Expanded subgenomic mRNA transcriptome and coding capacity of a nidovirus

Abstract

Members of the order Nidovirales express their structural protein ORFs from a nested set of 3' subgenomic mRNAs (sg mRNAs), and for most of these ORFs, a single genomic transcription regulatory sequence (TRS) was identified. Nine TRSs were previously reported for the arterivirus (SHFV). In the present study, which was facilitated by next-generation sequencing, 96 SHFV body TRSs were identified that were functional in both infected MA104 cells and macaque macrophages. The abundance of sg mRNAs produced from individual TRSs was consistent over time in the two different cell types. Most of the TRSs are located in the genomic 3' region, but some are in the 5' ORF1a/1b region and provide alternative sources of nonstructural proteins. Multiple functional TRSs were identified for the majority of the SHFV 3' ORFs, and four previously identified TRSs were found not to be the predominant ones used. A third of the TRSs generated sg mRNAs with variant leader-body junction sequences. Sg mRNAs encoding E', GP2, or ORF5a as their 5' ORF as well as sg mRNAs encoding six previously unreported alternative frame ORFs or 14 previously unreported C-terminal ORFs of known proteins were also identified. Mutation of the start codon of two C-terminal ORFs in an infectious clone reduced virus yield. Mass spectrometry detected one previously unreported protein and suggested translation of some of the C-terminal ORFs. The results reveal the complexity of the transcriptional regulatory mechanism and expanded coding capacity for SHFV, which may also be characteristic of other nidoviruses.

A planarian nidovirus expands the limits of RNA genome size

“…The net result of this mechanism is relatively high expression of the ORF1a- compared to ORF1b-encoded proteins, since PRF occurs at the ORF1a/1b junction in 15–60% of ORF1a translation events. In contrast, proteins encoded in the 3’ORFs region are produced by translation of subgenomic (sg) mRNAs, synthesized on specific minus-strand templates [ 51 – 53 ], which are in turn produced by discontinuous RNA synthesis on genomic templates. Discontinuous minus-strand template synthesis relies on lTRS and bTRS, which are nearly identical, short repeats at sites where RNA synthesis pauses (upstream of 3’ORFs) and resumes (in the 5’-UTR), respectively.…”

“…These and other proteins form a membrane-bound replication-transcription complex (RTC) [ 43 , 44 ] that invariably includes two key ORF1b-encoded subunits: the Nidovirus RdRp-Associated Nucleotidyltransferase (NiRAN) fused to an RNA-dependent RNA polymerase (RdRp) [ 45 , 46 ], and a zinc-binding domain (ZBD) fused to a superfamily 1 helicase (HEL1), respectively [ 47 – 50 ]. The RTC catalyzes the synthesis of genomic and 3’-coterminal subgenomic RNAs, the latter via discontinuous transcription that is regulated by leader and body transcription-regulating sequences (lTRS and bTRS) [ 51 – 53 ]. Subgenomic RNAs are translated to express virion and, in ExoN-positive viruses, accessory proteins encoded in the 3’ORFs [ 23 , 54 – 59 ].…”

Emergency Services of Viral RNAs: Repair and Remodeling

“…For example, an engineered 19-nt deletion affecting the hairpins controlling translation of the MS2 phage capsid protein could be ameliorated, upon passages in Escherichia coli, by either deletion of 6 more nucleotides, acquisition of an unrelated 18-nt insert, or both modifications, creating novel functional regulatory structures which included the Shine-Dalgarno sequence (451). A special case of mutational robustness was recently discovered in nidoviruses (452). Proteins of these viruses are largely translated from subgenomic mRNAs (sgRNAs), and the synthesis of each sgRNA is controlled by a distinct cis-element (TRS).…”

The transcriptional and translational landscape of equine torovirus

“…Our data suggests that transcription mechanisms in the Nidovirales fall into multiple categories, each requiring a distinct role of the TRS: (i) homology-driven reinitiation (canonical discontinuous RNA synthesis, as seen in coronaviruses and arteriviruses and to a low extent, EToV N protein-coding mRNAs); (ii) structure-driven discontinuous transcription (EToV S protein gene); and (iii) transcription termination (EToV M, HE and the majority of N protein-coding transcripts). These mechanisms all require a RdRp which is prone to translocating when even relatively short homologous sequences are present, potentially leading to a large number of irrelevant transcripts being produced (as previously observed in an arterivirus (36)) and also facilitating the production of defective interfering RNAs (34) and recombinant strains (7).…”