Coronavirus leader RNA regulates and initiates subgenomic mRNA transcription both in trans and in cis

Abstract

Mouse hepatitis virus (MHV), a coronavirus, utilizes a discontinuous transcription mechanism for subgenomic mRNA synthesis. Previous studies (C.-L. Liao and M. C. C. Lai, J. Virol. 68:4727-4737, 1994) have demonstrated that an upstream cis-acting leader sequence serves as a transcriptional enhancer, but the mechanism of transcriptional regulation is not clear. In this study, we constructed a series of defective interfering (DI) RNAs containing the chloramphenicol acetyltransferase (CAT) gene behind a differentially expressed transcription initiation (intergenic) sequence (for mRNA2-1). These DI RNAs had different copy numbers of the UCUAA pentanucleotide sequence at the 3' end of the leader. Transfection of these DI RNA constructs into cells infected with a helper MHV, which contains either two or three UCUAA copies at the 3' end of the leader, resulted in differential expression of CAT activities. We demonstrated that the copy number of UCUAA repeats in the leaders of both helper viral and DI RNAs affected the level of CAT activity, suggesting that MHV leader RNA could regulate both in trans and in cis the transcription of subgenomic mRNAs. The leader RNA of subgenomic mRNAs was derived from either the trans- or the cis-acting leader. Furthermore, insertion of a UA-rich sequence (UUUAUAAAC) immediately downstream of the leader in DI RNA, to match the sequence of helper viral RNA, enhanced the CAT activity by threefold, suggesting that this nine-nucleotide sequence is a cis-acting element. Interestingly, when the nine-nucleotide sequence was absent in DI RNA, the leaders of subgenomic mRNAs were exclusively derived from the helper virus. In contrast, when the nine-nucleotide sequence was present in DI RNA, the leaders were derived from both helper viral and DI RNAs. These results suggest that the nine-nucleotide sequence either is required for the leader RNA to initiate mRNA synthesis or, alternatively, serves as a transcription terminator for the leader RNA synthesis. However, when a constitutively expressed intergenic sequence (for mRNA7) was used, no difference in transcription efficiency was noted, regardless of the copy number of UCUAA in the DI RNA and helper virus. This study thus indicates that MHV subgenomic RNA transcription requires the interaction among the intergenic (promoter) sequence, a trans-acting leader, and a cis-acting leader sequence. A novel model of transcriptional regulation of coronavirus subgenomic mRNAs is presented.

Coronavirus cis-Acting RNA Elements

“…As mentioned earlier, the cis-acting RNA elements required for coronavirus replication (and transcription) are located in the 5 0 -and 3 0 -terminal genome regions and largely (but not exclusively) encompass noncoding regions (Chang et al, 1994;Dalton et al, 2001;Izeta et al, 1999;Kim et al, 1993;Liao and Lai, 1994;Lin et al, 1994Lin et al, , 1996Zhang et al, 1994). Additional cis-acting elements are located at internal positions and include the TRS elements involved in transcription as well as specific RNA signals required for genome packaging Escors et al, 2003;Morales et al, 2013;Penzes et al, 1994).…”

The presence of viral subpopulations in an infectious bronchitis virus vaccine with differing pathogenicity – A preliminary study

“…Interestingly, the coronavirus leader sequence, located at the 5 end of the genomic RNA, is able to regulate the transcription of other coronavirus genomes infecting the same cell during the template switch process (i.e. in cis or trans) [55,56]. Therefore, a possibility to be investigated further is whether the molecular machinery produced by VicS-del can be used for the replication of VicS-v in vivo, assuming both viruses can co-infect cells, which would go some way to explaining the concurrence of reduced growth of VicS-del and an increased growth of VicS-v in vivo, in cases when both subpopulations were present simultaneously.…”

Recombination and Coronavirus Defective Interfering RNAs

“…Furthermore, the UUUAUAAA-containing UUUAUAAAC 9-nt motif occurring just downstream of the MHV leader (Fig. 3A) has been shown to exert a powerful influence on the process of leader switching (37) and on the formation of the leader fusion sites in cis or in trans in MHV (68). This element is often missing in replicating MHV DI RNAs (37), but in BCV, the UUUAUAAA palindromic portion of this sequence is absolutely required for DI RNA replication (36).…”