Analysis of replication of tobacco mosaic virus by means of an in vitro transcription system

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The genome of tobacco mosaic virus (TMV) is a single-stranded, messenger-sense RNA composed of about 6,400 uncleotides. TMV RNA encodes 4 proteins: 130k, 180k, 30k and coat proteins. The 130k and 180k proteins are translated from the same initiatin codon and the latter is synthesized by read-through of the amber termination codon of the 130k protein gene. The purpose of this research is to lead to a bettwe understanding of TMV replication. First, we estabilished an in vitro transcription system that provides infectious TMV RNAs from cloned full-length cDNAs. By using this system, we constructed verious mutant TMV RNAs and analyzed their characters. Results showe that both the 130k and 180k proteins are involved in replication, only the 180k protein is the indispensable protein for replication, there are no cis-acting elements in the 30k and coat protein gene sequences, these two proteins are dispensable for replication, and that the relationship between the putative replicase (the 130k and 180k proteins) and 3' non-coding sequence is not so stringent. Besides these results, it was reveald that the 130k and 180k proteins are strongly localized in a cytplasmic structure formed specific to infection. In the course of the research, we developed an improved electroporation method by which tobacco protoplasts can be efficiently infected with a small amount of TMV RNA.