title: Analysis of second-site revertants of a murine coronavirus nucleocapsid protein deletion mutant and construction of nucleocapsid protein mutants by targeted RNA recombination.
abstract: The Alb4 mutant of the coronavirus mouse hepatitis virus (MHV) is both temperature sensitive and thermolabile owing to a deletion in the gene encoding its nucleocapsid (N) protein. The deletion removes 29 amino acids that constitute a putative spacer region preceding the carboxyl-terminal domain of the protein. As a step toward understanding the structure and function of the MHV N protein, we isolated multiple independent revertants of Alb4 that totally or partially regained the ability to form large (wild-type-sized) plaques at the nonpermissive temperature. The N proteins of these revertant viruses concomitantly regained the ability to bind to RNA in vitro at a temperature that was restrictive for RNA binding by Alb4 N protein. Sequence analysis of the N genes of the revertants revealed that each contained a single second-site point mutation that compensated for the effects of the deletion. All reverting mutations were clustered within a stretch of 40 amino acids centered some 80 residues on the amino side of the Alb4 deletion, within a domain to which the RNA-binding activity of N had been previously mapped. By means of a targeted RNA recombination method that we have recently developed, two of the reverting mutations were introduced into a wild-type MHV genomic background. The resulting recombinants were stable and showed no gross phenotypic differences from the wild type. A detailed analysis of one, however, revealed that it was at a selective disadvantage with respect to the wild type.
authors.affiliation: Department of Biomedical Sciences, State University of New York at Albany 12237, USA.
authors.forename: D
authors.initials: D
authors.lastname: Peng
authors.forename: C A
authors.initials: CA
authors.lastname: Koetzner
authors.forename: P S
authors.initials: PS
authors.lastname: Masters
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000235:genetics*; D002460:Cell Line; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics*; D009154:Mutation; D012367:RNA, Viral / Q000235:genetics*; D011995:Recombination, Genetic; D017384:Sequence Deletion; D014758:Viral Core Proteins / Q000235:genetics*
chemical_list: D012367:RNA, Viral; D014758:Viral Core Proteins