Abstract
Coronavirus sub-genomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving complementary transcription regulatory sequences (TRSs), one (TRS-L) encompassing the leader sequence of the 5′ untranslated region (UTR), and the other upstream of each structural and accessory gene (TRS-B). Several coronaviruses have an ORF located between the N gene and the 3′-UTR, an area previously thought to be non-coding in the Gammacoronavirus infectious bronchitis virus (IBV) due to a lack of a canonical TRS-B. Here, we identify a non-canonical TRS-B allowing for a novel sgmRNA relating to this ORF to be produced in several strains of IBV: Beaudette, CR88, H120, D1466, Italy-02 and QX. Interestingly, the potential protein produced by this ORF is prematurely truncated in the Beaudette strain. A single nucleotide deletion was made in the Beaudette strain allowing for the generation of a recombinant IBV (rIBV) that had the potential to express a full-length protein. Assessment of this rIBV in vitro demonstrated that restoration of the full-length potential protein had no effect on viral replication. Further assessment of the Beaudette-derived RNA identified a second non-canonically transcribed sgmRNA located within gene 2. Deep sequencing analysis of allantoic fluid from Beaudette-infected embryonated eggs confirmed the presence of both the newly identified non-canonically transcribed sgmRNAs and highlighted the potential for further yet unidentified sgmRNAs. This HiSeq data, alongside the confirmation of non-canonically transcribed sgmRNAs, indicates the potential of the coronavirus genome to encode a larger repertoire of genes than has currently been identified.
Highlights
Coronaviruses are members of the order Nidovirales, and possess the largest genomes of the currently identified RNA viruses
For the Infectious bronchitis virus (IBV) strain Beaudette, a canonical transcription regulatory sequences (TRSs)-B of either CUUAACAA, CUGAACAA or CUUAAUAA cannot be identified in the genome between the middle of the N gene and 5′ of the putative ORF, which raised the possibility that the mechanism of transcription was either similar to that of gene 4b [6], which uses a shortened non-c anonical TRS-B, or that a TRS-B of an unidentified sequence was being utilized
To determine whether a sub-genomic mRNA (sgmRNA) was produced during IBV infection that relates to the 3′ end of the genome, primary chicken kidney (CK) cells were inoculated with the recombinant IBV (rIBV) Beau-R [20]
Summary
Coronaviruses are members of the order Nidovirales, and possess the largest genomes of the currently identified RNA viruses. Notable members of the genus Betacoronavirus include the human pathogens severe acute respiratory syndrome coronavirus (SARSCoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), and the recent zoonotic virus SARS-CoV-2, the causative agent of COVID-19. Infectious bronchitis virus (IBV), the prototype of the genus Gammacoronavirus, is the aetiological agent of infectious bronchitis; an acute, highly contagious and economically important disease of poultry (reviewed elsewhere [1, 2]). As with all members of the family Coronaviridae, IBV possesses a large, ~27 kb, non-segmented, positive-sense, ssRNA genome, which is capped at the 5′ end and polyadenylated at the 3′ end [3]. General genome organization is shared between all coronaviruses, with the 5′
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