title: Functional and genetic analysis of coronavirus replicase-transcriptase proteins.
abstract: The coronavirus replicase-transcriptase complex is an assembly of viral and cellular proteins that mediate the synthesis of genome and subgenome-sized mRNAs in the virus-infected cell. Here, we report a genetic and functional analysis of 19 temperature-sensitive (ts) mutants of Murine hepatitis virus MHV-A59 that are unable to synthesize viral RNA when the infection is initiated and maintained at the non-permissive temperature. Both classical and biochemical complementation analysis leads us to predict that the majority of MHV-A59 ORF1a replicase gene products (non-structural proteins nsp1-nsp11) form a single complementation group (cistron1) while the replicase gene products encoded in ORF1b (non-structural proteins nsp12-nsp16) are able to function in trans and comprise at least three, and possibly five, further complementation groups (cistrons II-VI). Also, we have identified mutations in the non-structural proteins nsp 4, nsp5, nsp10, nsp12, nsp14, and nsp16 that are responsible for the ts phenotype of eight MHV-A59 mutants, which allows us to conclude that these proteins are essential for the assembly of a functional replicase-transcriptase complex. Finally, our analysis of viral RNA synthesis in ts mutant virus-infected cells allows us to discriminate three phenotypes with regard to the inability of specific mutants to synthesize viral RNA at the non-permissive temperature. Mutant LA ts6 appeared to be defective in continuing negative-strand synthesis, mutant Alb ts16 appeared to form negative strands but these were not utilized for positive-strand RNA synthesis, and mutant Alb ts22 was defective in the elongation of both positive- and negative-strand RNA. On the basis of these results, we propose a model that describes a pathway for viral RNA synthesis in MHV-A59-infected cells. Further biochemical analysis of these mutants should allow us to identify intermediates in this pathway and elucidate the precise function(s) of the viral replicase proteins involved.
authors.affiliation: Department of Medical Microbiology and Immunology, Medical University of Ohio, Toledo, Ohio, USA.
authors.forename: Stanley G
authors.initials: SG
authors.lastname: Sawicki
authors.forename: Dorothea L
authors.initials: DL
authors.lastname: Sawicki
authors.forename: Diane
authors.initials: D
authors.lastname: Younker
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Meyer
authors.forename: Volker
authors.initials: V
authors.lastname: Thiel
authors.forename: Helen
authors.initials: H
authors.lastname: Stokes
authors.forename: Stuart G
authors.initials: SG
authors.lastname: Siddell
mesh_terms: D003332:Coronaviridae / Q000201:enzymology* / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D009154:Mutation; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics
chemical_list: D014764:Viral Proteins; D012194:RNA-Directed DNA Polymerase; D012321:DNA-Directed RNA Polymerases