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^{▿ †}

Virus and cells.

DBT cells were maintained at 37°C and 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum (HyClone, Logan, UT). L2 cells were maintained at 37°C and 3% CO₂ in DMEM supplemented with 10% calf serum. Baby hamster kidney 21 cells expressing the MHV receptor (BHK-R) were grown in DMEM supplemented with 10% calf serum and G418 (800 μg/ml) to select for cells expressing the MHV receptor (37). MHV-A59 1000 (37) was used as a wild-type control virus for comparison with chimeric viruses.

Assembly of a full-length MHV-A59 infectious construct.

The reverse genetic system for MHV-A59 used in this study was initially described by Yount et al. (37). cDNAs representing the entire MHV-A59 genome with either the wild-type sequence or the MHV/SCoV chimeric sequences were constructed by ligation of the A (or G) fragments to fragments (A)B to G(F) as described previously (37). Chimeric and wild-type MHV genomes were transcribed and electroporated into cells as previously described (37). Cultures were observed for up to 72 h for the development of a cytopathic effect (CPE) (cell fusion) and harvested by freezing at −70°C. Cultures that did not develop a CPE were blind passaged three times through DBT cells in a further attempt to recover infectious virus. At least three independent experiments, including at least one experiment in which electroporated cells were incubated at 34°C and 40°C, were performed before a mutant genome was considered nonviable.

Plasmid constructions.

The primers used in this study are listed in Table 1. The A plasmid of the MHV reverse genetic system described previously by Yount et al. (37) was utilized as a basis for constructing a fusion of the SCoV 5′UTR to the MHV gene 1 coding sequence. The strategy employed to construct this fusion exploited “no see'm” technology to eliminate a BsmBI restriction site engineered into the end of DNA fragments by PCR (37). Briefly, the entire SCoV 5′UTR was amplified from purified SCoV RNA (Urbani strain) (27), obtained from the Center for Disease Control and Prevention (CDC), by reverse transcription (RT)-PCR using primers that contained additional BsmBI sites (S 5′UTR swap oligonucleotides 3 and 7). The resultant RT-PCR product was TA cloned into the pGEM-T vector (Promega) to produce plasmid 0A-1, maintaining the BmsBI sites. Plasmid 0A-2 was produced by subcloning a BamHI fragment from the A plasmid of the MHV reverse genetic system into the PCR-XL-TOPO vector (Invitrogen). This BamHI fragment contained a small portion of the A plasmid backbone, a T7 promoter, and the first 252 nt of the MHV genome, extending into the MHV gene 1 coding region. The MHV 5′UTR was removed from plasmid 0A-2 by inverse PCR with Pfx DNA polymerase (Invitrogen) using primers that contained BsmBI sites (S 5′UTR swap oligonucleotides 5 and 6), followed by self-ligation to produce plasmid 0A-3. The SCoV 5′UTR was excised from plasmid 0A-1 by BsmBI digestion and ligated with BsmBI-digested plasmid 0A-3 to produce plasmid 7074. This ligation eliminated the BsmBI sites and precisely fused the SCoV 5′UTR to the MHV gene 1 coding sequence. After sequencing to verify the construction, a restriction fragment exchange was performed to replace the 550-bp BamHI fragment in MHV plasmid A with the 620-bp BamHI fragment containing a T7 promoter plus the SCoV 5′UTR fused to 252 nt of the MHV gene 1 coding sequence. This chimeric plasmid was called pHK0A and could be used in the MHV reverse genetic system in place of plasmid A. A similar strategy was used to replace the MHV 3′UTR with the SCoV 3′UTR (27) in the MHV reverse genetic system G plasmid. The SCoV 3′UTR and 15 nt of the poly(A) tail were amplified from purified SCoV RNA (27) by RT-PCR using primers that contained BsmBI sites (oligonucleotides 1 and 2), which could subsequently be eliminated during ligation, and cloned (plasmid 0G-1). In plasmid 0G-1, the MHV-A59 sequences between an ApaI site at position 30072 (relative to the MHV-A59 genome) (GenBank accession no. NC_001846) (39) and a PacI site inserted just downstream of the poly(A) tail were derived from plasmid B36 (14). Inverse PCR (oligonucleotides 3 and 4) was utilized to produce plasmid 0G-2, replacing the MHV 3′UTR with two BsmBI sites separated by a small spacer sequence. After digestion by the BsmBI restriction enzyme, the plasmid 0G-1 fragment containing the SCoV 3′UTR was ligated into BsmBI-digested plasmid 0G-2 to eliminate the BsmBI sites, producing plasmid 0G-3. This plasmid contained a fusion of the last 969 nt of the N coding sequence to the SCoV 3′UTR. This was then transferred to the MHV genetic system G plasmid by restriction fragment exchange to produce a plasmid called pMF0G.

Site-directed mutagenesis.

Site-directed mutagenesis was performed to introduce mutations into pHK0A or plasmid A using an oligonucleotide assembly strategy (8). Overlapping oligonucleotides were designed to span the sequence regions between MluI and SacII (131 nt, which includes the MHV-A59 sequence at positions 1 to 106) in plasmid A or between MluI and AvrII (256 nt, which includes the SCoV sequence at positions 1 to 231) in pHK0A and, after annealing, to have MluI and SacII or MluI and AvrII overhangs at their 5′ and 3′ ends. The annealed oligonucleotides were ligated, and the resulting DNA fragment was purified and then ligated into MluI- and SacII-cut plasmid A or MluI- and AvrII-cut pHK0A to create chimeric plasmids. Schematic drawings of the various chimeric plasmids are shown in Fig. 2. pHK5A, pHK18A, and pHK12A were derived from plasmid A by replacing MHV sequences containing the predicted SL1, SL2, and SL3 structures with their predicted SCoV counterparts, respectively (Table 2). pHK4A was generated from pHK0A by replacing SCoV SL1, SL2, and SL3 with their MHV counterparts (Table 2). pHK8A was generated using “no see'm” technology that replaced MHV SL4 with its SCoV counterpart (Table 2) (37). To replace MHV SL4 with SCoV SL4, a 5′ DNA fragment spanning MHV SL1, SL2, and TRS and SCoV SL4 was amplified from pHK4A using primers containing MluI (sense) and BsmBI (antisense) restriction sites (MluI-T7-F and SL4-only-BsmBI-R). A 3′ DNA fragment spanning the sequence between the MHV SL4 and a BamHI site in ORF1a was amplified from plasmid A using primers containing BsmBI (sense) and BamHI (antisense) sites (7059 antisense oligonucleotide 8 and SL4-only-BsmBI-R). These 5′ and 3′ fragments were cloned into the pGEM-T easy vector, subsequently retrieved by double digestion of MluI and BsmBI or BamHI and BsmBI, and ligated into MluI and BamHI sites in plasmid A to eventually construct pHK8A. pHK11A was also generated using “no see'm” technology that replaced the MHV sequence between SL4 and the ORF1a start codon with its SCoV counterpart (Table 2). A 5′ DNA fragment spanning MHV SL1, SL2, TRS, and SL4 was amplified from plasmid A using primers containing MluI (sense primer MluI-T7-F) and BsmBI (antisense primer b/w SL4-AUG-BsmBI-R) sites. A 3′ DNA fragment spanning both (i) SCoV sequences between SL4 and the MHV ORF1a start codon and (ii) MHV sequences between the MHV ORF1a start codon and a BamHI site in MHV ORF1a was amplified from pHK4A using primers containing BsmBI (sense primer b/w SL4-AUG-BsmBI-F) and BamHI (7059 antisense oligonucleotide 8) sites. These 5′ and 3′ fragments were cloned into the pGEM-T easy vector, subsequently retrieved by double digestion of MluI and BsmBI or BamHI and BsmBI, and ligated into MluI and BamHI sites in plasmid A to eventually construct pHK11A. A frameshift mutation in ORF1b was created by digesting plasmid F (nt 398) with MunI, filling in the 4-base overhang with Klenow fragment, and religating the plasmid. This produced a plasmid called pLP0F, containing a frameshift mutation that should knock out the expression of functional NSP12 (RdRp) to NSP16 (Table 2).

Plaque purification, RT-PCR, and sequencing.

Mutant viruses were subjected to one round of plaque purification and were expanded once in DBT cells. The sequences of recovered viruses were confirmed by RT-PCR of the 5′UTR and 3′UTR, followed by direct sequencing of the amplified products.

Growth curves.

DBT cells were grown in 96-well plates, and replicate wells were infected at a multiplicity of infection (MOI) of 3 with mutant or wild-type MHV-A59 1000 virus. After washing away the inocula, cultures were incubated until 0, 4, 8, 12, 16, and 24 h postinfection, when they were frozen at −70°C. Triplicate samples were obtained at all time points. Virus production was quantitated by plaque assay on L2 cell monolayers.

Metabolic labeling.

DBT cells (2.25 × 10⁵ cells/well) were seeded into 24-well plates and incubated at 37°C for 15 h to reach approximately 2.5 × 10⁵ cells. The DBT cells were infected at an MOI of 1 (2.5 × 10⁵ PFU) or mock infected, further incubated for 6 h, washed two times with phosphate-free DMEM, fed with DMEM supplemented with 2% dialyzed calf serum and 10 μg/ml of actinomycin D, and incubated at 37°C for 15 min. At the end of 15 min, the medium was replaced with phosphate-free medium containing 10 μg/ml actinomycin D, 2% dialyzed serum, and 200 μCi/ml ³²PO₄ and incubated at 37°C for 5.5 h, by which time 90% of the cells infected with wild-type MHV-A59 1000 had formed syncytial giant cells. The labeled cultures were washed twice with cold phosphate-buffered saline, and RNA was extracted using an RNeasy Mini kit (QIAGEN). The amount of RNA in each sample was measured using the RediPlate 96 RiboGreen RNA quantitation kit (Invitrogen). Equal amounts of radiolabeled viral RNA (10 μg) were denatured in formaldehyde gel loading buffer containing ethidium bromide (20 μg/ml) at 65°C for 15 min and then electrophoresed in a 1% formaldehyde-agarose gel at 100 V for 5 h. Following electrophoresis, the gel was illuminated with UV light, the image was captured with a FluorChem 8900 (AlphaInotech) imaging system, and the relative amount of 18S rRNA bands was determined by densitometry. The gel was soaked in 70% methanol for 30 min, dried over a vacuum, and exposed to X-ray film. The amount of the individual subgenomic RNAs (sgRNAs) relative to genome-sized RNA (gRNA) and the relative amount of radiolabeled RNA in each sample were determined by exposing the dried gel to a Molecular Dynamics PhosphorImager equipped with Storm 8.2 software. The amount of 18S rRNA in each sample was used to normalize the PhosphorImager signals to account for small differences in the total amounts of RNA loaded per sample.

Detection of gRNA and sgmRNA.

A series of nested RT-PCR assays was performed to analyze RNAs produced by nonviable chimeric genomes. Chimeric genomes or wild-type MHV-A59 1000 RNA was electroporated into BHK-R cells, and total RNAs were extracted at 8 and 24 h postelectroporation (p.e.). In order to determine if the input RNAs, plus any replicated genome RNA, were present in the electroporated cells, the extracted RNAs were primed for reverse transcription by 7059 antisense oligonucleotide 8, followed by PCR using SCoV or MHV 5′ 1-20 (+) primers and 7059 (−) oligonucleotide 8. The RNA species present in cells electroporated with these chimeric genomes were characterized using nested RT-PCR methods described previously (16, 17, 21). For analyzing the synthesis of minus-sense gRNA by the chimeric viruses, the extracted RNAs were primed for reverse transcription by oligonucleotide A59(+) 14639-14658 and followed by the first PCR using oligonucleotide A59(+) 14639-14658 and oligonucleotide A59(−) 16596-16577. The resultant PCR products were further amplified by nested PCR using oligonucleotide A59(+) 16038-16059 and oligonucleotide A59(−) 16596-16577. Parallel reactions (without RT) in which reverse transcriptase was omitted from the cDNA step were always performed to ensure that the PCRs did not detect residual DNA transcription templates that entered the cells during electroporation. To detect plus- or minus-sense subgenomic mRNA7 (sgmRNA7), the extracted RNAs were primed for reverse transcription by antisense primer 7065 to detect minus-sense sgmRNA7 and by SCoV (GenBank accession no. AY278741) or MHV 5′ 1-20 sense primers to detect minus-sense sgmRNA7, respectively. The resultant cDNAs were used as templates for the first PCR using SCoV or MHV 5′ 1-20 sense primers and the 7065 antisense primer. The first PCR products were further amplified by nested PCR using SCoV or MHV 5′ 1-20 sense primers and the N antisense primer, and the nested PCR products were displayed by gel electrophoresis.

Comparison of secondary structures for the 5′-most 140 nt of the 5′UTRs of MHV and SCoV.

Consensus secondary structural models for the 5′-most 140 nt of the 5′UTR from MHV and SCoV were developed using the secondary structure prediction algorithms ViennaRNA 1.5 (12) and mfold 3.1 (40) that were informed by a multiple sequence alignment of nine CoV 5′UTRs (MHV [GenBank accession no. NC_001846], SCoV [accession no. NC_004718], 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], transmissible gastroenteritis virus (TGEV) [accession no. NC_002306], and infectious bronchitis virus (IBV) [accession no. NC_001451]) and examined for potential sequence covariations that might support the secondary structure prediction. The predicted secondary structural models from nine CoV genomes are strikingly similar and are characterized by three major putative helical stem-loops, named SL1, SL2, and SL4 (Fig. 1). The consensus model is anchored by SL2, which contains a highly conserved and previously unrecognized (C/U)UUG(U/C) U-turn containing a pentaloop (4) sequence stacked on a 5-bp stem of variable composition. While the predicted SL2 stem differs in sequence from virus to virus (Fig. 1B), SL2 is the most conserved structural element in the MHV and SCoV 5′UTR sequences (53.4% overall sequence identity in the first 140 nt) (21). Provided that the stem of SL2 is forced to be base paired, the consensus model is independent of the secondary structure prediction algorithm used (both mfold 3.1 and PKNOTS were tested) (26, 40). The existence of this base pairing is supported by covariation in the stem sequences (Fig. 1B; see Fig. S1 in the supplemental material). Extending these predictions to encompass the entire MHV and SCoV 5′UTRs also predicted the structures SL1 to SL4 (data not shown).

In contrast to SL2, the predicted structures of SL1 and SL4 differ in detail between MHV and SCoV. MHV SL1 has a longer hairpin loop and has two consecutive pyrimidine-pyrimidine mismatches in the stem that are not predicted to be present in SCoV SL1. The predicted MHV SL4 stem contains two internal loops not present in the SCoV SL4 stem. The SCoV 140-nt sequence is predicted to contain a fourth putative stem-loop, named SL3, spanning the SCoV TRS 5′-flanking sequence (5′FS) and CSs. Consistent with our data, van den Born et al. previously predicted that the SCoV TRS CS was contained in the loop portion of a stem-loop structure (33). The counterpart MHV TRS sequence is located between SL2 and SL4 but is predicted to be single stranded or weakly folded at 25°C. The predicted single-stranded region between SCoV SL1 and SL2 was longer (8 nt) than the corresponding region in MHV (1 nt). The SCoV sequence region between the putative SL4 and the start codon of nsp1 (nt 131 to 264) is 65 nt longer than its MHV counterpart (nt 141 to 209), and no significant similarity was found between the two RNA sequences in this region. ViennaRNA 1.5 predicted that this SCoV sequence region forms four stem-loop structures but that its MHV counterpart forms just two stem-loop structures.

The SCoV 5′UTR cannot functionally replace the MHV 5′UTR.

An examination of the sequences contained in the SCoV 5′UTR SL3 revealed that in addition to the SCoV TRS CS (ACGAAC [nt 67 to 72]), this structure also contained an overlapping MHV TRS CS (UCUAAAC [nt 62 to 67]) (Fig. 1). This led us to investigate whether the SCoV 5′UTR could functionally replace its MHV counterpart. In the reverse genetic system for MHV-A59 described previously by Yount et al. (37), plasmid A contains the 5′UTR plus an additional 4,672 nt of the ORF1a coding sequence. As described in Materials and Methods, we generated a modified plasmid A in which the entire MHV 5′UTR was precisely replaced by the SCoV 5′UTR (pHK0A) (Table 2 and Fig. 2). The chimeric cDNA contained in this plasmid was excised and ligated to cloned cDNAs B to G (37) and transcribed in vitro to generate chimeric MHV-A59 genome RNAs in which the SCoV 5′UTR had replaced the corresponding MHV sequences (MHV/SCoV-5′UTR). Three attempts to recover infectious virus after electroporation into MHV-permissive BHK-R cells were unsuccessful, including one attempt in which replicate electroporated cultures were incubated at 34°C and 40°C. To confirm that these chimeric genomes were nonviable, the electroporated cell cultures were frozen at −70°C to release cell-associated virus and blind passaged three times on DBT cells without developing cytopathic effects. Lysates from the third blind passage were subjected to plaque assay using L2 cells. No plaques were observed in all three independent experiments. Thus, we concluded that the MHV/SCoV-5′ UTR chimeric genome was nonviable (Fig. 3).

We next determined if a modified MHV/SCoV-5′UTR chimera in which the SCoV TRS CS was replaced by the MHV TRS CS (MHV/SCoV-5′UTR/MHV-TRS) (Fig. 2) was viable. Chimeric genomes were transcribed from ligated cDNAs and electroporated into BHK-R cells, and virus isolation from these cultures was attempted as described above. After three unsuccessful attempts to recover recombinant virus, we concluded that this chimeric genome was also not viable (Fig. 3).

SCoV 5′UTR SL1, SL2, and SL4 were functionally exchangeable with their MHV counterparts.

We next determined whether individual predicted stem-loop structures in the SCoV 5′UTR could functionally replace their MHV counterparts. The following MHV/SCoV chimeric genomes were generated as described in Materials and Methods (Table 2 and Fig. 2): (i) A59/SCoV-SL1 (MHV SL1 was replaced with SCoV SL1 using pHK5A), (ii) A59/SCoV-SL2 (MHV SL2 was replaced with SCoV SL2 using pHK18A), (iii) A59/SCoV-SL3 (MHV TRS CS and 8 nt of the 5′FS were replaced with SCoV SL3 using pHK12A), (iv) A59/SCoV-SL4 (MHV SL4 was replaced with SCoV SL4 using pHK8A), (v) A59/SCoV-b/w SL4&AUG (the MHV sequence region between SL4 and the start codon [AUG] of nsp1 was replaced with its SCoV counterpart using pHK11A), and (vi) MHV/SCoV-3′UTR (MHV 3′UTR was replaced with the SCoV 3′UTR using pMF1G). As a negative control, A59/nsp12-FS was generated using pLP1F, a plasmid that contained a cDNA construct harboring a frameshift mutation in the RdRp domain (nsp12) that should abrogate the translation of downstream nsp13 to nsp16. Cultures electroporated with A59/SCoV-SL1, A59/SCoV-SL2, A59/SCoV-SL4, and A59/SCoV-3′UTR chimeric genomes developed a CPE after 24 to 48 h. Viable virus was recovered from the medium, plaque purified, and expanded in DBT cells (Fig. 3). Sequencing analyses confirmed that there were no additional mutations in the 3′UTRs and 5′UTRs of the recovered viruses. The cultures electroporated with A59/SCoV-SL3 and A59/SCoV-b/w SL4&AUG chimeric genomes and the A59/nsp12-FS mutant genome failed to develop CPE. To confirm that these viral genomes were nonviable, the electroporated cell cultures were frozen at −70°C to release cell-associated virus and blind passaged three times on DBT cells. Lysates from the third blind passage were subjected to plaque assay using L2 cells. No plaques were observed in three independent experiments for each of these viruses. Thus, the A59/SCoV-SL3 and A59/SCoV-b/w SL4&AUG chimeric genomes and the A59/nsp12-FS mutant genome were judged to be nonviable (Fig. 3).

Phenotypic properties of the MHV/SCoV chimeric viruses.

Plaque size and growth kinetics of the recovered chimeric viruses were compared to those of parental MHV-A59 1000. As shown in Fig. 3, the MHV/SCoV chimeric viruses made smaller plaques than the parental MHV-A59 1000 virus. Average plaque sizes of A59/SCoV-SL1, A59/SCoV-SL2, A59/SCoV-SL4, and A59/SCoV-3′UTR chimeric viruses were 1.2 (±0.07), 1.8 (±0.09), 1.1 (±0.04), and 1.4 (±0.07) mm in diameter, respectively (Fig. 4). These sizes corresponded to 39% (P < 0.05) to 64% (P < 0.05) of the average plaque size of MHV-A59 1000, which is 2.8 (±0.07) mm in diameter. A59/SCoV-SL1, A59/SCoV-SL4, and A59/SCoV-3′UTR chimeric viruses grew to lower titers, had significantly delayed growth kinetics relative to those of MHV-A59 1000, and achieved maximal titers 15- to 40-fold less than those achieved by the parental MHV-A59 1000 virus (Fig. 5). However, A59/SCoV-SL2 grew almost as well as the parental virus, achieving a titer only fourfold lower than that of MHV-A59 1000 (Fig. 5).

To determine whether the altered growth phenotypes of the chimeric 5′UTR viruses are a result of deficits in genome replication or subgenomic RNA synthesis, virus-specific RNAs were metabolically radiolabeled and analyzed by gel electrophoresis (Fig. 6). DBT cells were either mock infected, infected with MHV-A59 1000 (WT), or infected with MHV/SCoV chimeric viruses. Starting at 5 h postinfection, MHV-specific RNAs were labeled for 5.5 h with ³²PO₄ in the presence of actinomycin D. After labeling, total viral and cellular RNAs were extracted from cell lysates and quantitated, and equal amounts of RNA were electrophoresed in formaldehyde-agarose gels. Total RNAs were visualized by ethidium bromide staining, and labeled MHV-specific RNAs were visualized by autoradiography. The amount of each MHV-specific RNA as well as the total amount of labeled virus-specific RNA in each sample were quantitated with a PhosphorImager (Fig. 6). All seven species of MHV-specific RNA were detected in cells infected with the MHV/SCoV chimeric viruses and MHV-A59 1000. The total amount of radiolabeled MHV-specific RNA (gRNA [genome] through sgmRNA7) in MHV/SCoV-SL2-infected cells was 92% of that detected in MHV-A59 1000-infected cells, a result that correlates with the relatively modest impairment of viral replication that we observed for this chimera. However, the amount of virus-specific RNA synthesis in cells infected with MHV/SCoV-SL1 (32% relative to WT), MHV/SCoV-SL4 (46%), and MHV/SCoV-3′UTR (42%) was significantly lower than that observed in cells infected with MHV-A59 1000 (Fig. 6). This decrease in RNA synthesis correlates with the decrease in replication efficiency that we observed in one-step growth curves (Fig. 5). The relative molar ratios of gRNA to sgmRNAs present in cells infected with MHV/SCoV chimeric viruses were generally similar to those found in cells infected with the wild type, with some exceptions for individual sgmRNAs (Table 3). The largest difference observed was an almost threefold increase in the relative molar ratio of sgmRNA2 to genome RNA in cells infected with A59/SCoV-SL1.

Nonviable MHV/SCoV chimeric genomes have defects in RNA synthesis.

We examined the stage at which RNA replication or transcription of the nonviable chimeric genomes was blocked using a series of nested RT-PCR assays (summarized in Table 4). Replicate cultures of BHK-R cells were separately electroporated with in vitro-synthesized genomic RNAs corresponding to A59/SCoV-5′UTR, A59/SCoV-5′UTR/MHV-TRS, and A59/SCoV-SL3 chimeric genomes; the A59/nsp12-FS mutant genome (a frameshift mutant expected to abrogate the expression of proteins required for RNA replication); and parental MHV-A59 1000. RNAs were extracted from the electroporated cultures at 8 and 24 h p.e. As expected, nested RT-PCR analyses of RNA extracted from cells electroporated with the A59/nsp12-FS (Fig. 7B, lanes 1 and 2) mutant genome failed to detect minus-sense gRNA at either time point, whereas this RNA species was readily detected at both time points in cells electroporated with the MHV-A59 1000 genome (Fig. 7B, lanes 9 and 10). Minus-sense gRNA was undetectable in cells electroporated with the A59/SCoV-5′UTR chimera (Fig. 7B, lanes 3 and 4) but was detectable in cells electroporated with the A59/SCoV-5′UTR/MHV-TRS and A59/SCoV-SL3 chimeras (Fig. 7B, lanes 5 to 8). Assays to detect plus- or minus-sense sgmRNA7s were similarly negative with RNAs obtained from cells electroporated with the A59/SCoV-5′UTR chimera (Fig. 7D, lanes 5 to 8) or the A59/nsp12-FS mutant (Fig. 7D, lanes 1 to 4), indicating that these genomes had global defects in RNA synthesis. The nested RT-PCR assay showed that at 24 h p.e., the A59/SCoV-5′UTR/MHV-TRS (Fig. 7D, lanes 9 to 12) and A59/SCoV-SL3 (Fig. 7D, lanes 13 to 16) chimeric genomes directed the synthesis of minus-sense gRNA at levels that appeared to be roughly similar to those observed in cells electroporated with wild-type MHV-A59 1000. Semiquantitative nested RT-PCR assays with serially diluted RNA templates confirmed that the cultures electroporated with the A59/SCoV-5′UTR/MHV-TRS and A59/SCoV-SL3 genomes contained amounts of minus-sense gRNA that were roughly equivalent to those of cells electroporated with MHV-A59 (data not shown). However, neither minus- nor plus-sense sgmRNA7 could be detected in cells electroporated with these two chimeric genomes (Fig. 7D, lanes 9 to 16), whereas these RNAs were easily detectable in cells electroporated with the parental MHV-A59 1000 genome (Fig. 7D, lanes 17 to 20).