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 (5'UTRs) 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 5'UTR 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 5'UTR with the corresponding SCoV sequence and by separately replacing MHV 5'UTR 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 5'UTR SL1, SL2, and SL4 were individually replaced by their SCoV counterparts were viable. Chimeras containing the complete SCoV 5'UTR or the predicted SCoV SL3 were not viable. A chimera containing the SCoV 5'UTR in which the SCoV TRS was replaced with the MHV TRS was also not viable. The chimera containing the entire SCoV 5'UTR failed to direct the synthesis of any virus-specific RNA. Replacing the SCoV TRS with the MHV TRS in the MHV/5'UTR 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.

FIG. 1.

Predicted secondary structures within the 140-nt 5′UTR of the MHV and SCoV genomes (see the text for details). Previously described predicted stem-loops 1 (SL1) (boldface and italic type), 2 (SL2) (large font and italics), and 4 (SL4) (large font) in the 5′UTRs are noted (17, 21). MHV leader TRS CS (boldface, underlined type) is contained within the linear sequence between SL2 and SL4. However, the SCoV leader TRS CS (boldface, underlined type) is contained within stem-loop 3 (SL3) (boldface type). The AUGs shown represent the start codons of nsp1 in the MHV (nt 210 to 212) and SCoV (nt 265 to 267) genomes. • indicates noncanonical base pairings. (B) SL2 sequence alignment of group 2 coronaviruses. The stem portion of SL2 is underlined; the U-turn motif is italicized; * indicates absolutely conserved nucleotides. SL2 sequences of MHV (GenBank accession no. NC_001846), SCoV (accession no. AY278741), BCoV (accession no. NC_003045), HCoV-OC43 (accession no. NC_005147), HCoV-HKU1 (accession no. NC_006577), HCoV-NL63 (accession no. NC_005831), HCoV-229E (accession no. NC_002645), TGEV (accession no. NC_002306), and IBV (accession no. NC_001451) are relative to the corresponding viral genomes in GenBank.

FIG. 2.

Schematic diagram depicting the major constructs used throughout this study. Both the names of the recombinant genomes and plasmids (in parentheses) used to construct these chimeric genomes are shown. The predicted stem-loop structures in the 5′ 140 nt of MHV and SCoV are indicated in the schematic. The positions of the first and last nucleotides are indicated below the boxes depicting the predicted stem-loops. The complete 5′UTR and 3′UTR of SCoV are represented by open rectangles. Single-stranded regions derived from SCoV and MHV are indicated by the thick and thin lines, respectively. The sequences of the 5′UTRs of MHV, SCoV, and each of the chimeric genomes used in this study are shown. SCoV-derived nucleotides are underlined. Boldface type indicates a predicted single-stranded region between the first nucleotide and the predicted 3′ end of SL4. Italicized sequenced are predicted to fold into the stem-loop structures as labeled above the italicized sequences. Bold dashes indicate transitions from predicted stem-loops to single-stranded regions. Sequences 3′ to SL4 are shown in lightface type and are not encompassed by our structural model.

FIG. 3.

Morphologies of plaques formed by MHV and SCoV chimeric genomes. Cultures of BHK-R cells were electroporated with MHV/SCoV chimeric or MHV-A59 full-length transcripts and seeded onto DBT cells in 75-cm² flasks. If a genome produced viable virus progeny, the progeny was plaque purified and amplified once in DBT cells. The plaques shown are wild-type MHV-A59 1000 virus (A), MHV/SCoV-SL1 (E), MHV/SCoV-SL2 (F), MHV/SCoV-SL4 (H), and MHV/SCoV-3′UTR (C) chimeric viruses. Nonviable chimeric genomes did not form any visible plaque (B, D, G, I, J, and K).

FIG. 4.

Average plaque sizes of wild-type MHV-A59 1000 and MHV/SCoV-SL1, MHV/SCoV-SL2, MHV/SCoV-SL4, and MHV/SCoV-3′UTR chimeric viruses. The middle bar in each column indicates the mean plaque size of the corresponding virus.

FIG. 5.

One-step growth kinetics for wild-type MHV-A59 1000 and MHV/SCoV-SL1 (A), MHV/SCoV-SL2 (B), MHV/SCoV-SL4 (C), and MHV/SCoV-3′UTR (D) chimeric viruses. Triplicate DBT cell cultures in a 96-well plate were infected at an MOI of 3 and harvested at the designated hours postinfection, and viral titers were determined by plaque assay.

FIG. 6.

RNA synthesis in cells infected with MHV/SCoV chimeric viruses. Cells were either mock infected or infected with MHV/SCoV-SL1, MHV/SCoV-SL2, MHV/SCoV-SL4, MHV/SCoV-3′UTR, or MHV-A59 1000, and viral RNAs were metabolically labeled as described in Materials and Methods. The labeled viral RNAs were resolved by formaldehyde agarose gel electrophoresis and visualized by autoradiography. Brightness and contrast have been adjusted to enhance the visibility of the bands of the MHV/SCoV chimeric viruses using Adobe Photoshop 6.0. Arrows indicate the positions of bands of virus-specific RNA1 to RNA7.

FIG. 7.

Nested RT-PCR assays for minus-sense gRNA and sgmRNA7. (A) Schematic drawing of the assay used to detect minus-sense (−) gRNA and the relative locations of the primers within the genome. (B) RT-PCR of RNAs extracted from MHV-A59 1000 (WT) and nonviable MHV/SCoV chimeric genome-electroporated cells at 8 and 24 h p.e. The arrow indicates the specifically amplified fragment from minus-sense gRNA. W/RT and W/O RT indicate products of nested RT-PCR (left) and the corresponding no-RT control reactions (right), respectively. (C) Schematic representation of the RT-nested PCR strategy and primers used to detect minus- and plus-sense sgmRNA7s. (D) Representative nested RT-PCR for minus- and plus-sense sgmRNA7s. The arrow indicates the specifically amplified sgmRNA7 fragment. The relative sizes of the leader sequences in sgmRNA7 and the body of sgmRNA7 are not to scale. The sizes of the marker DNAs in base pairs are shown to the right of electropherograms. The amplified products corresponding to minus-sense sgmRNA7 are shown in lanes 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19. Products corresponding to plus-sense sgmRNA7 are shown in lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20. The position of each primer in the MHV-A59 genome (GenBank accession no. NC_001846) is given in parentheses as part of the primer name. Primer sequences are listed in Table 1. A 1-kbp ladder was used as a molecular size marker.