Characterization of norovirus 3Dpol RNA-dependent RNA polymerase activity and initiation of RNA synthesis.
Norovirus (NV) 3D(pol) is a non-structural protein predicted to play an essential role in the replication of the NV genome. In this study, the characteristics of NV 3D(pol) activity and initiation of RNA synthesis have been examined in vitro. Recombinant NV 3D(pol), as well as a 3D(pol) active-site mutant were expressed in Escherichia coli and purified. NV 3D(pol) was able to synthesize RNA in vitro and displayed flexibility with respect to the use of Mg(2+) or Mn(2+) as a cofactor. NV 3D(pol) yielded two different products when incubated with synthetic RNA in vitro: (i) a double-stranded RNA consisting of two single strands of opposite polarity or (ii) the single-stranded RNA template labelled at its 3' terminus by terminal transferase activity. Initiation of RNA synthesis occurred de novo rather than by back-priming, as evidenced by the fact that the two strands of the double-stranded RNA product could be separated, and by dissociation in time-course analysis of terminal transferase and RNA synthesis activities. In addition, RNA synthesis was not affected by blocking of the 3' terminus of the RNA template by a chain terminator, sustaining de novo initiation of RNA synthesis. NV 3D(pol) displays in vitro properties characteristic of RNA-dependent RNA polymerases, allowing the implementation of this in vitro enzymic assay for the development and validation of antiviral drugs against NV, a so far non-cultivated virus and an important human pathogen.
Rohayem
Jacques
J
Institut für Virologie, The Calicilab, Medizinisch-Theoretisches Zentrum, Fiedlerstraße 42, D-01307 Dresden, Germany.
Jäger
Katrin
K
Institut für Virologie, The Calicilab, Medizinisch-Theoretisches Zentrum, Fiedlerstraße 42, D-01307 Dresden, Germany.
Robel
Ivonne
I
Institut für Virologie, The Calicilab, Medizinisch-Theoretisches Zentrum, Fiedlerstraße 42, D-01307 Dresden, Germany.
Scheffler
Ulrike
U
Institut für Virologie, The Calicilab, Medizinisch-Theoretisches Zentrum, Fiedlerstraße 42, D-01307 Dresden, Germany.
Temme
Achim
A
Institut für Immunologie, Medizinisch-Theoretisches Zentrum, Fiedlerstraße 42, D-01307 Dresden, Germany.
Rudolph
Wolfram
W
Institut für Virologie, The Calicilab, Medizinisch-Theoretisches Zentrum, Fiedlerstraße 42, D-01307 Dresden, Germany.
D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D002413:Cations, Divalent / Q000494:pharmacology; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D029322:Norovirus / Q000201:enzymology* / Q000378:metabolism / Q000472:pathogenicity; D012330:RNA, Double-Stranded / Q000096:biosynthesis / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D002413:Cations, Divalent; D004279:DNA, Viral; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D011994:Recombinant Proteins; D012324:RNA-Dependent RNA Polymerase