Sequences Required for Replication and Packaging of IBV RNA

Abstract

We have used a naturally occurring IBV defective RNA (D-RNA), CD-91 (9.1 kb; Penzes et al., 1994) to investigate IBV replication and packaging signals. This D-RNA comprised the 5’-most 1133 nucleotides (domain I, including the 528 nucleotide 5’ untranslated region, UTR) and 3’-most 1626 nucleotides (domain III) of the genome and a middle section (domain II) comprising discontinuous parts (6322 nucleotides in total) of the 1b open reading frame (ORF) of gene 1, the Polymerase gene. In a limited study (Penzes et al., 1996) 3 kb of the ORF 1b sequence was removed to produce CD-61 which was replicated and packaged as efficiently as CD-91. Removal of a further 1.4 kb from ORF 1b produced D-RNA CD-44 which was not rescued (replicated and packaged; detected by Northern blot analysis) by helper IBV. This led us to propose that the 1.4 kb sequence was essential either for replication or, more likely, for packaging of the RNA. We have extended that investigation to an additional 26 modified D-RNAs. Previously we relied upon several passages of the rescued D-RNAs to produce sufficient RNA to be detectable by Northern blot analysis. The incorporation of a chloramphenicol acetyltransferase (CAT) reporter gene into the D-RNAs (Stirrups et al., 2000b) has allowed us to detect replication of IBV D-RNA constructs in transfected cells, without reliance on packaging to indicate that replication had occurred. Thus we have been able to distinguish the processes of replication and packaging.