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.

中文翻译：

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禽冠状病毒传染性支气管炎病毒产生的多种新型非规范转录亚基因组 mRNA。

冠状病毒亚基因组 mRNA (sgmRNA) 的合成是通过不连续转录过程发生的，该过程涉及互补转录调控序列 (TRS)，其中一个 (TRS-L) 包含 5' 非翻译区 (UTR) 的前导序列，另一个上游包含每个结构和辅助基因 (TRS-B)。几种冠状病毒的 ORF 位于 N 基因和 3'-UTR 之间，该区域以前被认为是 Gammacoronavirus 的非编码区域传染性支气管炎病毒 (IBV) 由于缺乏典型的 TRS-B。在这里，我们确定了一个非规范的 TRS-B，允许在几种 IBV 菌株中产生与该 ORF 相关的新型 sgmRNA：Beaudette、CR88、H120、D1466、Italy-02 和 QX。有趣的是，由该 ORF 产生的潜在蛋白质在 Beaudette 菌株中被过早截断。在 Beaudette 菌株中进行了单核苷酸缺失，从而产生了具有表达全长蛋白质潜力的重组 IBV (rIBV)。在体外评估这种 rIBV证明全长潜在蛋白的恢复对病毒复制没有影响。对 Beaudette 衍生的 RNA 的进一步评估确定了位于基因 2 内的第二个非规范转录的 sgmRNA。对 Beaudette 感染的含胚卵的尿囊液进行深度测序分析，证实了新发现的非规范转录的 sgmRNA 的存在，并强调了潜在的用于进一步尚未识别的 sgmRNA。该 HiSeq 数据以及非规范转录 sgmRNA 的确认表明，冠状病毒基因组有可能编码比目前已确定的更大的基因库。