Expression and characterization of RNA polymerase of the norovirus recombinant GII.P12/GII.3 strain isolated in China

Abstract:

[Background] Norovirus is the primary foodborne pathogen causing acute gastroenteritis in humans.There are currently no licensed vaccines and drugs available for this virus.RNA-dependent RNA polymerase (RdRp) of norovirus is a major target for the development of antivirals.[Objective] This study intended to express the RdRp of norovirus recombinant GII.P12/GII.3 and systematically study its replication characteristics.[Methods] The high-purity soluble RdRp of GII.P12/GII.3 was expressed and purified in Escherichia coli.The effects of temperature,template,substrate,and salt concentration on the function of RdRp were determined by in vitro RNA synthesis experiments.[Results] The RdRp of GII.P12/GII.3 showed the highest activity at 30℃ and 1 mmol/L MnCl₂.The K_m values of RdRp binding substrate GTP and template polyC were 79.0µmol/L and 10.6µg/mL,respectively.In vitro inhibitory assays indicated that ribavirin,favipiravir,and NF023 in the micromolar range inhibited RdRp,with half-maximal inhibitory concentrations of 23µmol/L,59µmol/L,and 11µmol/L,respectively.[Conclusion] This study firstly reported the RdRp properties of norovirus recombinant genotype GII.P12/GII.3.Fluorescence-based enzyme activity tests showed that RdRp had catalytic activity in vitro,which provided technical and theoretical support for the screening of norovirus RdRp inhibitors and the treatment of norovirus infections.

Key words:norovirus;RNA-dependent RNA polymerase;GII.P12/GII.3;inhibitors