A naturally occurring 2.2-kb defective interfering (DI) RNA of the bovine coronavirus, structurally a simple fusion of the genomic termini, contains a single contiguous open reading frame (ORF) or 1.7 kb composed of the 5'-terminal 288 nucleotides of polymerase gene 1a and all 1,344 nucleotides of the nucleocapsid protein (N) gene. The ORF must remain 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 naturally occurring internal I protein ORF, accomplished by deleting the first base in the N start codon which leads to a +1 frameshift, nor could it be replaced by the chloramphenicol acetyltransferase ORF. Furthermore, 3'-terminal truncations of the N gene leaving less than 85% of its total length were likewise not tolerated. Small in-frame deletions and 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 that 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 RNA and suggest that a similar requirement may exist for viral genome replication.