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. 2004 Jan;78(2):980-94.
doi: 10.1128/jvi.78.2.980-994.2004.

Sequence motifs involved in the regulation of discontinuous coronavirus subgenomic RNA synthesis

Sonia Zúñiga  1 , Isabel Sola, Sara Alonso, Luis Enjuanes
Affiliations

Sequence motifs involved in the regulation of discontinuous coronavirus subgenomic RNA synthesis

Sonia Zúñiga et al. J Virol. 2004 Jan.

Abstract

Coronavirus transcription leads to the synthesis of a nested set of mRNAs with a leader sequence derived from the 5' end of the genome. The mRNAs are produced by a discontinuous transcription in which the leader is linked to the mRNA coding sequences. This process is regulated by transcription-regulating sequences (TRSs) preceding each mRNA, including a highly conserved core sequence (CS) with high identity to sequences present in the virus genome and at the 3' end of the leader (TRS-L). The role of TRSs was analyzed by reverse genetics using a full-length infectious coronavirus cDNA and site-directed mutagenesis of the CS. The canonical CS-B was nonessential for the generation of subgenomic mRNAs (sgmRNAs), but its presence led to transcription levels at least 10(3)-fold higher than those in its absence. The data obtained are compatible with a transcription mechanism including three steps: (i) formation of 5'-3' complexes in the genomic RNA, (ii) base-pairing scanning of the nascent negative RNA strand by the TRS-L, and (iii) template switching during synthesis of the negative strand to complete the negative sgRNA. This template switch takes place after copying the CS sequence and was predicted in silico based on high base-pairing score between the nascent negative RNA strand and the TRS-L and minimum DeltaG.

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Figures

FIG. 1.
FIG. 1.
Diagram of the elements involved in coronavirus transcription. (A) The scheme represents all of the sequence elements probably involved in the discontinuous negative-strand synthesis model. CS-L, leader CS; CS-B, body CS. TRS-L and TRS-B, transcription-regulating sequences from the leader and body, respectively. An, poly(A). (B) Representation of the discontinuous transcription during negative-strand synthesis. cCS-B and cTRS-B represent the CS-B and TRS-B complementary sequences, respectively. Un, poly(U). (C) Leader and body sequences are probably located close to one another in higher-order structures maintained by RNA-protein and protein-protein interactions.
FIG. 2.
FIG. 2.
Mutations introduced in the TGEV full-length cDNA and virus recovery. Nucleotide substitutions were introduced in the 3a gene CS (CS-B mutants [A]), the leader CS (CS-L mutants [B]), in both the CS-L and CS-B (double mutants [D]), and leader CS mutants with changes allowing non-Watson-Crick base pairing with the body cCS (non-Watson-Crick mutants [C]). Virus titers (PFU per milliliter) obtained for the passage 0 supernatant are indicated in the figure.
FIG. 3.
FIG. 3.
Northern blot analysis of rTGEVs. ST cells were infected with rTGEV at an MOI of 0.5 (for the wild type [wt] and CS-B mutants) or 1 (for CS-L and double mutants). Total RNA was extracted at 20 hpi and analyzed by Northern blotting with a probe complementary to the 3′ end of the gRNA. To normalize the amount of viral RNA in the gel, lanes L and D were loaded with three times the amount of the other lanes. L, CS-L mutant; B, CS-B mutant; D, double mutant. Viral mRNAs are indicated on the left side of the figure, and new sgmRNAs that have been clearly identified are indicated on the right (some of them correspond to the alternative sgRNAs analyzed in this work, indicated by the same number). n.i., still unidentified sgmRNAs.
FIG. 4.
FIG. 4.
RT-PCR analysis of the CS-B mutants. (A) Scheme of the RT-PCR strategy for testing the gRNA and the mRNA-3a. Arrows indicate the approximate oligonucleotide position in the genome and sgmRNA. UTR, 3′ untranslated region. (B) mRNA-3a specific RT-PCR products were resolved in an agarose gel. mRNA-3a species were numbered 3a.1 (wild type [wt]), 3a.2, and 3a.3. MW, molecular weight markers. (C) Sequence analysis of the leader-body junction sites in the three mRNA-3a species. The sequence in the light-gray box corresponds to the leader (L) sequence. The CS appears as white letters in a dark-gray box in all cases. The sequence on top corresponds to the gRNA sequence in the fusion site; the sequence at the bottom is the mRNA sequence with nucleotides from the leader in a light-gray box. CS in white letters in a dark-gray box represents the mutated CS in each case; two examples of leader-to-body junction sites generating mRNA-3a.1 are presented: the B-C1G and B-A3C mutants. The GAA motif appears in a medium-gray box. Vertical bars represent the identity between the sequences, with thick bars at the possible fusion site. Dotted vertical bars represent the possible non-Watson-Crick interaction. Crossover should occur in any of the nucleotides above the arrow.
FIG. 5.
FIG. 5.
Effect of CS-B mutations in the transcription of other TGEV mRNAs. mRNAs from genes S, 3a, E and 7 were analyzed by RT-PCR using specific oligonucleotides (Table 2). WT, wild-type virus; B-C1G and B-A3C, CS-3a mutants with mutation at positions 1 and 3, respectively.
FIG. 6.
FIG. 6.
In silico analysis of the identity between TRS-L and the TGEV genome. As indicated in Materials and Methods, a continuous line graph was selected to facilitate visualization of the data. (A) Graphical plot of the potential base-pairing score versus the genome position. All peaks assigned to the viral CSs are indicated as the peaks corresponding to the new 3a sgmRNA species. (B) Graphical plot of the potential base-pairing score versus the genome position around CS-3a. Each three-dimensional line represents either the wild-type (wt) situation or the body mutants. The peaks assigned to each 3a sgmRNA species are indicated.
FIG. 7.
FIG. 7.
mRNA-3a quantification by real-time RT-PCR. (A) Amount of mRNA-3a.1, quantified by real-time RT-PCR, in the body mutants relative to the wild-type (wt) levels. Shown is a graphical representation of the ΔG (as −ΔG in kilocalories per mole) of the CS-L with cCS-B duplex and the relative amount of mRNA-3a.1 (represented as log [mRNA-3a.1] in relative units) for each virus. The data presented are the average of six independent experiments with duplicates in each case. Error bars represent the standard deviation in each case. (B) Graphical plot of the amounts of mRNA-3a.1 and mRNA-3a.2 relative to the level of gRNA, expressed as [mRNA] in relative units.
FIG. 8.
FIG. 8.
Analysis by RT-PCR of viral sgmRNAs generated by rTGEVs with CS-L substitutions. After ST cell infection with rTGEVs, total RNA was analyzed by RT-PCR with specific oligonucleotides to detect all viral mRNAs. Viruses with CS-L substitutions are indicated on top of the figure. The viral mRNA detected is shown to the left of the figure. The titer (PFU per milliliter) of each virus is shown at the bottom.
FIG. 9.
FIG. 9.
RT-PCR analysis of the S mRNA species present in leader mutants. (A) mRNA S detection by RT-PCR in leader and double mutants. sgmRNA species are named mRNA S.1, S.2, S.3, S.4, and S.5, as shown to the right of the panel. The oligonucleotides used for the analysis did not allow the detection of sgmRNAs S.6 and S.7. (B) Sequence analysis of the leader-to-body fusion site in all of the S gene sgmRNAs generated. The sequence in the light-gray box at the bottom represents the wild-type (wt) or mutated leader; the sequence on top is the gRNA sequence in the junction sites. CS is in white letters in a dark-gray box. The GAA motif is in a medium-gray box. Vertical bars represent the identity between the sequences; thick bars correspond to the possible fusion site, because crossover should occur in any nucleotide above the arrow. Dotted vertical bars represent the possible non-Watson-Crick interaction. Numbers indicate the position in the TGEV genome.
FIG. 10.
FIG. 10.
Analysis of 3a and 7 sgmRNAs present in leader mutants. (A) mRNA-3a detection by RT-PCR. sgmRNA species are named as mentioned before. (B) In silico analysis of the identity between the wild-type (wt) or mutated TRS-L and the TGEV genome surrounding the 3a gene CS. Data are graphically plotted as potential base-pairing score versus the genome position. (C) mRNA-7 detection by RT-PCR. The sgRNA species are named mRNA 7.1 and 7.2. (D) Sequence analysis of the leader-to-body junction sites in all of the 7 gene sgRNAs generated. The sequence at the bottom (light-gray box) represents the wild-type or mutated leader, and the one on top represents the gRNA in the fusion site context. CS is in white letters in a dark-gray box. Vertical bars show the identity between the sequences, and thick bars represent the possible fusion site, because strand transfer should occur in any of the nucleotides above the arrow. Dotted vertical bars represent the possible non-Watson-Crick interaction. Numbers indicate the position in the TGEV genome.
FIG. 11.
FIG. 11.
CS adjacent flanking sequences identity. Identity between the TRS-L sequence and TRS-Bs for all TGEV sgmRNAs is shown in the figure. The CS sequence is in white letters in a black box. White boxes highlight the identity in the sequences immediately flanking CS both at the 5′ and 3′ ends.
FIG. 12.
FIG. 12.
Three-step working model of coronavirus transcription. (A) The 5′-3′ complex formation step. Proteins binding the 5′- and 3′-end TGEV sequences are represented by the green ovals. The leader sequence is red, and CS sequences are yellow. An, poly(A) tail. (B) Base-pairing scanning step. Negative-strand RNA is in a lighter color than positive-strand RNA. The transcription complex is represented by the hexagon. Vertical dotted bars represent the base-pairing scanning by the TRS-L sequence in the transcription process. Vertical solid bars indicate complementarity between gRNA and the nascent negative strand. Un, poly(U) tail. (C) Template switch step. The thick arrow indicates the switch in the template made by the transcription complex to complete the synthesis of negative sgRNA.
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