The long-term goal of this project is to define the location, synthesis and function of the polymerase proteins of the murine coronavirus mouse hepatitis virus (MHV). Mouse hepatitis virus strain A59 (MHV-A59) is capable of causing in mice either an acute, rapidly fatal encephalitis or a persistant infection which causes recurrent demyelination similar in pathology and presentation to multiple sclerosis (MS) in humans. This reproducible, bimodal nature of MHV A59 infection can also be demonstrated in mouse cell culture, either as a lytic infection or a persistent one with no cell changes. Thus MHV presents a model system for the study of viral persistence. This model is ever more intriguing on a molecular level. The virion does not contain polymerase, and thus the first event ofter uncoating is presumed to be synthesis of an RNA dependent, RNA polymerase from gene A of the input positive stand RNA. This polymerase then directs transcription of negative strand RNA as well as subgenomic and genomic positive strand RNA. Since initiation of persistence may include reduction in replication of infections genome, the polymerase may play an important role in the outcome of MHV infection. This protocol proposes a detailed study of the synthesis and processing of the putative polymerase proteins of MHV-A59. Initial studies will involve translation of genomic RNA in vitro in a cell free system. This system will be used to analyze the size, kinetics, processing of gene A translation products and will be modified to favor synthesis of very large proteins for which the polymerase gene potentially codes. Processing will be assayed by studies to identify endogenous and virus encoded proteases as well as membrane directed modifications. These results will be compared with lysates of MHV infected mouse cells. Subsequently, antibodies directed against products of the 5' and 3' portions of gene A will be produced in rabbits from fusion proteins of gene A cDNA subclones or from synthetic oligopeptides based upon the sequences of these clones. Antibodies will be directed against in vitro and in vivo translation products by radioimmunoprecipitation and western blots of electrophoresed proteins to identify minor proteins against a high host cell protein background in vivo, and to assess their times of synthesis and precursor/product relationships. These results will allow mapping of products to specific transcriptional events. The long term goal of this project is to then use antibodies in vivo to examine the contribution of individual transcriptional events on the outcome of MHV A59 infection, and thus increase our understanding of the molecular mechanisms of viral persistence.
