MOLECULAR BASIS OF PERSISTENT REOVIRUS INFECTION

持续性呼肠孤病毒感染的分子基础

• 批准号: 2067435
• 负责人: TERENCE S. DERMODY
• 金额: $ 10.24万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2067435
• 关键词: L cell; Orthoreovirus; Reoviridae disease; ammonium chloride; cell growth regulation; central nervous system; double stranded RNA; drug resistance; endocytosis; gene mutation; growth inhibitors; histopathology; host organism interaction; laboratory mouse; laboratory rabbit; microorganism growth; nervous system infection; newborn animals; nucleic acid sequence; protein structure function; recombinant DNA; tissue /cell culture; virus cytopathogenic effect; virus genetics; virus infection mechanism; virus protein; virus replication

The objective of this research is to understand what determines whether a viral infection is lytic or persistent. The mammalian reoviruses have been used as a model system to explore the molecular determinants of viral replication in the cell and viral pathogenesis in the host. Reoviruses are typically cytolytic, but can establish persistent infection in L cells if high-passage (HP) stocks are used to initiate infection. A model has been proposed which suggests that specific gene segments play important roles in reovirus persistence. The L2 gene is important for the generation of mutations during high passage, the S4 gene is important for initiation of persistence, and the S1 gene is important for maintenance of the persistent state. These genes each encode capsid proteins with well-characterized functions. In order to study the mechanism of persistent reovirus infection in cell culture, we will determine the S4 and S1 nucleotide sequences of viruses isolated from independent persistently infected L-cell cultures initiated with HP stocks of wild-type reovirus strain type 3 Dearing (T3D). Changes in the deduced amino acid sequences of the proteins encoded by these genes will be compared to the corresponding T3D sequences in order to develop a model to explain how mutations in S4 and S1 can affect the outcome of viral infection. In addition, we have found that an ammonium chloride (AC) sensitive step in reovirus replication is subject to change during reovirus persistence. Ac acts to inhibit the intralysosomal digestion of virions following receptor-mediated endocytosis. Viruses isolated from persistently infected cultures are resistant to growth inhibition in cells treated with AC, suggesting that modifications of the intralysosomal digestion of virions can regulate the lytic potential of the virus and lead to the establishment of persistent infection. We will conduct experiments to better understand the nature of the AC-sensitive step in reovirus replication by using other reagents which inhibit the acidification of endosomes and lysosomes, and we will determine the genetic basis of AC-resistance through the use of reassortant viruses. Finally, we have found the CNS clearance of viruses isolated from persistently infected cultures is delayed in comparison to T3D, suggesting that there is an in vivo correlate to in vitro persistence. We will investigate the pathology produced by CNS infection with viruses isolated from persistently infected cultures, and we will determine the genetic basis of delayed clearance from the CNS by using reassortant viruses. Furthermore, we will serially passage viruses isolated from persistently infected cultures in the newborn mouse brain in order to determine whether viruses which manifest delayed CNS clearance can be adapted to establish persistence in the animal. Our approach, which utilizes molecular studies of genes known to be altered in reovirus persistence in addition to studies of the pathogenesis of reoviruses isolated from persistently infected cultures, will allow us to gain insight into the molecular basis of viral persistence and to explore the mechanism of viral-mediated cellular injury.

这项研究的目的是了解是什么决定了 病毒感染是溶解性的或持续性的。哺乳动物呼肠孤病毒 已被用作模型系统，以探索分子决定因素， 细胞中的病毒复制和宿主中的病毒发病机制。 呼肠孤病毒通常是溶细胞的，但可以建立持久的 如果使用高传代（HP）储备液启动，则L细胞中会发生感染 感染已经提出了一个模型，表明特定基因 片段在呼肠孤病毒持久性中起重要作用。L2基因是 对于在高传代期间产生突变很重要，S4 基因是重要的启动持久性，和S1基因是 这对于持久状态的维护是非常重要的。每一个基因 编码具有良好表征功能的衣壳蛋白。为了 为了研究呼肠孤病毒在细胞培养中持续感染的机制， 将确定分离的病毒的S4和S1核苷酸序列， 来自HP启动的独立持续感染L细胞培养物 野生型呼肠孤病毒毒株3型Dearing（T3D）的原种。变化 由这些基因编码的蛋白质的推导的氨基酸序列将 与相应的T3D序列进行比较，以开发 模型来解释S4和S1的突变如何影响 病毒感染此外，我们还发现，氯化铵 (AC)呼肠孤病毒复制中的敏感步骤在 呼肠孤病毒持久性。Ac的作用是抑制溶酶体内消化 受体介导的内吞作用后的病毒体。病毒分离自 持续感染的培养物对生长抑制具有抗性， 用AC处理的细胞，这表明 溶酶体内消化病毒粒子可以调节 病毒并导致持续感染的建立。我们将 进行实验，以更好地了解交流敏感的性质 通过使用其它抑制呼肠孤病毒复制的试剂， 核内体和溶酶体的酸化，我们将确定 通过使用抗性病毒的AC抗性的遗传基础。 最后，我们发现从以下组织分离的病毒可以被中枢神经系统清除 与T3D相比，持续感染的培养物被延迟， 这表明存在与体外持久性相关的体内相关性。 我们将研究中枢神经系统感染病毒所产生的病理学 从持续感染的培养物中分离出来，我们将确定 使用抑制剂延迟CNS清除的遗传基础 病毒此外，我们将连续传代从 在新生小鼠脑中持续感染培养物， 确定表现出CNS清除延迟的病毒是否可以 适应于在动物体内建立持久性。我们的方法， 利用已知在呼肠孤病毒中改变的基因的分子研究， 持久性除了研究呼肠孤病毒的发病机制 从持续感染的培养物中分离出来，将使我们获得 深入了解病毒持久性的分子基础，并探索 病毒介导的细胞损伤机制。