MOLECULAR BASIS OF PERSISTENT REOVIRUS INFECTION

这项研究的目的是了解是什么决定了 病毒感染是裂解性或持续性的。哺乳动物呼肠孤病毒有 被用作模型系统来探索分子决定因素 病毒在细胞内的复制和病毒在宿主中的致病机制。 呼肠孤病毒通常是溶细胞性的，但可以建立持久的 用高传代(HP)菌种引发L细胞感染的研究 感染。已经提出了一个模型，该模型认为特定的基因 片段在呼肠孤病毒的持久性中起着重要的作用。L2基因是 在高传代过程中，S4对突变的产生很重要 基因对持久力的启动很重要，而S1基因是 对于持久状态的维护很重要。这些基因各自 编码具有良好功能的衣壳蛋白。为了 研究呼肠孤病毒在细胞培养中持续感染的机制 将确定分离病毒的S4和S1核苷酸序列 从幽门螺杆菌开始的独立持续感染L细胞培养 野生型呼肠孤病毒3型毒株的库存(T3D)。的变化 这些基因编码的蛋白质的推导氨基酸序列将 与相应的T3D序列进行比较，以便开发出 模型来解释S4和S1的突变如何影响预后 病毒感染。此外，我们还发现一种氯化铵 (Ac)呼肠孤病毒复制的敏感步骤可能会在 呼肠孤病毒持续存在。AC作用于抑制溶酶体内消化 受体介导的内吞作用后的病毒粒子。从以下地点分离的病毒 持续感染的培养物能抵抗生长抑制。 用AC处理的细胞，表明对 溶酶体内消化病毒粒子可以调节病毒的裂解能力 并导致病毒的建立和持续感染。我们会 进行实验以更好地了解交流敏感的本质 通过使用其他抑制呼肠孤病毒复制的试剂 内切体和溶酶体的酸化，我们将确定 通过使用重组病毒获得AC抗性的遗传基础。 最后，我们发现了从中国分离的病毒对中枢神经系统的清除 与T3D相比，持续感染的培养被延迟， 这表明在体内存在与体外持久性相关的因素。 我们将研究中枢神经系统感染病毒所产生的病理 从持续感染的培养中分离出来，我们将确定 使用重配子延迟清除中枢神经系统的遗传学基础 病毒。此外，我们将连续传代从中国分离的病毒 在新生小鼠大脑中持续感染培养物，以便 确定显示中枢神经系统清除延迟的病毒是否可以 适应于在动物身上建立毅力。我们的方法，即 利用已知在呼肠孤病毒中改变的基因的分子研究 除了呼肠孤病毒致病机制的研究外，还要持之以恒 从持续感染的文化中分离出来，将使我们能够获得 洞察病毒持续存在的分子基础并探索 病毒介导的细胞损伤机制。

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.