Cells and transfection.

Baby hamster kidney (BHK) cells stably transformed with the porcine amino peptidase N gene (4) and with the Sindbis virus replicon pSINrep21 (8), expressing TGEV N protein (BHK-N), were grown in Dulbecco's modified medium supplemented with 5% FCS and G418 (1.5 mg/ml) as a selection agent. BHK-N cells were grown to 95% confluence on 35-mm-diameter plates and transfected with 4 μg of each TGEV replicon, representing on average 100 molecules per cell, by using 12 μg of Lipofectamine 2000 (Invitrogen) according to the manufacturer's specifications.

Plasmid constructs.

cDNAs of TGEV-derived replicons including mutated sequences of TRS-N and the coding sequence of the TGEV N gene were generated by PCR-directed mutagenesis. Mutants TRS-N and E-TRS-N were generated by PCR using the plasmid pBAC-TGEV (2), containing the TGEV genome (GenBank accession no. AJ271965), as a template, with the same reverse primer and specific forward oligonucleotides (Table ​(Table1).1). PCR fragments including AscI restriction sites were cloned into the plasmid pBAC-REP-1 (1), previously digested with MluI and AscI enzymes, generating compatible ends. To generate replicon E-TRS-GFP, two overlapping PCR fragments were obtained using pcDNA3-GFP and pBAC-TGEV as templates and specific oligonucleotides given in Table ​Table1.1. The final amplification product (2,068 bp), which includes the TRS from the N gene fused to the green fluorescent protein (GFP)-coding sequence, was generated with outer oligonucleotides E-800 AvrII VS and 3′AscI GFP RS, digested with AvrII and AscI, and cloned into the same restriction sites of the plasmid pBAC-REP-1. To obtain mutant E-TRS3a-N, a PCR fragment was amplified using pBAC-TGEV as a template and oligonucleotides E-800 AvrII VS and E-120 AvrII RS. The amplification fragment (680 bp) was digested by AvrII and cloned into the same restriction sites of intermediate plasmid pBAC-REP-TRS3a-N (J. L. Moreno, I. Sola, and L. Enjuanes, unpublished).

To generate replicons including different sequences from the distal 5′ TRS-N region, PCR fragments of the same size (360 bp) were amplified with specific oligonucleotides containing AvrII restriction sequences (Table ​(Table1)1) and pBAC-TGEV as a template. PCR products were digested with AvrII and cloned into the same restriction sites of plasmid TRS-N, leading to plasmids E1-TRS-N, E2-TRS-N, and E3-TRS-N. To obtain mutations in the complementary 9-nt sequences, overlapping PCR fragments were amplified by using oligonucleotides given in Table ​Table11 and pBAC-TGEV as a template. The final PCR product (340 bp), amplified with outer oligonucleotides E-740 AvrII VS and E-160 AvrII RS, was digested with AvrII and cloned into the same restriction site of the plasmid TRS-N, leading to the mutants M1-TRS-N, M4-TRS-N, M5-TRS-N, M6-TRS-N, and M7-TRS-N. The mutant M2-TRS-N was engineered using TRS-N as a template. Overlapping PCR fragments including mutations were amplified by using specific oligonucleotides (Table ​(Table1).1). The final amplification product (1,645 nt), generated with outer oligonucleotides E-740 AvrII VS and 3′AscI RS, was digested with AvrII and AscI and cloned into the same restriction sites of the plasmid pBAC-REP-1. To generate mutant M3-TRS-N, the AvrII digestion product (340 bp) from plasmid E2-TRS-N was cloned into the same restriction sites of the plasmid M2-TRS-N. All cloning steps were checked by sequencing the PCR-amplified fragments and cloning junctions.

RNA analysis by real-time RT-PCR.

Total intracellular RNA was extracted at 24 h posttransfection (h p.t.) from transfected BHK-pAPN-N cells or at 12 h p.i. from infected ST cells as previously described. cDNAs were synthesized at 50°C for 1.5 h with avian myeloblastosis virus reverse transcriptase (ThermoScript; Invitrogen) and the reverse primers given in Table ​Table2.2. The cDNAs generated were used as templates for specific PCR amplification. Real-time reverse transcription-PCR (RT-PCR) was used for quantitative analysis of genome RNA (gRNA; used as an endogenous standard) and sg mRNAs from TGEV and TGEV-derived replicons. Oligonucleotides used for RT and PCRs (Table ​(Table2)2) were designed by Primer Express software. Sybr green PCR master mix (Applied Biosystems) was used in the PCR step, according to the manufacturer's specifications. Detection was performed with an ABI PRISM 7000 sequence detection system (Applied Biosystems). Data were analyzed with ABI PRISM 7000 SDS version 1.2.3 software.

Pulse and chase assay.

Viral RNAs were metabolically labeled as described above. At 9 h p.i. cells were washed with fresh medium containing unlabeled phosphate and 2% dialyzed FCS. Cells were incubated at 37°C, and the total intracellular RNA was isolated and quantitated at different chase hours as described above.

Study of TGEV sg mRNA stability.

To analyze whether a higher stability of sg mRNA N might explain its high levels during TGEV infection, pulse and chase experiments were performed. Viral RNAs were metabolically labeled with [³³P]orthophosphate, and RNA from infected cells was isolated at different chase times. RNAs were separated by electrophoresis, and labeled sg mRNAs were quantified by gel densitometry (Fig. ​(Fig.22 and data not shown). As observed from autoradiography and quantification, the stability of sg mRNA N was not higher than that of the other viral sg mRNAs. Therefore, the exceptional high levels of mRNA N during transcription must be caused by another regulatory mechanism.

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FIG. 2.

TGEV sg mRNA stability as determined by pulse-chase experiments. Viral sg mRNAs were metabolically labeled with [³³P]orthophosphate. At 9 h p.i., cells were incubated with fresh medium containing unlabeled phosphate and the total intracellular RNA was harvested and analyzed by electrophoresis in a denaturing gel at the indicated times.

Localization of signals responsible for transcriptional activation of sg mRNA N.

The increase in transcription levels over those predicted by the ΔG value of base pairing between TRS-L and cTRS-B was observed only for the N gene; therefore, we decided to investigate potential regulatory sequences near this gene. The presence of signals responsible for the specific transcriptional activation of sg mRNA N was analyzed within the N gene coding sequence and within the distal and proximal 5′ TRSs of the N gene, comprising the M gene, by using a TGEV-derived replicon (REP-1). The cDNA of this replicon includes the N gene in the same sequence context and at the same distance from the 3′ end as in the whole viral genome (1). In addition, since efficient RNA synthesis activity of this replicon is associated with the presence of the nucleoprotein, and the expression of this protein is prevented in some designed mutants, the analysis of their functionality was performed with BHK cells expressing in trans TGEV N protein. A set of three mutants was engineered using the TGEV-derived replicon (Fig. ​(Fig.3A).3A). The E-TRS-N mutant contained the coding sequence of the N gene and 800 nt from the 5′ TRS of the N gene, representing the complete M gene. This sequence context was identical to that of the N gene in the wild-type (wt) virus. In the E-TRS-GFP mutant, the N gene coding sequence from E-TRS-N was replaced by the GFP coding sequence (600 nt) plus a nonrelated 500-nt region to mimic the length of original N gene (1,100 bp). To study the effect of the proximal 5′ TRS of the N gene in the levels of sg mRNA N, a replicon mutant TRS-N that includes only the N gene coding sequence and 120 nt of the 5′ TRS was engineered. BHK-N cells were transfected with the cDNAs encoding mutant replicons, and the levels of intracellular sg mRNA N were analyzed by real-time RT-PCR. The amount of sg mRNA N was expressed in relation to that of sg mRNA 7. The relative level of sg mRNA N in the E-TRS-N mutant was similar to that obtained during TGEV infection (Fig. ​(Fig.3B),3B), indicating that similar transcription-regulating mechanisms most likely were operating in the replicon and in the whole virus and validating the study of transcriptional activation of the N gene in the replicon system. The transcriptional activity of E-TRS-GFP was similar to that of E-TRS-N, including the same regulating sequences and the N gene coding sequence, indicating that the signals responsible for transcriptional activation of the N gene were not included within the N gene coding sequence and were mostly located within the 5′ TRS. In contrast, the amount of sg mRNA N from mutant TRS-N was significantly reduced, indicating that the proximal 5′ TRSs comprising 120 nt immediately upstream of CS-N were not sufficient to promote transcription of the N gene up to the levels observed in a standard virus infection and that the distal 5′ TRS-N sequences, including nt −800 to −120 upstream of the CS-N, were necessary for transcriptional activation of the N gene.

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FIG. 3.

Localization of signals responsible for transcriptional activation of mRNA N. (A) Scheme showing the genetic structure of TGEV-derived replicons. The TGEV genome and the genetic structure of TGEV-derived replicon REP-1 are shown in the first and second lines, respectively. TGEV genes are represented by letters above the boxes. The scheme of different mutant replicons generated to localize the signals responsible for transcriptional activation of sg mRNA N is shown at the bottom, and their names are indicated to the left. Numbers indicate distances in nucleotides upstream of CS-N. Distal 5′ TRS-N includes nt −800 to −120 upstream of the CS of the N gene (larger black box). Deletion mutants from this region are represented with smaller black boxes. 5′ TRS-N represents the first 120 nt upstream of the CS of the N gene (dark gray boxes). 3′ TRS-N includes the 3′ TRS of gene N. N indicates the N gene coding sequence (light gray boxes). 5′ TRS-3a includes the first 120 nt upstream of the CS of gene 3a. 3′ TRS-3a includes the 3′ TRS of gene 3a. UTR, untranslated region. (B) Quantification of sg mRNA N by real-time RT-PCR. The amounts of sg mRNA N expressed from each mutant replicon are shown relative to that of sg mRNA 7. The data are the averages of four independent experiments performed in triplicate in each case. Error bars represent standard deviations.

To localize the signals responsible for sg mRNA N transcription activity within the distal 5′ TRS-N region, three deletion mutants were engineered (Fig. ​(Fig.3A).3A). These mutants, E1-TRS-N, E2-TRS-N, and E3-TRS-N, included overlapping sequences from the distal 5′ TRS-N region, nt −800 to −460, nt −630 to −290, and nt −460 to −120, respectively, preventing the interruption of potential elements responsible for transcriptional activation of the N gene at the level of primary sequence or secondary structure. These sequences were fused to the proximal region included in the TRS-N mutant, comprising 120 nt immediately upstream of the CS-N and 3′ TRS-N. To study whether the effect in transcriptional activation of the distal 5′ TRS-N region was independent of the proximal 5′ TRS-N region, these sequences were replaced in mutant E-TRS-N by the proximal 5′ TRS from gene 3a (TRS-3a), generating the E-TRS3a-N mutant. BHK-N cells were transfected with cDNAs encoding the mutant replicons, and the levels of sg mRNA N were analyzed by real-time RT-PCR. The levels of sg mRNA N in the E2-TRS-N mutant were even higher than those in the replicon containing the complete 5′ TRS-N regulating sequences, probably as a consequence of approaching the distal and proximal 5′ TRS-N domains containing the elements involved in the transcriptional activation of the N gene. In contrast, the amounts of sg mRNA N in mutants E1-TRS-N and E3-TRS-N were significantly lower than those in E-TRS-N and TGEV (Fig. ​(Fig.3B).3B). Therefore, the sequences from the distal 5′ TRS-N region included in the E2-TRS-N mutant were required for high-level transcription of the N gene. Nevertheless, the sg mRNA N levels in the E-TRS3a-N mutant, containing the complete distal 5′ TRS-N region fused to the proximal 5′ TRS from gene 3a, were lower than those in E-TRS-N and TGEV, indicating that the distal 5′ TRS-N regions were not able to enhance the transcription of the N gene when associated with other proximal 5′ TRSs (Fig. ​(Fig.3B).3B). All together, these results indicated that transcriptional activation of gene N requires the presence of sequences located within the distal 5′ TRS-N region (nt −630 to −290) in addition to sequences located just upstream of the CS of gene N (the first 120 nt).

Long-distance RNA-RNA interaction is required for transcriptional activation of gene N.

Sequence analysis of the distal 5′ TRS-N region included in the E2-TRS-N mutant and the proximal 5′ TRS-N region revealed the presence of a 9-nt sequence (5′-AUAUGUAAU-3′) within the distal 5′ TRS-N region, 449 nt upstream of CS-N, unique along the whole genome of the TGEV-PUR46-MAD strain, which was complementary to another 9-nt segment (5′-AUUACAUAU-3′) located 7 nucleotides upstream of CS-N (Fig. ​(Fig.4A).4A). To test whether the transcriptional activation of the N gene was regulated by the putative base-paring interaction between the distal and proximal 9-nt sequences, a set of mutants in which the predicted interaction was disrupted was constructed (Fig. ​(Fig.4A).4A). The distal 9-nt sequence is located into a transmembrane domain of the M protein, according to previous publications (7). In the M1-TRS-N mutant, the 9-nt sequence in the distal 5′ TRS-N region was replaced by a mutant sequence (5′-AUUACAUAU-3′) that disrupted the potential base-pairing interaction in 5 out of 9 nt, whereas in the M2-TRS-N mutant, the 9-nt sequence located in the proximal 5′ TRS-N region was replaced by another sequence (5′-AUAUGUAAU-3′) that similarly disrupted the predicted base-pairing interaction. Mutant M3-TRS-N included complementarity 9-nt mutant sequences at distal and proximal locations that restored potential base pairing. BHK-N cells were transfected with the cDNA of mutant replicons, and sg mRNA N levels were determined by real-time RT-PCR (Fig. ​(Fig.3B).3B). The sg mRNA N levels in the M1-TRS-N and M2-TRS-N mutants were significantly lower than those in the original E2-TRS-N replicon. In contrast, the amount of sg mRNA N in the M3-TRS-N mutant increased almost to levels similar to those observed in the E2-TRS-N replicon. The remaining activities of these mutants were probably due to the retention of partial base pairing between the mutant sequence and its complementary element. These results reinforce the functional relevance of the proposed long-distance base pairing between proximal and distal 9-nt elements for N gene transcriptional activation.

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FIG. 4.

Long-distance RNA-RNA interaction required for transcriptional activation of the N gene. (A) Scheme showing the genetic structure of TGEV-derived replicons. The 9-nt sequence segments are represented by small white boxes within the proximal and distal 5′ TRS-N regions. The scheme of different mutant replicons generated to analyze the functional relevance of the putative RNA-RNA interaction is shown at the bottom, and their names are indicated to the left. The sequences of the 9-nt motif mutants located within the proximal and distal 5′ TRS-N regions are represented in smaller black boxes. Asterisks represent nucleotides identical to those in E2-TRS-N. Potential base pairing between proximal and distal sequences is shown in the right column by vertical lines. See the legend to Fig. ​Fig.33 for more details. UTR, untranslated region. (B) Quantification of sg mRNA N by real-time RT-PCR. The amounts of sg mRNA N expressed by each mutant replicon are shown relative to that of sg mRNA 7. The data are the averages of four independent experiments performed in triplicate in each case. Error bars represent standard deviations.

Influence of ΔG duplex formation between the distal and proximal 9-nt elements on sg mRNA N synthesis.

To study the effect of duplex stability between the distal and proximal 9-nt sequences in the sg mRNA N synthesis, four mutants with nucleotide substitutions within the distal 9-nt sequence were generated (Fig. ​(Fig.5A).5A). In the M4-TRS-N and M5-TRS-N mutants, the most 5′ nucleotide at the distal 9-nt sequence was mutated to a G and a C, respectively, decreasing the stability, measured as ΔG of duplex between the distal and proximal 9-nt elements (Fig. ​(Fig.5A).5A). The ΔG value of duplex formation in the M4-TRS-N mutant was higher than that in the M5-TRS-N mutant because a non-Watson-Crick base pair could be established between the mutated G and the U in the proximal sequence. In the M6-TRS-N mutant, two point mutations at the 5′ and 3′ ends of the distal sequence severely decreased the ΔG. In the M7-TRS-N mutant, in addition to an internal mutation that disrupts continuous complementarity, two nucleotide substitutions that increased the complementarity of the distal 9-nt element with the proximal element up to a ΔG value similar to that of the wt sequence were introduced just upstream of the distal 9-nt element. BHK-N cells were transfected with cDNAs encoding the mutant replicons, and the levels of sg mRNA N were analyzed by real-time RT-PCR (Fig. ​(Fig.5B).5B). The amounts of sg mRNA N in replicons M4, M5, and M6, including mutations that decreased duplex stability between proximal and distal elements, were lower than that in the E2-TRS-N replicon. In contrast, the level of sg mRNA N in the M7-TRS-N mutant, with a ΔG for duplex formation similar to that of the E2-TRS-N replicon, was similar to that observed in the wt replicon, in spite of the internal mutation that disrupts complementarity between sequence elements. A good correlation was observed between the amount of sg mRNA N and duplex formation ΔG in the distal and proximal 9-nt elements, indicating that the stability of the duplex between these elements may be a decisive factor for transcriptional activation of the TGEV N gene.

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FIG. 5.

Influence of ΔG duplex formation between the distal and proximal elements on sg mRNA N synthesis. (A) Scheme showing the genetic structures of REP-1, E-TRS-N, and E2-TRS-N TGEV-derived replicons. The names of mutant replicons are indicated in the left column. Sequences of the proximal and distal 9-nt motifs in mutant replicons are shown in the central columns. Asterisks represent nucleotides identical to those in replicon E2-TRS-N. Letters within the light gray box in mutant M7-TRS-N represent nucleotides increasing the complementarity with the proximal 9-nt element. Potential base pairing between proximal and distal sequences are indicated in the right column. Solid vertical bars represent Watson-Crick base pairing. Dotted vertical bars represent non-Watson-Crick interactions. See the legend to Fig. ​Fig.44 for more details. UTR, untranslated region. (B) Quantification of sg mRNA N by real-time RT-PCR. The mutants are represented in decreasing order of ΔG duplex formation between the distal and proximal 9-nt sequences. The data are the averages of four independent experiments performed in triplicate in each case. Error bars represent standard deviations.

We thank J. C. Oliveros for the in silico analysis and M. González for technical assistance.

This work was supported by grants from the European Union (Frame VI, DISSECT PROJECT, SP22-CT-2004-511060, and RiViGene PROJECT, SSPE-CT-2005-022639) and a grant (CPE03-021-C4-3) from the National Institute of Agronomics Research (INIA), Spain. J.L.M., S.Z., and I.S.G. received fellowships from Consejo Superior de Investigaciones Científicas (CSIC).