ANALYSIS OF A RECOMBINANT MOUSE HEPATITIS-VIRUS EXPRESSING A FOREIGN GENE REVEALS A NOVEL ASPECT OF CORONAVIRUS TRANSCRIPTION

We have inserted heterologous genetic material into the nonessential g ene 4 of the coronavirus mouse hepatitis virus (MHV) in order to test the applicability of targeted RNA recombination for site-directed muta genesis of the MHV genome upstream of the nucleocapsid (N gene and to develop further genetic tools for site-directed mutagenesis of structu ral genes other than N, Initially, a 19-nucleotide tag was inserted in to the start of gene 4a of MHV strain A59 with the N gene deletion mut ant AIb-4 as the recipient virus. In further work, the entire gene for the green fluorescent protein (GFP) was inserted in place of gene 4, creating the currently largest known RNA virus. The expression of GFP was demonstrated by Western blot analysis of infected cell lysates; ho wever, the level of GFP expression was not sufficient to allow detecti on of fluorescence of viral plaques. Northern blot analysis of transcr ipts of GFP recombinants showed the expected alteration of the pattern of the nested MHV subgenomic mRNAs. Surprisingly, though, GFP recombi nants also produced an RNA species that was the same size as wild-type mRNA4. Analysis of the 5' end of this species revealed that it was ac tually a collection of mRNAs originating from 10 different genomic fus ion sites, none possessing a canonical intergenic sequence. The findin g of these aberrant mRNAs suggests that long-range RNA or the ribonucl eoprotein structure of the MHV genome can sometimes be the sole determ inant of the site of initiation of transcription.