cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts

Abstract

The entire genome of tobacco mosaic virus (TMV) was copied into a series of subgenomic cDNA clones. cDNA sequences of the 5' and 3' ends of TMV were cloned separately. A synthetic oligonucleotide primer was used to generate a Pst I site at the 5' terminus, whereas a different primer was used to generate an Nde I site at the 3' terminus.This strategy permitted removal of non-TMV sequences from doned cDNA inserts by treatment with exonuclease VII following restriction endonuclease cleavage. Pst I linkers were added to TMV 3' terminal cDNAs. Subgenomic cDNA fragments were ligated together into several independent fullgenomic constructions from which TMV cDNA sequences could be cleanly excised as a single fragment by Pst I digestion.Full-genomic TMV cDNA was ligated immediately downstream from the X phage promoter from pPM1 and transcribed in vitro with Escherichia coli RNA polymerase. RNA transcripts from three of four full-genomic cDNA constructions were infectious, even though they contained 6 non-TMV nucleotides at the 3' end. Transcripts from a construction with 6 extra nucleotides at the 5' end also were infectious. Progeny virus from plants infected with cDNA transcripts appeared identical to the parental vinrus. Restriction maps of independent cDNA clones of the same regions of the genome were identical to each other and as predicted from the reported nucleotide sequence of TMV. Also, sequences of the 200 nucleotides proximal to the 5' termini of four independent cDNA clones were identical to each other and to published sequences, suggesting that independent isolates of TMV may have remarkably similar sequences.Tobacco mosaic virus (TMV) is a virus with worldwide distribution that infects -200 plant species (1). Numerous strains of TMV cause serious losses in tobacco, tomato, and other crop plants. The virion is a rod-shaped particle 18 x 300 nm. The genome of TMV resides in a single strand of messenger-sense RNA encoding at least four proteins, two of which are translated from the same reading frame and two from processed mRNA fragments.Historically, TMV has contributed to the understanding of the genetics of RNA viruses, including the first studies on biological variability, mutability and protein sequence variation of viral genomes (2-4). TMV continues to offer numerous advantages as a genetic system. Variants of this virus express a large number of easily scored phenotypic characters useful in studying specific viral functions. Isolates of TMV that vary in symptomology, host range, elicitation of host defense mechanisms, cross-protection, encapsidation, cell-to-cell and long-distance movement within the host, and several functions of RNA replication have been described. TMV strains infecting tobacco and tomato have been most intensely investigated. Of these, two viral strains and a variant have been fully sequenced (5-7). However, in spite of our understanding of the general organization and replication of this virus, only a few viral functions have been assigned to specific sequences. The inabil...

Biologically Active Transcripts of a Large Satellite RNA from Arabis Mosaic Nepovirus and the Importance of 5' End Sequences for its Replication

“…However, in most recorded instances the biological activity of synthetic transcripts has drastically diminished with increasing numbers of 'extra', non-viral nucleotides at the 5' end of the molecule (Dawson et al, 1986;Janda et al, 1987;Ziegler-Graffet al, 1988;Masuta et al, 1988;Heaton et al, 1989;Eggen et al, 1989). In our experiments by contrast, there was no noticeable difference in the biological activity of MIR and M3R, which have either six or 29 extra nucleotides (derived from the cloning vectors) at the 5' terminus.…”

Mechanistic study of the hydrothermal reduction of palladium on the Tobacco mosaic virus

“…In 1985, the genome of TMV was copied into a series of subgenomic cDNA clones which were used as inoculant for Tobacco plants 40 . The clones were confirmed to be infectious resulting in the first incident of artificial production of TMV.…”

Replication of tobacco mosaic virus RNA

“…The importance of the 3'-terminal region of TMV RNA has been shown by the demonstration that a mutant lacking the 3'-terminal 346 nucleotides is not infectious (Dawson et al 1986). The 3'-untranslated region of TMV vulgare (approximately 200 nucleotides) can be folded into three major structural domains, connected by a central core.…”