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Identification and characterization of RNA-dependent RNA polymerase activity in recombinant Japanese encephalitis virus NS5 protein.

Identification and characterization of RNA-dependent RNA polymerase activity in recombinant Japanese encephalitis virus NS5 protein. Research Abstract Details 

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  • Identification and characterization of RNA-dependent RNA polymerase activity in recombinant Japanese encephalitis virus NS5 protein. Abstract Text:

    f yuF Yu,f hasebeF Hasebe,s inoueS Inoue,e g m mathengeE G M Mathenge,k moritaK Morita,

    The complete nonstructural NS5 gene of Japanese encephalitis virus (JEV) was amplified and cloned into an expression vector. The NS5 protein was expressed in Escherichia coli and purified by His-tag based affinity chromatography. This recombinant NS5 protein exhibited RNA-dependent RNA polymerase (RdRp) activity in vitro in the absence of other viral or cellular factors. The RNA polymerase activity was dependent on divalent cations, and Mn(2+) was found to be 20 times more effective than Mg(2+) in coordinating the catalytic reaction of RdRp, while Ca(2+) inhibited enzyme activity. The optimal reaction conditions for the in vitro RdRp reaction were established. Characterization of the RdRp reaction products demonstrated that the JEV NS5 protein can initiate RNA synthesis through a de novo initiation mechanism in our in vitro reaction system. Comparing the efficiency of different RNA templates, we found that JEV NS5 protein was more efficient in using negative-strand RNA templates, indicating that the JEV NS5 protein is involved in regulating the ratio of positive- to negative-strand RNA. Four amino acid sequence motifs crucial for RdRp activity were also identified using site-directed mutagenesis analysis. All substitutions of the conserved residues within these motifs led to a complete inactivation or severe loss of enzyme activity.

    Identification and characterization of RNA-dependent RNA polymerase activity in recombinant Japanese encephalitis virus NS5 protein. Publishing Authors By Initials

    f yuF Yu,f hasebeF Hasebe,s inoueS Inoue,eg mathengeEG Mathenge,k moritaK Morita,

    For similar proteins: viral proteins: viral nonstructural proteins research abstracts see: proteins: viral proteins: viral nonstructural proteins research

    PUBMED ID PMID:

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    Identification and characterization of RNA-dependent RNA polymerase activity in recombinant Japanese encephalitis virus NS5 protein. Journal Published:

    PUBLICATION TYPE: Research Support, Non-U.S. Gov

    Journal: Archives of virology

    VOLUME: 152

    Page Numbers: 1859-69

    Journal Abbreviation: Arch. Virol.

    ISSN: 0304-8608

    DAY: 18

    MONTH: 06

    YEAR: 2007

    Identification and characterization of RNA-dependent RNA polymerase activity in recombinant Japanese encephalitis virus NS5 protein. Information

    Number of References:

    LANGUAGE: eng

    NlmUniqueID: 7506870

    Identification and characterization of RNA-dependent RNA polymerase activity in recombinant Japanese encephalitis virus NS5 protein. Keywords Mesh Terms:

    KEYWORDS: Viral Nonstructural Proteins

    MESH TERMS: metabolism

    Chemical & Substance for Abstract: Identification and characterization of RNA-dependent RNA polymerase activity in recombinant Japanese encephalitis virus NS5 protein. Information

    Substance Name: RNA Replicase

    Registry Number: EC 2.7.7.48

    Grant and Affiliation Information for Identification and characterization of RNA-dependent RNA polymerase activity in recombinant Japanese encephalitis virus NS5 protein.

    AFFILIATION: Center for International Collaborative Research, Nagasaki University, Nagasaki, Japan. yufuxun1@nagasaki-u.ac.jp

    Country: Austria

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    MEDLINETA: Arch Virol

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