Comparison of the replication properties of murine and human calicivirus RNA-dependent RNA polymerases.
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 MnCl(2) 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.
School of Biotechnology and Biomolecular Sciences, Faculty of Science, The University of New South Wales, Sydney, NSW 2052, Australia. r.bull@unsw.edu.au
Rowena A
RA
Bull
Jennifer
J
Hyde
Jason M
JM
Mackenzie
Grant S
GS
Hansman
Tomoichiro
T
Oka
Naokazu
N
Takeda
Peter A
PA
White
D000595:Amino Acid Sequence; D000818:Animals; D002139:Caliciviridae / Q000201:enzymology* / Q000502:physiology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D020539:Sequence Analysis, Protein; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism; D014779:Virus Replication
D012367:RNA, Viral; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase