Based on a full-length infectious cDNA clone, gene modifications of Tobacco necrosis virus A Chinese isolate (TNV-AC) were made by site-directed mutagenesis or nucleotide deletions for in vitro transcrip-tion of mutant viral RNAs. Mechanical inoculations of Chenopodium amaranticolor with in vitro tran-scripts, containing a single nucleic acid substitution at the presumed transcriptional start sites for the two subgenomic (sg) RNAs, showed that the sgRNA1 and sgRNA2 of TNV-AC were initiated at G2184 or G2460, respectively. Mutagenesis of the translational initiation-codons for the open-reading frame (ORF) P8 or P6 encoded by sgRNA1 indicated that each of the two genes was essential for formation of local lesions on C. amaranticolor leaves, perhaps by blocking virus cell-to-cell movement, but were not necessary for viral RNA replication in the protoplast of tobacco cell BY-2. Results of prokaryotic expression showed that the ORF coding for coat protein on TNV-AC sgRNA2 was initiatively translated by the first AUG codon at nucleotides 2612―2614. Site-directed mutation of translational start codons, and deletion of the entire coding region, showed that the intact TNV-AC coat protein was dispensable for establishment of TNV-AC infection in C. amaranticolor, otherwise the numbers of local lesions and the viral RNA accumulation level were reduced, or the time to symptom appearance significantly delayed. These results suggested that the nucleotide sequence around the translational start codon coding for TNV-AC coat protein gene may play an important role in the local symptoms. Aspects of the involve-ment of the coat protein in the TNV-AC life cycle were discussed.
LI Jiang LI Min LI Cui GAO Yang LI DaWei HAN ChengGui YU JiaLin
