Nucleotide sequence and infectious in vitro transcripts of RNA 3 of tomato aspermy virus pepper isolate

The nucleotide sequence of a cDNA clone corresponding to the entire RNA 3 of a pepper isolate of tomato aspermy cucumovirus (P-TAV) was determined. The 2222 nucleotides (nt) long RNA 3 is dicistronic and codes a 3a (280 amino acids) and a coat protein (cp, 218 amino acids). The nucleotide sequence of P-TAV RNA 3 shows a high sequence identity (99%) with that of C-TAV RNA 3 and the amino acid sequences of the 3a and the coat proteins between these two TAV strains are also very similar. The transcription start site of the subgenomic RNA 4 was mapped to the G at the position of 1148 of RNA 3. A full-length cDNA copy of P-TAV RNA-3 was cloned downstream of the phage T7 promoter. Transcripts synthesized in vitro had no extra nucleotides at the 5' end, but there were nine non-viral nucleotides at the 3' terminus. The transcripts of TAV RNA 3 were infectious when coinoculated into test plants in the presence of purified RNA 1 and 2 of P-TAV or Trk7-CMV respectively. Northern blot analysis of RNA extracted from inoculated plants showed the expected genomic and subgenomic RNAs.