Optimization of targeted RNA recombination and mapping of a novel nucleocapsid gene mutation in the coronavirus mouse hepatitis virus.

We have recently described a method of introducing site-specific mutations into the genome of the coronavirus mouse hepatitis virus (MHV) by RNA recombination between cotransfected genomic RNA and a synthetic subgenomic mRNA (C. A. Koetzner, M. M. Parker, C. S. Ricard, L. S. Sturman, and P. S. Masters, J. Virol. 66:1841-1848, 1992). By using a thermolabile N protein mutant of MHV (Alb4) as the recipient virus and synthetic RNA7 (the mRNA for the nucleocapsid protein N) as the donor, we selected engineered recombinant viruses as heat-stable progeny resulting from cotransfection. We have now been able to greatly increase the efficiency of targeted recombination in this process by using a synthetic defective interfering (DI) RNA in place of RNA7. The frequency of recombination is sufficiently high that, with Alb4 as the recipient, recombinants can be directly identified without using thermal selection. The synthetic DI RNA has been used to demonstrate that the lesion in another temperature-sensitive and thermolabile MHV mutant, Alb1, maps to the N gene. Sequencing of the Alb1 N gene revealed two closely linked point mutations that fall in a region of the N molecule previously noted as being the most highly conserved region among all of the coronavirus N proteins. Analysis of revertants of the Alb1 mutant revealed that one of the two mutations is critical for the temperature-sensitive phenotype; the second mutation is phenotypically silent.