CONSTRUCTION OF MURINE CORONAVIRUS MUTANTS CONTAINING INTERSPECIES CHIMERIC NUCLEOCAPSID PROTEINS

Targeted RNA recombination was used to construct mouse hepatitis virus (MHV) mutants containing chimeric nucleocapsid (N) protein genes in w hich segments of the bovine coronavirus N gene were substituted in pla ce of their corresponding MHV sequences. This defined portions of the two N proteins that, despite evolutionary divergence, have remained fu nctionally equivalent. These regions included most of the centrally lo cated RNA-binding domain and two putative spacers that link the three domains of the N protein. By contrast, the amino terminus of N, the ac idic carboxy-terminal domain, and a serine- and arginine-rich segment of the central domain could not be transferred from bovine coronavirus to MHV, presumably because these parts of the molecule participate in protein-protein interactions that are specific for each virus (or, po ssibly, each host). Our results demonstrate that targeted recombinatio n can be used to make extensive substitutions in the coronavirus genom e and ean generate recombinants that could not otherwise be made betwe en two viruses separated by a species barrier. The implications of the se findings for N protein structure and function as well as for corona virus RNA recombination are discussed.