Comparative Analysis of Gene Expression in Virulent and Attenuated Strains of Infectious Bronchitis Virus at Subcodon Resolution

ABSTRACTLike all coronaviruses, avian infectious bronchitis virus (IBV) possesses a long, single-stranded, positive-sense RNA genome (∼27 kb) and has a complex replication strategy that includes the production of a nested set of subgenomic mRNAs (sgmRNAs). Here, we used whole-transcriptome sequencing (RNASeq) and ribosome profiling (RiboSeq) to delineate gene expression in the IBV M41-CK and Beau-R strains at subcodon resolution. RNASeq facilitated a comparative analysis of viral RNA synthesis and revealed two novel transcription junction sites in the attenuated Beau-R strain, one of which would generate a sgmRNA encoding a ribosomally occupied open reading frame (dORF) located downstream of the nucleocapsid coding region. RiboSeq permitted quantification of the translational efficiency of virus gene expression and identified, for the first time, sites of ribosomal pausing on the genome. 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 to 40%). Triplet phasing of RiboSeq data allowed precise determination of reading frames and revealed the translation of two ORFs (ORF4b and ORF4c on sgmRNA IR), which are widely conserved across IBV isolates. Analysis of differential gene expression in infected primary chick kidney cells indicated that the host cell response to IBV occurs primarily at the level of transcription, with global upregulation of immune-related mRNA transcripts following infection and comparatively modest changes in the translation efficiencies of host genes. Cellular genes and gene networks differentially expressed during virus infection were also identified, giving insights into the host cell response to IBV infection.IMPORTANCEIBV 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 virus, 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 cells infected with both pathogenic and attenuated forms of IBV and expand our understanding of gammacoronaviral gene expression. We demonstrate that gene expression shows considerable intraspecies variation, with single nucleotide polymorphisms being associated with altered production of sgmRNA transcripts, and our RiboSeq data sets enabled us to uncover novel ribosomally occupied ORFs in both strains. The numerous cellular genes and gene networks found to be differentially expressed during virus infection provide insights into the host cell response to IBV infection.

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

10.1101/612614 ◽

2019 ◽

Cited By ~ 1

Author(s):

Adam M. Dinan ◽

Sarah Keep ◽

Erica Bickerton ◽

Paul Britton ◽

Andrew E. Firth ◽

...

Keyword(s):

Gene Expression ◽

Host Cell ◽

Infectious Bronchitis Virus ◽

Gene Networks ◽

Ribosome Profiling ◽

Species Variation ◽

Messenger Rnas ◽

High Resolution Data ◽

Infectious Bronchitis

ABSTRACTInfectious bronchitis virus (IBV) is a member of the genusGammacoronavirusand 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 within vitromeasurements. 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.IMPORTANCEIBV 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.

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