1997 Fiscal Year Final Research Report Summary
Mechanisms of viral RNA replication in the Sindbis-virus superfamily
| Project/Area Number |
08457092
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Virology
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| Research Institution | The University of Tokyo |
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Principal Investigator |
SHIRAKO Yukio Asian Natural Environmental Science Center, Professor, アジア生物資源環境研究センター, 教授 (90143023)
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| Project Period (FY) |
1996 – 1997
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| Keywords | RNA virus / Virus Replication / RNA polymerase / Viral genome analysis / infectious cDNA clone |
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| Research Abstract |
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.
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