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Virus Genes

, Volume 42, Issue 1, pp 16–27 | Cite as

Comparison of the replication properties of murine and human calicivirus RNA-dependent RNA polymerases

  • Rowena A. BullEmail author
  • Jennifer Hyde
  • Jason M. Mackenzie
  • Grant S. Hansman
  • Tomoichiro Oka
  • Naokazu Takeda
  • Peter A. WhiteEmail author
Article

Abstract

The human caliciviruses (CV), norovirus (NoV) and sapovirus (SaV), are major causes of outbreak gastroenteritis worldwide. To date, the investigation of human NoV and SaV replication cycles has been impeded as neither is culturable. Consequently, the recently discovered murine NoV (MNV) has been adopted as a surrogate replication model for the human CVs. In this study, we sought to compare the biochemical properties of the MNV RNA-dependent RNA polymerase (RdRp) with related human NoV and SaV-RdRps to address the suitability of MNV as a model for the human CVs. Three human NoV-RdRps (GII.b, GII.4 and GII.7), an MNV-RdRp and two human SaV-RdRps (GI and GII) were overexpressed in Escherichia coli, purified and their enzymatic activity and fidelity compared. Despite ~70% amino acid variation between the RdRp from the two different CV genera, the majority of the physiological characteristics of the RdRps were similar. All RdRps exhibited co-operative dimerisation and had optimal activity at 25°C, a pH range between 7 and 8, required 2–5 mM MnCl2 and were inhibited with increasing NaCl concentrations. We observed RdRp activity at temperatures as low as 5°C and as high as 65°C. Using an in vitro fidelity assay, similar mutation rates were observed for the separate RdRps (1 × 10−4–1 × 10−5). This is the first report to compare the physiological, biochemical and mutational properties of the MNV-RdRp to those of the human CV-RdRps and it suggests that MNV may be directly applicable to the study of human NoV.

Keywords

Norovirus Sapovirus Gastroenteritis RNA-dependent RNA polymerase 

Notes

Acknowledgments

RAB was supported by a National Health and Medical Research Council Postdoctoral Fellowship. We thank Professor William Rawlinson for useful discussions and Dr Leighton Clancy for technical support.

Supplementary material

11262_2010_535_MOESM1_ESM.tif (726 kb)
Fig. S1 SDS-PAGE analysis of purified recombinant RdRps expressed in E. coli. RdRps were expressed with a C-terminal hexahistidine tag and purified in a nickel affinity column with increasing concentrations of imidazole. Three microlitres of purified enzyme was loaded into each lane. Lane 1, Precision Plus Standards Protein marker, kDa (Biorad); lane 2, NoV GII.4-RdRp; lane 3, NoV GII.b-RdRp; lane 4, NoV GII.7-RdRp; lane 5, NoV GII.7GAA-RdRp; lane 6, MNV-RdRp; lane 7, SaV GI-RdRp; lane 8, SaV GII-RdRp; Lane 9, HCV3a-RdRp.(TIFF 726 kb)

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Rowena A. Bull
    • 1
    • 2
    Email author
  • Jennifer Hyde
    • 3
  • Jason M. Mackenzie
    • 3
  • Grant S. Hansman
    • 4
  • Tomoichiro Oka
    • 4
  • Naokazu Takeda
    • 4
  • Peter A. White
    • 1
    Email author
  1. 1.School of Biotechnology and Biomolecular Sciences, Faculty of ScienceThe University of New South Wales (UNSW)SydneyAustralia
  2. 2.School of Medical Sciences, Faculty of ScienceThe University of New South Wales (UNSW)SydneyAustralia
  3. 3.Department of MicrobiologyLa Trobe UniversityBundooraAustralia
  4. 4.Department of Virology IINational Institute of Infectious DiseasesTokyoJapan

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