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.