具有重要意义，但对鼻病毒感染的发病机制知之甚少 因为没有方便的动物模型。本提案中的实验涉及广泛的 关于鼻病毒生物学的实验问题，包括细胞培养中的复制， 宿主范围的基础，对感染的先天反应，以及在小鼠中建立复制。这些目标将是 通过以下四个具体目标来实现。1.确定在鼻病毒复制中的作用 与病毒2B和3A蛋白相互作用的细胞蛋白。我们已经证明， HRV 39与GCP 60和FinGERS相互作用，这些蛋白质参与囊泡运输，并与一种参与 在脂质生物合成中，CDIPT（磷脂酰肌醇合成酶）。与2B相互作用的细胞蛋白质也将被 鉴定我们将评估这些细胞蛋白质在病毒复制、膜改变和细胞凋亡中的作用。 超微结构，并通过降低细胞中这些蛋白质的水平来抑制ER到高尔基体的运输， 干扰它们的功能与显性负形式的蛋白质，或通过产生非相互作用 变异病毒2.确定鼻病毒IRES在病毒宿主范围内的作用。我们发现 HRV 2的IRES不介导成年小鼠的大多数器官中的报告蛋白的翻译， 肺。然而，HRV 2 IRES介导的翻译发生在新生小鼠的许多器官中。实验 在这个特定的目标，旨在确定是否IRES的RV 16，RV 39，和RV 1A可以在 小鼠肺的细胞。这些细胞培养物将用于确定细胞的特定结构化区域是否是细胞的主要结构化区域。 IRES阻止成年小鼠原代肺细胞的内部启动。实验还计划 确定在培养的小鼠细胞中缺乏HRV 2 IRES介导的活性是否是由于抑制剂或 缺乏所需的蛋白质，并提出了鉴定这些因子的实验。3.确定角色 鼻病毒复制中的模式识别分子。针对这一特定目标设计了实验 以确定鼻病毒复制中特定模式识别分子的作用。康贝特人将以 RIG-I、MDA-5、TLR 3和TLR 7在感测培养细胞中鼻病毒复制中的作用。我们发现 RIG-I和MDA-5在感染鼻病毒的细胞中被切割。实验计划确定 这些传感器分子的裂解是否有利于病毒复制。此外，我们将确定是否 鼻病毒可以在树突状细胞中复制，而TLR 3或TLR 7是否在感受病毒的免疫反应中起作用， 在这些细胞中。对在鼻病毒复制中起作用的细胞蛋白的鉴定可能揭示 普通感冒治疗干预的新目标。

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 aredesigned 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.