The overall objective of this proposal is to continue our studies into the mechanism of coronavirus transcription, replication, and RNA recombination utilizing MHV-A59 as a model. The mechanism for MHV transcription is unclear and needs to be reexamined. During mixed MHV infection, RNA recombination occurs at frequencies approaching the reassortment frequencies of RNA viruses with segmented genomes. Currently, little information is available concerning the genetics of MHV transcription or the mechanism of RNA recombination. In this proposal, we shall study the genetics of MHV transcription and replication, and examine the mechanism of RNA recombination. Specifically, we shall: Ill Isolate temperature sensitive (ts) mutants of MHV and characterize these mutants by RNA phenotype and complementation analyses. (2) Identify genetic functions of MHV-A59 which act in regulating MRNA and/or genome synthesis, genome length and subgenomic negative strand synthesis, and the synthesis of the small leader RNAs by temperature shift experiments. (3) Develop a genetic approach to distinguish between the mechanisms for MHV transcription; (4) Determine if the MHV complementation groups encode phenotypically distinct subgroups. (5) Extend the current genetic recombination map for MHV and fine map the location of individual ts mutations in each complementation group; and (6] analyze the mechanism of RNA recombination by: (a) determining the RNA recombination frequency for the MHV genome, (b) determine if hot spots of recombination are present in the genome and map the location and sequences involved, (c) determine if recombination occurs early and/or late in the virus growth cycle and during positive and/or negative strand synthesis, (d) examine the role of subgenomic minus strands in RNA recombination, (e) isolate and sequence crossover sites in recombinant viruses, (f) clone and sequence crossover sites in recombinant MRNA and genome utilizing the polymerase chain reaction technique and specific oligodeoxyribonucleotide primers. These experiments will increase our understanding of the genetics of MHV transcription and provide insight into the mechanism of high frequency RNA recombination.
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