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

作者：PENG D; KOETZNER CA; MASTERS PS

来源：Journal of Virology, 1995, 69(6): 3449-3457.

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 shelved 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.

• 出版日期1995-6