Full-length tobacco mosaic virus RNAs and defective RNAs have different 3 ' replication signals

The viral replicase complex of positive-stranded RNA viruses interacts with cis-acting elements that are usually located at the termini of the viral R NAs. On comparison of the replication requirement of a tobacco mosaic virus (TMV)-based defective RNA (dRNA) and its helper virus, we found different requirements for replication of TMV RNAs in cis and in trans. The level of replication of full-length TMV RNA-decreased substantially in the absence o f pseudoknot(pk) 1 and/or 2, whereas identical deletions in dRNAs did not a ffect their replication. However, pk3 was required for replication of both full-length TMV RNAs and dRNAs. The requirements for homologous sequences w ere greater for dRNA replication than for replication of full-length TMV RN As. Defective RNAs with heterologous 3' nontranslated regions (NTRs) failed to be replicated or replicated minimally, whereas replication of similarly mutated full-length RNAs was much less affected, increasing amounts of con tiguous heterologous sequences in the dRNAs compensated for the impaired in teractions between the replicase and 3' NTR. The precision requirement appe ared to involve the terminal 28 nucleotides, specifically the pseudoknot in the aminoacyl acceptor arm of the tRNA like structure, which was important in replication of both dRNAs and full-length TMV RNAs. (C) 2000 Academic P ress.