4.1. Construction of PSTVd Dimeric cDNA Clones and Transcription of RNA Transcripts

Dimeric PSTVd-I_wt (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"M16826","term_id":"333352","term_text":"M16826"}}M16826), PSTVd-D_wt (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB623143","term_id":"343403737","term_text":"AB623143"}}AB623143), and their mutants at positions 42, 43, 64, 310, and 311/312 (hereafter abbreviated as 312) were created by reciprocally exchanging individual nucleotide between PSTVd-I_wt and PSTVd-D_wt on the pBluescript II (SK–) (Stratagene, Japan) plasmid as described elsewhere [42,62,69]. The mutants are named as: PSTVd-I:C42U, PSTVd-I:U43C, PSTVd-I:64U, PSTVd-I:A310C, PSTVd-I:312UU, PSTVd-D:U42C, PSTVd-D:C43U, PSTVd-D:64Δ, PSTVd-D:C310A and, PSTVd-D:312UUΔ. An additional mutant, PSTVd-I:C42U/64U, a PSTVd-I-based mutant with two PSTVd-D-type mutations at positions 42 and 64, was constructed by inserting a chemically synthesized unit-length cDNA copy with BamHI termini under the control of T7 RNA polymerase promoter sequence in pUC57 plasmid vector (GENEWIZ Japan Corp., Kawaguchi, Japan). Plasmid DNA (about 2 μg) containing the dimeric cDNA was digested with the AscI or NotI restriction enzyme (Takara Bio Inc., Otsu, Shiga, Japan) at 37 °C overnight, and used for in vitro transcription at 37 °C for 2–4 h using T7 RNA polymerase (Invitrogen, Carlsbad, CA, USA) in a 20-μL reaction mixture according to the manufacturer’s instructions. The transcribed RNAs were recovered with ethanol precipitation, dissolved in 50 μL sterilized distilled water. Integrity of in vitro transcripts were examined by agarose gel electrophoresis followed by quantification using UV spectrophotometry [74].