5′-UTR sequence of DHV-1 ZJ strain (GenBank No. EF382778) was aligned with other DHV-1 isolates from GenBank using DNAstar (DNASTAR Inc.) software. Secondary structure elements were predicted in Mfold.
Plasmid construction
DNA preparations were performed using standard methods as described in manufacturers’ instructions. The reporter plasmids pGEM-CAT/EMC/LUC, containing the EMCV IRES cDNA, and pGEM-CAT/LUC were kindly offered by Ian Goodfellow (Imperial College, London, UK). These plasmids allow T7 promoter-directed expression of dicistronic mRNAs encoding chloramphenicol acetyl transferase (CAT) and firefly luciferase (LUC). For the construction of the DHV-1 5′-UTR-containing plasmid, a pair of primers (DHV-1 UTRF: 5′-GAGGATCCTTTGAAAGCGGGTGCATG-3′; DHV-1 UTRR: 5′-CGGGATTCTGCATGAAAG TCTACTGGT-3′) were used to amplify the DHV-1 5′-UTR from wild DHV-1 strain (ZJ-V, GenBank No. EF382778). The purified PCR fragments were digested with BamHI, and then inserted into similarly digested and phosphatased pGEM-CAT/LUC between the two open reading frames (ORFs). The plasmid, containing the DHV-1 5′-UTR cDNA, was designated pGEM-CAT/DHV-1/LUC (Figure 1A). The further constructs containing the cDNA corresponding to the DHV-1 5′-UTR plus 10, 40, 60 nt of coding sequence were generated similarly using three pairs of specific primers and the resulting plasmids were termed pGEM-DHVUTR+10nt, pGEM-DHVUTR+40nt, pGEM-DHVUTR+60nt, respectively. Initiating AUG codon of the DHV-1 IRES was mutated to allow the expression of the luciferase from its own AUG.
Mutagenesis of the DHV-1 cDNA
In order to introduce mutations in the predicted IIIe region, Quick-Change site-direct mutagenesis kit (Stratagene) was used in order to change the sequence in the loop region 570-573 from GAUA to AAAA. The plasmid pGEM-CAT/DHV-1UTR+60/LUC was used as the template with two specific primers: P1 (sense primer): 5′-CCTACACTGCCTAAAAGGGTCGCGGCTGGT-3′; P2 (antisense primer): 5′- CAGCCGCGACCCTTTTAGGCAGTGTAGGTT-3′. The resultant plasmid was named pGEMDHV IIIe mut. Similar strategies were used to introduce mutations within the stem sequences of the predicted pseudo-knots (termed Stem1-mut and Stem2-mut). For the generation of the Stem1-mutant, the nts 447-449 (TGT) were changed to CCC. The mutagenic primers were as follows: P3 (sense primer): 5′-TGTAGGTGAGTGCCCGGTCTAGAGTAGGC-3′; P4 (anti-sense primer): 5′-CTACTCTAGACCGGGCACTCACCTACAAC-3′. For the generation of the Stem2 mutant, the nts 604-605 (CC) were changed to GG. The mutagenic primers were as follows: P5 (sense primer): 5′-TGATAGGGTCGCCCCTGGTCGAGTCCCA-3′; P6 (antisense primer): 5′-GGACTCGACC AGGGGCGACCCTATCAGG-3′. The presence of all the expected mutations in the plasmids (pGEMDHV IIIe mut, pGEMDHV S1 mut and pGEMDHV S2 mut) was confirmed by DNA sequencing. Similar strategies were used to introduce the compensatory mutations in Stem1 mut’ and Stem2 mut’ constructs.
In vitro translation reactions
For in vitro translation reactions, transcription of capped dicistronic mRNA was performed from the above plasmids, linearized with XhoI, using the Megascript transcription system (Ambion). Addition of the 7-mGTP cap 0 structure was performed using ScriptCap™m7G Capping System (Epicentre Biotechnologies) and mRNA was poly-adenylated using poly-A polymerase (PAP) following the suppliers’ recommendations. In vitro transcribed mRNAs were added to the Flexi rabbit reticulocyte lysate (RRL) system (Promega) to a final concentration of 10 μg/ml. This concentration of RNA was previously determined to give a linear yield of the translated product over the time course of the experiment (90 min). In reactions that required the addition of either recombinant 4E-BP1, L-protease, the dominant negative mutant forms of eIF4A or hippuristanol, the RRL were pre-incubated with the recombinant proteins or with the eIF4A inhibitor at 30°C for 15 min prior to the addition of the different plasmids. After 90 min, the reactions were terminated by the addition of SDS-PAGE sample buffer and subsequently resolved on 12.5% polyacrylamide gels.