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。为了研究甲病毒属病毒RNA复制的机制，构建了土传小麦花叶病毒（Japan-Tochigi isolation）RNA 1和RNA 2的全长cDNA克隆pJS 1和pJS 2。在这两个克隆中，cDNA插入片段的5 ′端直接置于SP 6启动子的下游，3 ′端之后是用于质粒线性化的独特SpeI位点。将pJS 1和pJS 2的离体转录物混合后机械接种到藜叶上，接种7 ~ 10 d后，叶片上出现直径2 ~ 3 mm的典型褪绿斑。从褪绿斑点，通过Western印迹分析检测衣壳蛋白，并从汁液中回收感染性病毒。从pJS 1和pJS 2的体外转录物也感染小麦植株，通过Western分析，从未接种的上部叶片中检测到衣壳蛋白，表明该病毒是系统传播的。在体外转录的全长cDNA克隆pSAG 2是感染BHK 21细胞和病毒的野生型表型后，从培养基中回收RNA横切。

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.