Structure and Functional Relevance of a Transcription-Regulating Sequence Involved in Coronavirus Discontinuous RNA Synthesis

Abstract

Transmissible gastroenteritis coronavirus (TGEV) genomic RNA transcription generates 5-and 3-coterminal subgenomic mRNAs. This process involves a discontinuous step during the synthesis of minus-sense RNA that is modulated by transcription-regulating sequences located at the 3 end of the leader (TRS-L) and also preceding each viral gene (TRS-Bs). TRSs include a highly conserved core sequence (CS) (5-CUAAAC-3) and variable flanking sequences. It has been previously proposed that TRS-Bs act as attenuation or stop signals during the synthesis of minus-sense RNAs. The nascent minus-stranded RNA would then be transferred by a template switch process to the TRS-L, which acts as the acceptor RNA. To study whether the TRS-L is structured and to determine whether this structure has a functional impact on genomic and subgenomic viral RNA synthesis, we have used a combination of nuclear magnetic resonance (NMR) spectroscopy and UV thermal denaturation approaches together with site-directed mutagenesis and in vivo transcriptional analyses. The results indicated that a 36-nucleotide oligomer encompassing the wild-type TRS-L forms a structured hairpin closed by an apical AACUAAA heptaloop. This loop contains most of the CS and is isolated from a nearby internal loop by a short Watson-Crick base-paired stem. TRS-L mutations altering the structure and the stability of the TRS-L hairpin affected replication and transcription, indicating the requirement of a functional RNA hairpin structure in these processes.Transmissible gastroenteritis virus (TGEV) is an enveloped virus of the Coronaviridae family containing a single-stranded positive-sense RNA genome of 28.5 kb. The first two-thirds of the genome encodes two replicase polyproteins, while the last third comprises the genes encoding structural and nonstructural viral proteins (Fig. 1A) (10, 18).Coronaviruses (CoVs) share with other plus-stranded RNA viruses the synthesis of subgenomic mRNAs (sgmRNAs) as a mechanism to regulate the expression of the proteins encoded at the 3Ј end of the genome (9,10,18,22,27). CoV sgmRNAs have a terminal leader sequence of around 60 to 93 nucleotides (nt) derived from the 5Ј end of the genome fused to distant RNA coding sequences. This implies a discontinuous step during the synthesis of sgRNAs (22,29,35). This step is guided by transcription-regulating sequences (TRSs) found at the 3Ј end of the leader (TRS-L) and also preceding each gene (TRS-Bs). TRSs comprise a common core sequence (CS) (5Ј-CUAAAC-3Ј), together with 5Ј-and 3Ј-flanking nucleotide sequences that vary depending on the gene but that also are essential for sgmRNA production (29,35).According to the current model of discontinuous transcription in CoVs (26,29,35) (Fig. 1B) and related arteriviruses (23,24,32), the TRS-Bs would act as slow-down and detachment signals for the transcription complex during the synthesis of minus-stranded RNA, possibly by forming a precomplex with the TRS-L and proteins (29). Then, base pairing between the complement of TRS-B (cTRS-B) in the nascent min...

Structural and functional conservation of cis-acting RNA elements in coronavirus 5'-terminal genome regions

“…1, nucleotides of the TRS-L core sequence and nucleotides adjacent to the TRS region were accessible to DMS modification, confirming that they are part of single-stranded regions. In conclusion, our structure model is consistent with previous betacoronavirus studies (Kang et al, 2006;Liu et al, 2007; and supports the idea that, in most coronaviruses, TRS-L is part of an unstructured region rather than a stable SL structure (Van Den Born et al, 2004;Dufour et al, 2011) (see also Discussion).…”

Continuous and Discontinuous RNA Synthesis in Coronaviruses

“…Secondary structure analysis of the TRS-L region from transmissible gastroenteritis virus (TGEV) (23) and bovine coronavirus (BCoV) (24) showed that the CS-L is exposed in the loop of a structured hairpin that is relevant for replication and transcription (23). 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.…”

Transmissible gastroenteritis virus infection induces cell cycle arrest at S and G2/M phases via p53-dependent pathway

“…Quantitative analysis of genomic RNA (gRNA) and subgenomic mRNAs (sgRNAs) from TGEV-derived replicons was performed by real-time RT-PCR using Bio-Rad iQ5 Real Time PCR System. The primers for qRT-PCR in this study were described in previous study (Dufour et al, 2011). Reactions were carried out in 25 l volume containing 1× SYBR Premix Ex TaqTM II (Takara, Dalian, China), sense and anti-sense primers and target cDNA.…”