Comparative analysis of gene expression in virulent and attenuated strains of infectious bronchitis virus at sub-codon resolution

Dinan, Adam M.; Keep, Sarah; Bickerton, Erica; Britton, Paul; Firth, Andrew E.; Brierley, Ian

Infectious bronchitis virus (IBV) is a member of the genus Gammacoronavirus and the causative agent of avian infectious bronchitis. IBV has a single-stranded, positive-sense RNA genome ~27 kb in length and, like all coronaviruses, produces a set of sub-genomic messenger RNAs (sgmRNAs) synthesised via the viral polymerase. Here, we used RNA sequencing (RNASeq) and ribosome profiling (RiboSeq) to delineate gene expression in the IBV M41-CK and Beau-CK strains at sub-codon resolution. Quantification of reads flanking the programmed ribosomal frameshifting (PRF) signal at the genomic RNA ORF1a/ORF1b junction revealed that PRF in IBV is highly efficient (33–40%), consistent with in vitro measurements. Triplet phasing of the profiling data allowed precise determination of reading frames and revealed the translation of two intergenic genes (4b and 4c on sgmRNA4), which are widely conserved across IBV isolates. RNASeq revealed two novel transcription junction sites in the attenuated Beau-CK strain, one of which would generate a sgmRNA encoding a ribosomally occupied ORF in the viral 3’ untranslated region (dORF). Within IBV transcripts, the nucleocapsid (N) protein was unexpectedly found to be inefficiently translated, despite being an abundant structural component of mature IBV virions. Finally, we demonstrate that the host cell response to IBV occurs primarily at the level of transcription, with a global up-regulation of immune-related mRNA transcripts following infection, and comparatively modest changes in the translation efficiencies of host genes. IMPORTANCE IBV is a major avian pathogen and presents a substantial economic burden to the poultry industry. Improved vaccination strategies are urgently needed to curb the global spread of this pathogen, and the development of suitable vaccine candidates will be aided by an improved understanding of IBV molecular biology. Our high-resolution data have enabled a precise study of transcription and translation in both pathogenic and attenuated forms of IBV, and expand our understanding of gammacoronaviral gene expression. We demonstrate that gene expression shows considerable intra-species variation, with single nucleotide polymorphisms associated with altered production of sgmRNA transcripts, and our RiboSeq data sets enabled us to uncover novel ribosomally occupied ORFs in both strains. We also identify numerous cellular genes and gene networks that are differentially expressed during virus infection, giving insights into the host cell reponse to IBV infection.