Mechanisms of viral RNA replication in the Sindbis-virus superfamily

甲病毒超家族包括几个正链、植物和动物RNA病毒家族。为了研究甲型病毒超家族中病毒RNA复制的机制，构建了全长cDNA克隆，并在体外产生感染性RNA转录物。在细菌质粒中构建了小麦土传花叶病毒（日本-枥木分离株）pJS1和pJS2的RNA 1和RNA 2的cDNA克隆。在这两个克隆中，cDNA插入的5‘端被直接放置在SP6启动子的下游，3’端后面是一个独特的SpeI位点，用于质粒线性化。将pJS1和pJS2的离体转录本混合，机械接种于藜麦叶片上，接种7 ~ 10 d后，叶片上出现典型的2 ~ 3mm直径的绿斑。免疫印迹法检测到pJS1和pJS2的体外转录本也可侵染小麦植株，免疫印迹法检测到pJS1和pJS2的体外转录本可侵染小麦植株，免疫印迹法检测到pJS1和pJS2的体外转录本在未接种的小麦上部叶片中检测到该病毒的衣壳蛋白，表明该病毒具有系统性传播。在SP6或T7启动子下游的细菌质粒中构建了1956年在日本分离到的一种α病毒Sagiyama病毒的全长cDNA克隆。全长cDNA克隆pSAG2的体外转录物可感染BHK21细胞，经RNA横切后从培养基中获得野生型病毒。

The alphavirus superfamily includes several families of positive-stranded, plant and animal RNA viruses. To study mechanisms of viral RNA replication in the alphavirus superfamily, full-length cDNA clones from which infectious RNA transcripts are produced in vitro were constructed.The two full-length cDNA clones to RNA 1 and RNA 2 of soil-borne wheat mosaic virus (Japan-Tochigi isolate), pJS1 and pJS2 respectively, were constructed in a bacterial plasmid. In both clones, the 5' end of the cDNA insert was placed immediately downstream of the SP6 promoter and the 3'end was followed by a unique SpeI site for plasmid linearization. When in vitro transcripts from pJS1 and pJS2 were mixed and inoculated mechanically on Chenopodium quinoa leaves, typical chlorotic spots with 2 to 3 mm in diameter appeared on the inoculated leaves in 7 to 10 days. From the chlorotic spots, the capsid protein was detected by Western blot analysis and the infectious virus was recovered from the sap. In vitro transcripts from the pJS1 and pJS2 were also infectious to wheat plants and the capsid protein was detected from uninoculated upper leaves by Western analysis, indicating that the virus spread systemically.The full-length cDNA clones of Sagiyama virus, an alphavirus isolated in Japan in 1956, were also constructed in a bacterial plasmid downstream of the SP6 or T7 promoter. In vitro transcripts from a full-length cDNA clone pSAG2 was infectious to BHK21 cells and a virus with the wild-type phenotype was recovered from the medium after RNA transection.