Trans complementation of virus-encoded replicase components of tobacco mosaic virus.
ABSTRACT We examined whether the 130K and 180K proteins of tobacco mosaic virus (TMV), the putative virus-encoded replicase components, produced by a replication-competent TMV mutant could complement a replication-defective mutant in a single cell. The replication-competent mutant (LDCS29) had a deletion in the coat protein gene and the replication-defective mutant (LDR28) had a large deletion in the gene encoding the 130K and 180K proteins. Neither the replication of LDR28 nor the production of the coat protein from LDR28 or LDCS29 was detected when the mutants were inoculated separately into tobacco protoplasts. However, when the two mutants were co-inoculated, the production of the LDR28 genomic RNA and the subgenomic RNA for the coat protein and accumulation of the coat protein were observed. These results show that the virus-encoded replicase components of TMV complemented the replication-defective mutant in trans.
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ABSTRACT: The viral replicase complex of positive-stranded RNA viruses interacts with cis-acting elements that are usually located at the termini of the viral RNAs. 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 of pseudoknot (pk) 1 and/or 2, whereas identical deletions in dRNAs did not affect their replication. However, pk3 was required for replication of both full-length TMV RNAs and dRNAs. The requirements for homologous sequences were greater for dRNA replication than for replication of full-length TMV RNAs. 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 contiguous heterologous sequences in the dRNAs compensated for the impaired interactions between the replicase and 3′ NTR. The precision requirement appeared 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.Virology 08/2000; · 3.37 Impact Factor
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ABSTRACT: Hibiscus latent Singapore virus (HLSV) is a member of Tobamovirus and its full-length cDNA clones were constructed. The in vitro transcripts from two HLSV full-length cDNA clones, which contain a hepta-adenosine stretch (pHLSV-7A) and an octo-adenosine stretch (pHLSV-8A), are both infectious. The replication level of HLSV-7A in Nicotiana benthamiana protoplasts was 5-fold lower, as compared to that of HLSV-8A. The replicase proteins of HLSV-7A were produced through programmed −1 ribosomal frameshift (−1 PRF) and the 7A stretch was a slippery sequence for −1 PRF. Mutations to the downstream pseudoknot of 7A stretch showed that the pseudoknot was not required for the frameshift in vitro. The stretch was found to be extended to 8A after subsequent replication cycles in vivo. It is envisaged that HLSV employs the monotonous runs of A and −1 PRF to convert its 7A to 8A to reach higher replication for its survival in plants.Virology 01/2014; 449:229–234. · 3.35 Impact Factor
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ABSTRACT: Tobacco mosaic virus produces two proteins that contain domains similar to the methyltransferase (MT) and helicase (HEL)-like domains of the replicase-associated proteins of other RNA viruses. The more abundant 126-kDa protein contains only the MT and HEL-like domains, whereas the 183-kDa readthrough protein additionally contains the polymerase domain. We examined the functions of these proteins by constructing a bipartite system to express the 126- and 183-kDa proteins from separate RNAs. Mutants expressing the 183-kDa protein recognized promoters for negative- and positive-stranded RNA synthesis, transcribed subgenomic mRNAs, capped RNAs, synthesized proteins, moved cell to cell within the plant, and replicated defective RNAs (dRNAs). The principal function of the 126-kDa protein was to increase the rate of replication approximately tenfold. The 126-kDa protein appeared to function primarily in cis, and production of the 126-kDa protein in trans did not enhance replication of the helper virus. dRNAs producing a functional 126-kDa protein were replicated efficiently by helper viruses that produced only the 183-kDa protein but not by wild-type virus, suggesting that efficient replication required the 183-kDa protein to form a heterodimer with the 126-kDa protein already bound to the target dRNA.Virology 06/2000; 271(1):90-8. · 3.37 Impact Factor