CIS REQUIREMENT FOR N-SPECIFIC PROTEIN-SEQUENCE IN BOVINE CORONAVIRUSDEFECTIVE INTERFERING RNA REPLICATION

A naturally occurring 2.2-kb defective interfering (DI) RNA of the bov ine coronavirus, structurally a simple fusion of the genomic termini, contains a single contiguous open reading frame (ORF) of 1.7 kb compos ed of the 5'-terminal 288 nucleotides of polymerase gene la and all 1, 344 nucleotides of the nucleocapsid protein (N) gene. The ORF must rem ain open throughout most of its sequence for replication to occur. To determine the qualitative importance of the N portion of the chimeric ORF in DI RNA replication, transcripts of mutated reporter-containing constructs were tested for replication in helper virus-infected cells. It was determined that the N ORF could not be replaced by the natural ly occurring internal I protein ORF, accomplished by deleting the firs t base in the N start codon which leads to a +1 frameshift, nor could it be replaced by the chloramphenicol acetyltransferase ORF. Furthermo re, 3'-terminal truncations of the N gene leaving less than 85% of its total length were likewise not tolerated. Small in-frame deletions an d in-frame foreign sequence insertions of up to 99 nucleotides within certain regions of the N ORF were tolerated, however, but the rate of DI RNA accumulation in these cases was lower. These results indicate t hat there is a requirement for translation of most if not all of the N protein in cis for optimal replication of the bovine coronavirus DI R NA and suggest that a similar requirement may exist for viral genome r eplication.