Role of nucleotides immediately flanking the transcription-regulating sequence core in coronavirus subgenomic mRNA synthesis

Abstract

The generation of subgenomic mRNAs in coronavirus involves a discontinuous mechanism of transcription by which the common leader sequence, derived from the genome 5' terminus, is fused to the 5' end of the mRNA coding sequence (body). Transcription-regulating sequences (TRSs) precede each gene and include a conserved core sequence (CS) surrounded by relatively variable sequences (5' TRS and 3' TRS). Regulation of transcription in coronaviruses has been studied by reverse-genetics analysis of the sequences immediately flanking a unique CS in the Transmissible gastroenteritis virus genome (CS-S2), located inside the S gene, that does not lead to detectable amounts of the corresponding mRNA, in spite of its canonical sequence. The transcriptional inactivity of CS-S2 was genome position independent. The presence of a canonical CS was not sufficient to drive transcription, but subgenomic synthesis requires a minimum base pairing between the leader TRS (TRS-L) and the complement of the body TRS (cTRS-B) provided by the CS and its adjacent nucleotides. A good correlation was observed between the free energy of TRS-L and cTRS-B duplex formation and the levels of subgenomic mRNA S2, demonstrating that base pairing between the leader and body beyond the CS is a determinant regulation factor in coronavirus transcription. In TRS mutants with increasing complementarity between TRS-L and cTRS-B, a tendency to reach a plateau in DeltaG values was observed, suggesting that a more precise definition of the TRS limits might be proposed, specifically that it consists of the central CS and around 4 nucleotides flanking 5' and 3' the CS. Sequences downstream of the CS exert a stronger influence on the template-switching decision according to a model of polymerase strand transfer and template switching during minus-strand synthesis.

FIG. 1.

Sequences of TRS-S2 mutants that extend the complementarity with the leader TRS. Mutations introduced into the nucleotides immediately flanking the CS-S2 are shown in the figure (mutants M1 to M10). The right column shows the titers of each mutant when it was grown on ST cells after three plaque purification steps. L, sequences adjacent to the CS-L in the leader region; S1, sequences adjacent to the native CS-S1; S2, sequences adjacent to the native CS-S2. Virus titers (PFU per milliliter) are indicated in the right column.

FIG. 2.

Synthesis of sgmRNA-S2 by TRS-S2 mutants. (A) Diagram showing the insertion site in the TGEV genome (at the position of nonessential genes 3a and 3b) of the TRS-S2 transcription unit, which consists of CS-S2 and 30 nt from both the 5′ TRS and the 3′ TRS, including a series of nucleotide substitutions that extend the complementarity with the leader TRS. The structure of the expected sgmRNA-S2 is shown under the gRNA structure. Arrows indicate the approximate positions of primers used for RT-PCR. UTR, 3′ untranslated region. (B) Specific detection by RT-PCR of mRNA-S2 in TRS-S2 mutants. The mRNA 3a.2 species corresponds to the alternative mRNA generated from a noncanonical leader-to-body junction located at the 3′ end of ORF S, 57 nt upstream of the CS-S2 engineered at the 3ab site. TRS-S1, transcription unit containing the sequence of the active TRS that drives the synthesis of sgmRNA S; TRS-S2 wt, transcription module containing the wt sequence of the inactive TRS, including CS-S2. (C) Sequence analysis of the body-to-leader junction sites in the two mRNA species detected. As an example, the case of M8 is shown. The sequence on the top corresponds to gRNA in the fusion site. The bottom sequence in the light-gray box corresponds to the leader (L) sequence. The CS appears as white letters in a black box. CGAA and GAAA motifs are shown in dark-gray boxes. 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 interactions. Crossover should occur in any nucleotides above the arrow.

FIG. 3.

In silico analysis of the identity between TRS-L (5′-CGAACUAAACGAAA-3′) and the TGEV genomic sequences in the region of TRS-S2 insertion, the place previously occupied by genes 3a and 3b. A graphical plot of the potential base-pairing score versus the genome position is shown. Each of the three-dimensional lines represent the identity between TRS-L and the sequences corresponding to TRS-S1, TRS-S2, or the different TRS-S2 mutants with increasing complementarity with the leader (M1 to M10). The peaks assigned to the two sgmRNA species are indicated.

FIG. 4.

Northern blot analysis of intracellular RNA from TRS-S2 mutants. ST cells were infected with rTGEVs at a multiplicity of infection of 3. Total RNA was extracted at 16 hpi and analyzed by Northern blotting with a probe complementary to the 3′ end of the gRNA as described in Materials and Methods. The difference in the sizes of mRNA S and wt TGEV is due to the deletion of ORFs 3a and 3b (918 bp), which have been replaced by the TRS-S1 or TRS-S2 transcription unit (80 bp). Viral mRNAs are indicated on the left side of the figure, and the mRNA-S2 detected in mutants M9 and M10 is indicated on the right.

FIG. 5.

Quantification of sgmRNA-S2 by real-time RT-PCR. Shown are the amounts of sgmRNA-S2 in TRS-S2 mutants relative to the wt mRNA-S2 level. The log levels of mRNA-S2 are represented. The data presented are the averages of results from six independent experiments carried out in triplicate in each case. Error bars represent standard deviations. The ΔG value (shown as -ΔG) of the formation of the TRS-L-cTRS-S2 duplex is represented for each virus.

FIG. 6.

Relation between mRNA-S2 transcription levels and the ΔG of the formation of the TRS-L-TRS-B duplex. Shown is a graphical representation of the ΔG of the formation of the TRS-L-cTRS-S2 duplex (shown as -ΔG) versus the amount of mRNA-S2 in TRS-S2 mutants, relative to the wt mRNA-S2 level. The upper curve represents the TRS-S2 mutants in which the complementarity with the TRS-L was extended by replacing nucleotides at the 5′ TRS (mutants M1, M2, M3, and M4). The lower curve represents the mutants in which the complementarity with the TRS-L was extended by replacing the nucleotides at the 3′ TRS (M5, M6, M7, and M8) or at both the 5′ and 3′ TRSs (M9 and M10). The scale for the relative amounts of mRNA in both representations is significantly shifted.

FIG. 7.

Identity between the TRS-L and TRS-Bs of all TGEV sgmRNAs. The CS sequence is in white letters in a black box. White boxes highlight the identity in the sequences immediately flanking the CS both at the 5′ and 3′ ends. The transcriptional activity of each TRS is shown in the right column.

FIG. 8.

Three-step working model of coronavirus transcription for subgenomic mRNA synthesis in TRS-S2 mutants extending complementarity with TRS-L either at the 3′ TRS or 5′ TRS. (A) 5′-3′ complex formation step. Proteins binding the 5′- and 3′-end TGEV sequences are represented by the green ovals. The leader sequence, represented as the predicted secondary structure, is in red, and CS sequences are in yellow. (A)n, poly(A) tail. (B) Base-pairing scanning step. Negative-strand RNA is in a lighter color than positive-strand RNA. The replication complex is represented by the hexagon. Vertical dotted bars represent the base-pairing scanning by the TRS-L during synthesis of nascent mRNA at attenuation sites. Vertical solid bars indicate complementarity between gRNA and the nascent negative-strand. (U)n, 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.