Biological activity of transcripts synthesized in vitro from full-length and mutated DNA copies of tobacco rattle virus RNA 2.

Full-length cDNA clones of RNA 2 of tobacco rattle virus (TRV) strain PLB have been cloned into the transcription vector pPM1. Products of in vitro transcription by Escherichia coli RNA polymerase, either capped or uncapped, were as infectious as native RNA 2 when coinoculated with RNA 1 of TRV strain TCM. At least 70% of the internal sequence of the cDNA could be deleted without reduction of the replication efficiency of the transcripts. Sequences of 340 nucleotides at the 5' end and 405 nucleotides at the 3' end of PLB RNA 2 were found to be sufficient for replication. The encapsidation of deletion mutants of PLB RNA 2 was investigated after addition of native PLB RNA 1 and RNA 2. Accumulation of these mutants was distinguished from that of wild-type RNA 2 by insertion of nonviral sequences in the deleted parts. Three mutant forms of RNA 2 with extensive deletions in the coat protein (CP) gene were replicated but failed to encapsidate, while mutants with nonviral sequences inserted downstream from the CP gene showed a large reduction in replication efficiency.