ANALYSIS OF 2ND-SITE REVERTANTS OF A MURINE CORONAVIRUS NUCLEOCAPSID PROTEIN DELETION MUTANT AND CONSTRUCTION OF NUCLEOCAPSID PROTEIN MUTANTS BY TARGETED RNA RECOMBINATION

The Alb4 mutant of the coronavirus mouse hepatitis virus (MHV) is both temperature sensitive and thermolabile owing to a deletion in the gen e 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 st ructure and function of the MHV N protein, we isolated multiple indepe ndent revertants of Alb4 that totally or partially regained the abilit y to form large (wild-type-sized) plaques at the nonpermissive tempera ture. The N proteins of these revertant viruses concomitantly regained the ability to bind to RNA in vitro at a temperature that was restric tive for RNA binding by Alb4 N protein. Sequence analysis of the N gen es 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, with in 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 int o a wild-type MHV genomic background; The resulting recombinants were stable and shelved no gross phenotypic differences from the wild type, A detailed analysis of one, however, revealed that it was at a select ive disadvantage with respect to the wild type.