1997 Fiscal Year Final Research Report Summary
Analysis of HCV derived RNA polymerase expressed in mammalian cell live
| Project/Area Number |
07807054
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Gastroenterology
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| Research Institution | The University of Tokyo |
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Principal Investigator |
ABURATANI Hiroyuki The University of Tokyo, Dept of Genatries Assistant Professor, 医学部・付属病院, 助手 (10202657)
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| Co-Investigator(Kenkyū-buntansha) |
GUNJI Toshiake The University of Tokyo, Dept of Genatries Assistant, 医学部・付属病院, 医員
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| Project Period (FY) |
1995 – 1997
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| Keywords | Hepatitis C Virus / RNA polymerase |
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| Research Abstract |
Hepatitis C virus(HOV) is a single strand RNA virus with positive polarity. In replication process of HOV, it is assumed that NS5b protein acts as RNA-dependent RNA polymerase(RdRp), which produces negative strand RNA as replicative intermediate. From these aspects, anti-RdRp agents are expected to inhibit HCV replication. AIM To establish mammalian cell lines expressing NS5b protein with activity of RdRp reaction and screen anti-RdRp agents using these cell lines.
METHOD The GFP-NS5b expression plasmid, pEGFP-NS5b, was transfected into a mammalian cell line(CHO cells), and stable transformat expressing GFP-NS5b fusion protein in the cells was established. RdRp activity of NS5b protein expressed in CHO cell was determined by RNA-dependent RNA synthesis using DCoH RNA template (in vitro RdRp assay ; Behrens S-E.et al, EMBO J.1996) and by presence of negative strand HCV-RNA (negative strand assay ; Gunji T.et al, Arch. Virol. 1994).
RESULT
(1) The GFP-NS5b protein expressed in the CHO cell did not exert apparent RNA synthesis in vitro. However, when the GFP-NS5b protein was cleaved between GFP and NS5b protein, the sole NS5b protein retained activity for RNA-dependent RNA synthesis.
(2) Negative strand HCV RNA was detected in the cell expressing the GFP-NS5b protein. The titer of negative strand RNA was 10-100 fold less than that of positive strand RNA by end point dilution method. These results indicate that the expressed GFP-NS5b protein retains activity for synthesizing anti-sense strand RNA from positive strand RNA template.
CONCLUSION
We established the mammalian cell line (CHO cell) expressing the GFP-NS5b fusion protein which retains RNA-dependent RNA synthesis activity in vitro, Such cell line as presented here would be useful and important for screening agents inhibitting HCV infection and replication in vivo.
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