Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences

Mutagenesis of Full-Length cDNA Clone pEAV030.

Nucleotide substitutions in the leader TRS (nucleotides 207–211 of the EAV genome; GenBank accession no. Y07862) and the RNA7 body TRS (nucleotides 12,252–12,256) were engineered in shuttle vectors by PCR mutagenesis as described before (17). Fully sequenced restriction fragments containing the mutations were introduced into EAV full-length cDNA clone pEAV030HNB, a derivative of clone pEAV030H (17) containing engineered NcoI (nucleotides 223–228) and BspEI (nucleotides 12,228–12,233) restriction sites.

RNA Transfection.

Full-length transcripts (12.8 kb) were generated in vitro from XhoI-linearized plasmid DNA as described before (17). Baby hamster kidney cells (BHK-21; ATCC CCL10) were used for all transfection experiments by using the previously described protocol (17).

Immunofluorescence Assays (IFAs).

Transfected BHK-21 cells were seeded on coverslips and fixed with paraformaldehyde at 12 h posttransfection (19). The immunofluorescence double staining for EAV nonstructural protein 2 (nsp2) (20) and the large glycoprotein (G_L) [mAb 6D10; (21)] has been described before (17). The double labeling for nsp2 and the EAV nucleocapsid protein (N) was carried out by using an anti-nsp2 rabbit serum and an anti-N mouse mAb (mAb 3E2; ref. 22). As secondary antibodies, a Cy3-conjugated donkey anti-rabbit IgG antibody and a FITC-conjugated donkey anti-mouse IgG antibody were used.

RNA Analysis.

At 12 h posttransfection, total intracellular (i.c.) RNA was isolated from BHK-21 cells transfected with transcripts from wild-type or mutant (Table 1) EAV full-length cDNA clones (17, 23). RNAs were separated in denaturing 1.5% agarose gels and hybridized (18) to a radiolabeled oligonucleotide probe complementary to the 3′ end of the genome (nucleotides 12,680–12,704), which recognizes all viral mRNAs (Fig. 1).

The sequence at the mRNA7 leader–body junction was determined by using reverse transcription–PCR (RT-PCR) and dideoxynucleotide sequencing, essentially as described previously (23). First, cDNA was generated by using a primer complementary to nucleotides 12,680–12,704 of the EAV genome and Moloney murine leukemia virus reverse transcriptase. Subsequently, a PCR was carried out by using a primer complementary to genome position 12,623–12,646 (in the mRNA7 body) and a primer corresponding to genome position 63–89 (leader). The PCR consisted of 25 cycles, each composed of 1-min denaturation at 95°C, 1-min annealing at 58°C, and 1-min extension at 72°C, after which samples were incubated at 72°C for an additional 10 min.

A similar RT-PCR was used to determine the sequences at the mRNA3 and 3.1 junction sites. In this case, the RT primer was complementary to nucleotides 10,307–10,321 and the antisense PCR primer was complementary to nucleotides 10,301–10,318. The sense PCR primer and PCR protocol were identical to those for the RNA7-specific RT-PCR, with the exception that the annealing temperature was 45°C instead of 58°C.

RNA Structure Predictions.

The structure of the 5′ terminal region of the genomes of EAV, porcine reproductive and respiratory syndrome virus, lactate-dehydrogenase elevating virus, and simian hemorrhagic fever virus (GenBank accession nos. Y07862, M96262, U15146, and L39091) were predicted by using a previously described genetic algorithm implemented in the program star (24, 25).

TRS Mutagenesis Severely Affects EAV Structural Protein Expression.

To analyze the role of the TRS in EAV sg mRNA synthesis, mutations were introduced into the full-length cDNA clone (Table 1), either in the leader TRS (L series mutants), in the RNA7 body TRS (B series mutants), or in both (LB series mutants). The RNA7 TRS was chosen because RNA7 is the most abundant sg mRNA produced by EAV. EAV replication and transcription in transfected cells initially was analyzed indirectly by using IFAs. As described before (17), a double staining with a rabbit antiserum recognizing one of the EAV replicase subunits and a mouse mAb directed against one of the viral structural proteins can be used to monitor genome replication and sg mRNA transcription, respectively. Cells were labeled for replicase cleavage product nsp2 and for either the ORF7-encoded N protein (Fig. 2a) or the ORF5-encoded glycoprotein G_L (Fig. 2b), which are expressed from mRNA7 and mRNA5, respectively.

As an example, Fig. 2 shows the results obtained for one set of mutants (L3, B3, and LB3) in which the TRS was changed from 5′-UCAAC-3′ to 5′-UGAAG-3′. For all mutants, the nsp2 signal was identical to that of the wild-type control, indicating that they replicated their genome efficiently. However, striking effects at the level of sg mRNA transcription were revealed by the labeling for the structural proteins G_L and N. Leader TRS mutant L3 appeared to be unable to generate mRNA5 and mRNA7, because G_L and N synthesis could not be detected. Mutant B3, in which the mRNA7 body TRS has been changed, did not express N whereas it still expressed wild-type levels of G_L, a result that suggested that mRNA7 synthesis was specifically affected in this mutant. Strikingly, mutant LB3, containing mutant leader and mRNA7 body TRSs but a wild-type mRNA5 TRS, showed efficient restoration of N expression but not of G_L synthesis. Essentially similar IFA results were obtained for cells transfected with two other sets of mutants, L1/B1/LB1 and L2/B2/LB2 (data not shown).

Base Pairing Between the (+) Leader TRS and the (−) Body TRS Plays a Key Role in Arterivirus sg mRNA Transcription.

Our IFA data strongly suggested that mutations in either the genomic leader TRS or the body TRS severely affected sg mRNA synthesis. However, as illustrated by the results obtained with mutant LB3 (Fig. 2), transcription apparently could be rescued by introducing identical mutations in leader and body TRSs. Subsequently, the same set of nine mutants was subjected to a direct RNA analysis. Total i.c. RNA was isolated from transfected cells at 12 h posttransfection, and viral mRNAs were visualized by hybridization to a radiolabeled oligonucleotide probe (Fig. 3a). For the wild-type construct, the genome (mRNA1) and the six sg mRNAs (mRNAs 2–7) were readily detected. Mutants L1–L3, in which either one or both of the C residues in the leader TRS had been substituted, replicated with wild-type efficiency, which confirmed that these mutations in the 5′ end of the viral genome did not interfere with replication (Fig. 3a, lanes L1–L3). However, in agreement with our IFA data (Fig. 2), sg mRNA transcription from the 3′ terminal region of these mutant genomes was severely affected or completely abolished, which corroborated the crucial role of the leader TRS in mRNA synthesis. Remarkably, mutant L2 continued to synthesize only mRNA3 (see below) and two larger additional RNAs that were not analyzed in detail. The introduction of mutations into the RNA7 body TRS (Fig. 3a, lanes B1–B3) severely reduced or completely abolished the transcription of mRNA7, but not that of the other sg mRNAs. Finally, the simultaneous introduction of the same mutation(s) in the leader and RNA7 body TRSs (Fig. 3a, lanes LB1–LB3) specifically restored mRNA7 transcription to wild-type levels, but the synthesis of other sg mRNAs was not restored. From the results obtained with the LB double mutants, we concluded that base pairing between the (+) leader TRS and the (−) body TRS complement is an essential step in arterivirus sg mRNA transcription.

The double C → G replacement in the leader and body TRSs of mutant LB3 did not have a detectable effect on its capacity to produce sg mRNA7 (Fig. 3a). This suggested that the sequence of the TRS per se is not crucial, as long as the possibility for TRS base pairing is maintained. Therefore, we constructed a fourth set of mutants in which the entire TRS (5′-UCAAC-3′) was replaced by the sequence 5′-AGUUG-3′. As expected, mutant L4 did not synthesize any of the sg mRNAs, whereas mutant B4 produced only mRNAs 2–6 (Fig. 3b). Remarkably, the simultaneous mutagenesis of leader TRS and body TRS in construct LB4 again specifically restored RNA7 synthesis, albeit not to wild-type levels. Finally, by using RT-PCR amplification and sequence analysis, we confirmed that in all double mutants (LB1–LB4) the mRNA7 leader and body indeed had been fused at the expected position and not at an alternative site (Table 1). As an example, the results for mutant LB1 are shown in Fig. 4a.

Continued RNA3 Synthesis by Mutants L2 and LB2.

The continued synthesis of mRNA3 by mutants L2 and LB2 (Fig. 3a) can be explained by our previous observation (13) that EAV produces two major mRNA3 species, one (mRNA3) derived from a consensus TRS and one (mRNA3.1) derived from a nonconsensus TRS (5′-UCAAUACCC-3′). The latter TRS lacks the C residue at position 5, but contains 5 downstream nucleotides (UACCC) that match the corresponding sequence 3′ of the leader TRS. Thus, the residue at position 5 of the mRNA3.1 TRS apparently is not involved in base pairing. Consequently, mutants L2 and LB2, which have mutations at TRS position 5, are not affected in the synthesis of mRNA3.1. An RT-PCR and sequence analysis for mutants L2 and LB2 confirmed the continued synthesis of mRNA3.1 when the leader TRS has the sequence 5′-UCAAG-3′ (data not shown).

The TRS in the sg mRNA Is Derived Exclusively from the Body TRS.

Mutant B1 and, to a lesser extent, mutants L1, L2, and B2 still produced low levels of mRNA7. The RNA7 produced by mutant B1 was detectable via hybridization (Fig. 3a, lane B1). Also, for mutants B2, L1, and L2, some RNA7 could be detected by hybridization after long exposure and, more convincingly, by RT-PCR analysis (data not shown).We took advantage of this residual mRNA7 synthesis to address another important issue. Is the TRS that forms the mRNA leader–body junction derived from the leader or from the body sequence? Because leader and body TRS normally are identical, this question cannot be answered by using wild-type sequences. However, because we mutated either the leader TRS or the RNA7 body TRS in constructs L1, L2, B1, and B2, sequence analysis of sg mRNA7 enabled us to determine the origin of the sequence at the leader–body junction site. Sequences were determined by direct analysis of mRNA7-specific RT-PCR products generated by using i.c. RNA from cells transfected with one of these four mutants (Table 1). As shown for mutants L1 and B1 in Fig. 4b, the sequences of the mRNA7 leader–body junction sites obtained in this manner were unambiguous and did not show any heterogeneity. For mutants L1 and L2, we established that point mutations in the leader TRS were not transferred to sg mRNA7, which contained the wild-type 5′-UCAAC-3′ sequence. Conversely, mRNA7 transcripts from mutants B1 and B2 contained exclusively a mutagenized TRS at their leader–body junction. Thus, in all these mutants, the mRNA7 junction site sequence was derived from the body TRS and not from the leader TRS. The implications of these findings for the mechanism of discontinuous sg RNA transcription will be discussed below.