Dynamics of arenavirus gene expression in vivo

描述（由申请方提供）：淋巴细胞性脉络丛脑膜炎病毒（LCMV）是一种单链双义RNA病毒，能够在其天然宿主家鼠中建立非细胞溶解性持续感染。该建议旨在描述LCMV在建立和维持持续感染期间用于调节其复制的机制。我们将测试，在持续感染LCMV期间，（a）病毒是否能够通过以链特异性方式维持其基因组而变得复制静止（基因组意义），（B）病毒锌结合（Z）蛋白是否与基因组的这种链特异性维持相关，和（c）含有小末端缺失的病毒RNA片段的积累是否与病毒蛋白表达和感染性病毒产生的减少有关。为了实现这些目标，我们将用LCMV，Armstrong株感染细胞系或Balb/c小鼠，并在选定的急性和持续时间点收集细胞或组织。从这些样本中，我们将通过实时定量RT-PCR定量（a）感染期间产生的每种完整（基因组、反基因组或亚基因组）或末端缺失（基因组或反基因组）病毒RNA种类的滴度，（B）通过噬斑试验定量感染性病毒的滴度，以及（c）通过Western印迹和免疫组织化学定量病毒蛋白（Z、糖蛋白或核蛋白）表达水平。RNA病毒（逆转录病毒除外）建立和维持持续感染的机制尚未完全了解。通过这一提议，我们希望为LCMV建立和维持持续感染的机制提供新的见解。我们的发现可能适用于其他导致持续感染的RNA病毒。

DESCRIPTION (provided by applicant): Lymphocytic choriomeningitis virus (LCMV) is a single-stranded, ambisense RNA virus that is able to establish a noncytolytic, persistent infection in its natural host, the house mouse. This proposal is designed to delineate the mechanism(s) utilized by LCMV to regulate its replication during the establishment and maintenance of persistent infection. We will test, during persistent infection with LCMV, (a) whether the virus is able to become replicatively quiescent via maintenance of its genome in a strand-specific manner (genomic-sense), (b) whether the viral zinc binding (Z) protein is associated with this strand-specific maintenance of the genome, and (c) whether the accumulation of viral RNA segments containing small terminal deletions is associated with reduced viral protein expression and infectious virus production. To accomplish these aims, we will infect cell lines or Balb/c mice with LCMV, strain Armstrong, and collect cells or tissues at selected acute and persistent time points. From these samples, we will quantitate (a) the titers of each of the intact (genomic, antigenomic, or subgenomic) or terminally-deleted (genomic or antigenomic) viral RNA species produced during infection via real-time quantitative RT-PCR, (b) the titers of infectious virus via plaque assay, and (c) the levels of viral protein (Z, glycoprotein, or nucleoprotein) expression via Western blot and immunohistochemistry. The mechanisms by which RNA viruses (with the exception of retroviruses) establish and maintain persistent infection have yet to be fully understood. Through this proposal, we hope to provide novel insight into the mechanism(s) utilized by LCMV to establish and maintain persistent infection. Our findings could be applicable to other RNA viruses that cause persistent infection.