Coronavirus transcription model
Experimental data on transcription in coronaviruses and the related arteriviruses can be integrated into a transcription model that includes three steps.
- First, gRNA forms transcription initiation precomplexes, bringing into physical proximity the distal TRS-L and TRS-B. RNA-RNA, RNA-protein, and protein-protein interactions might maintain these precomplexes in a dynamic equilibrium.
- These precomplexes act as slowdown and detachment signals for the transcription complex during the synthesis of negative-strand RNA
- Once the TRS-B has been copied, if the ΔG of duplex formation between the cTRS-B (in the nascent negative-strand RNA) and the TRS-L exceeds a minimum treshold, a template switch to the leader takes place, adding a copy of the TRS-L exceeds a minimum treshold, a template switch to the leader takes place, adding a copy of the TRS-L to complete the negative-strand sgRNA. These negative-strand sgRNAs subsequently serve as templates for the synthesis of multiple copies of sgmRNAs
The secondary structure of the active domain and the high-order structure formed by the RNA-RNA interactions could also promote the slowdown and stoppage of the transcription complex at the CS-N, as described for tombusvirus transcription. The sequences and seccondary structures of the RNA motifs involved in these long-distance interactions are conserved among members of the species Alphacoronavirus I, suggesting a functional similarity.
This interaction could bring into physical proximity the leader sequence, at the genome 5’end, and the TRS-N, which would promote the template switch during synthesis of the negative-strand sgRNAs. This long-distance RNA-RNA interaction provided for the first time experimental support of the physical proximity between TRS-L and a TRS-B during discontinuous transcription in order to promote efficient RdRp transfer.
The amount of sgmRNA N produced is directly proportional to the extent of the complementarity between pE and dE and inversely proportional to the distance between them. This interaction is probably necessary to relocate the active domain, another cis-acting RNA motif, consisting of a 173-nt region at the 5’flank of dE, immmediately preceding the CS-N. The second long-distance RNA-RNA interaction is held between a 10-nt sequence within the active domain and a complementary RNA motif located at the 5’end of the viral genome (nucleotides 477 to 486), more than 25,000 nt apart, and represents the longest-distance RNA-RNA interaction reported so far in the RNA virus world.
In TGEV, two intragenomic, long-distance RNA-RNA interactions have been described to regulate the transcription of sgmRNA [coding for the nucleocapsid protein (N protein)], which is the most abundant sgmRNA during viral infection despite its low ΔG value for TRS-L-TRS-B duplex formation. The first interaction is established between two complementary 9-nt cis-acting elements preceding the CS of the N gene, the proximal element (pE) and the distal element (dE), which are located 7 and 449 nt upstream of the CS-N, respectively.
The coronavirus discontinuous transcription process implies a premature termination during the synthesis of the negative-strand RNAs and a template switch of the nascent negative strand RNA to the leader. This switch requires long-distance RNA-RNA interactions, probably assisted by RNA-protein complexes that would bring into close proximity the 5′ end TRS-L and TRS-B preceding each gene. These complexes, presumably formed prior to the template switch, might contribute to the stoppage of negative strand RNA synthesis at the TRS-B.
These observations provided experimental evidence for the selection of the TRS-L during the template switch, excluding other genome TRS-Bs that contain the CS. Only the CS-L, located in a sequence context with optimal secondary structure and stability for template switching, may act as a landing site for the newly synthesized negative-strand RNA.
Coronavirus transcription resembles high-frequency, similarity-assisted copy-choice RNA recombination, requiring sequence identity between donor and acceptor RNAs and hairpin structures present in the acceptor RNA, in which the TRS-L would act as an acceptor for the cTRS-B donor sequence. Secondary structure analysis of the TRS-L region from transmissible gastroenteritis virus (TGEV) and bovine coronaviruses (BCoV) showed that the CS-L is exposed in the loop of a structured hairpin that is relevant for replication and transcription.
By engineering the base-pairing between the CS-L and cCS-B in infectious genomic cDNAs of coronaviruses and arteriviruses, it was normally demonstrated that the discontinuous step of transcription occurs during the synthesis of the negative-strand RNA, and base-pairing between the CS-L and cCS-B is required to drive the template switch of the nascent negative-strand RNA to the leader. Additionally, the stability (free energy, ΔG) of the extended duplex between the TRS-L and the complement of the TRS-B(cTRS-B), including 5′ and 3′ TRS flanking sequences , was confirmed as a critical regulatory factor for the synthesis of sgmRNAs.