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...

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Inoculation of six brome mosaic virus (BMV) RNA3 transcripts with defined deletions in the coat protein (CP) gene to three Chenopodium spp demonstrated that synthesis of a functional, encapsidation-competent CP is required for the induction of local lesions. The BMV CP open reading frame contains two in-frame AUG codons separated by seven amino acids, resulting in the synthesis of two CPs (CP1 and CP2). To elucidate the biological significance of the N-terminal basic region of BMV CP, RNA3 variants capable of producing either CP1 or CP2 but not both were constructed. Infection phenotypes elicited on three Chenopodium spp by each RNA3 variant revealed that amino-terminal residues 1 to 7 are required to establish chlorotic local lesions and systemic infection in Chenopodium quinoa. Deletion of this region has no effect on infection in barley plants but resulted in the induction of the hypersensitive response on the inoculated leaves of C. quinoa and blocked systemic spread. Analysis of seven additional RNA3 variant transcripts, each having a six-base deletion (two amino acids) in the sequence encoding the N-terminal seven residues, indicated that variants that share a common deletion of positively charged lysine rendered the CP encapsidation-incompetent and failed to establish infection. Taken together, these results suggest that residues 1 to 7 of the BMV CP play an important role in virus-host interactions and contribute differently to the virulence phenotype in different host plants.

ABSTRACTa-Trichosanthin, a eukaryotic ribosomeinactivating protein from Trichosanthes kirilowii, inhibits the replication of the human immunodeficiency virus (HIV) in vitro. The a-trichosanthin gene was placed under the transcriptional control of a tobamovirus subgenomic promoter in a plant RNA viral vector. Two weeks after inoculation, transfected Nicotiana benthamiana plants accumulated a-trichosanthin to levels of at least 2% of total soluble protein. The recombinant a-trichosanthin was purified and its structural and biological properties were analyzed. The 23-amino acid signal peptide was recognized by N. benthamiana and the processed enzyme caused a concentration-dependent inhibition of protein synthesis in vitro. The high level of heterologous gene expression observed in these studies is due to the unique features of the RNA viral-based transfection system.