Putative cis -Acting Stem-Loops in the 5′ Untranslated Region of the Severe Acute Respiratory Syndrome Coronavirus Can Substitute for Their Mouse Hepatitis Virus Counterparts

Abstract

Consensus covariation-based secondary structural models for the 5 140 nucleotides of the 5 untranslated regions (5UTRs) from mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SCoV) were developed and predicted three major helical stem-loop structures, designated stem-loop 1 (SL1), SL2, and SL4. The SCoV 5UTR was predicted to contain a fourth stem-loop, named SL3, in which the leader transcriptional regulatory sequence (TRS) is folded into a hairpin loop. cDNAs corresponding to MHV/SCoV chimeric genomes were constructed by replacing the complete MHV 5UTR with the corresponding SCoV sequence and by separately replacing MHV 5UTR putative SL1, putative SL2, TRS, and putative SL4 with the corresponding SCoV sequences. Chimeric genomes were transcribed in vitro, and viruses were recovered after electroporation into permissive cells. Genomes in which the MHV 5UTR SL1, SL2, and SL4 were individually replaced by their SCoV counterparts were viable. Chimeras containing the complete SCoV 5UTR or the predicted SCoV SL3 were not viable. A chimera containing the SCoV 5UTR in which the SCoV TRS was replaced with the MHV TRS was also not viable. The chimera containing the entire SCoV 5UTR failed to direct the synthesis of any virus-specific RNA. Replacing the SCoV TRS with the MHV TRS in the MHV/5UTR SCoV chimera permitted the synthesis of minus-sense genome-sized RNA but did not support the production of positive-or minus-sense subgenomic RNA7. A similar phenotype was obtained with the MHV/SCoV SL3 chimera. These results suggest a role for the TRS in the replication of minus-sense genomic RNA in addition to its known function in subgenomic RNA synthesis.

Continuous and Discontinuous RNA Synthesis in Coronaviruses

“…These RNA elements mainly consist of stem-loop (SL) structures that present a varying degree of conservation among different coronaviruses (9, 101–103). In order to consolidate the information from various publications, we adopt a uniform nomenclature for the 5′ cis -acting RNA elements (Figure 4) based on that used by the Leibowitz, Giedroc, and Olsthoorn laboratories (102, 103). SL1 and SL2 are conserved in all coronaviruses (101, 102).…”

RNA structure analysis of alphacoronavirus terminal genome regions

“…Subsequent studies suggested (a varying degree of) conservation of 5 cis-acting elements among betacoronaviruses and even the more distantly related alpha-and gammacoronaviruses (Chen and Olsthoorn, 2010;Kang et al, 2006). To facilitate the discussion of data obtained in studies of different viruses by different laboratories, we will use in this article a uniform nomenclature for the main RNA structural elements in the 5 -proximal genome region (SL1, SL2, [SL3 if present], SL4 and SL5).…”

“…To facilitate the discussion of data obtained in studies of different viruses by different laboratories, we will use in this article a uniform nomenclature for the main RNA structural elements in the 5 -proximal genome region (SL1, SL2, [SL3 if present], SL4 and SL5). The nomenclature is based on SL designations used by the Leibowitz and Giedroc laboratories and in predictions of genus-and lineage-specific conservation patterns of 5 cis-acting elements in alpha-, beta-and gammacoronaviruses (Chen and Olsthoorn, 2010;Kang et al, 2006;Liu et al, 2006Liu et al, , 2007. The proposed functional and structural conservation of 5 cis-acting elements among betacoronaviruses received strong support by reverse-genetic data demonstrating that SARS-CoV SL1, SL2, and SL4 can functionally replace their counterparts in the MHV genome when introduced individually (Kang et al, 2006).…”

“…The nomenclature is based on SL designations used by the Leibowitz and Giedroc laboratories and in predictions of genus-and lineage-specific conservation patterns of 5 cis-acting elements in alpha-, beta-and gammacoronaviruses (Chen and Olsthoorn, 2010;Kang et al, 2006;Liu et al, 2006Liu et al, , 2007. The proposed functional and structural conservation of 5 cis-acting elements among betacoronaviruses received strong support by reverse-genetic data demonstrating that SARS-CoV SL1, SL2, and SL4 can functionally replace their counterparts in the MHV genome when introduced individually (Kang et al, 2006). By contrast, replacement of the entire MHV 5 UTR with that of SARS-CoV did not produce a viable MHV mutant, suggesting a requirement for additional stable or transient long-range RNA-RNA interactions of the 5 UTR with other genome regions.…”

Effect of mutations in a simian virus 40 PolyA signal enhancer on green fluorescent protein reporter gene expression

“…The untranslated regions (UTRs) of the murine hepatitis virus (MHV) contain cisacting sequences and secondary structures, which are known to have essential roles in RNA synthesis, gene expression, and virion assembly (Kang et al, 2006). Base 35 (A35), located in the stem middle of the 5' UTR first stem-loop (SL1) in the MHV genome, is an extrahelical nucleotide that forms a bulge that destabilizes the SL1 structure.…”