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

烟草花叶病毒(TMV)基因组是由约6400个非密码子组成的单链信使RNA。TMV RNA编码4种蛋白：130k、180k、30k和外壳蛋白。130k和180k蛋白由同一起始密码子翻译而来，后者是通过通读130k蛋白基因的琥珀终止密码子合成的。这项研究的目的是为了更好地了解TMV的复制。首先，我们建立了一个体外转录系统，可以从克隆的全长cDNA中提供具有感染性的TMV RNA。利用该体系构建了多种突变体TMV RNAs，并对其特性进行了分析。结果表明，130k和180k蛋白都参与了复制，只有180k蛋白是复制所必需的蛋白，30k和外壳蛋白基因序列中没有顺式作用元件，这两种蛋白是复制所必需的，推测的复制酶(130k和180k蛋白)与3‘非编码序列之间的关系并不那么严格。除了这些结果外，还发现130k和180k蛋白强烈定位于感染所形成的细胞质结构中。在研究过程中，我们开发了一种改进的电穿孔方法，通过这种方法可以有效地将少量TMV RNA感染烟草原生质体。

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.