In vivo analysis of the TSWV cap-snatching mechanism: single base complementarity and primer length requirements

Requirements for capped leader sequences for use during transcription initi ation by tomato spotted wilt virus (TSWV) were tested using mutant alfalfa mosaic virus (AMV) RNAs as specific cap donors in transgenic Nicotiana taba cum plants expressing the AMV replicase proteins. Using a series of AMV RNA 3 mutants modified in either the 5'-non-translated region or in the subgeno mic RNA4 leader, sequence analysis revealed that cleaved leader lengths cou ld vary between 13 and 18 nucleotides. Cleavage occurred preferentially at an A residue, suggesting a requirement for a single base complementarity wi th the TSWV RNA template, which could be confirmed by analyses of host mRNA s used in vivo as cap donors.