我国诺如病毒易重组基因型GII.P12/GII.3毒株RNA聚合酶的表达与功能表征

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

摘要:

【背景】诺如病毒是引起人类急性胃肠炎的主要食源性病原体。目前尚无获批的诺如病毒疫苗和药物，诺如病毒RNA依赖性RNA聚合酶(RdRp)是当前抗诺如病毒药物开发的主要靶点。【目的】表达我国诺如病毒易重组基因型GII.P12/GII.3毒株的RdRp并系统地表征其复制特征。 【方法】基于大肠杆菌系统表达并纯化得到高纯度可溶性的GII.P12/GII.3诺如病毒RdRp蛋白，通过体外RNA合成实验确定温度、模板、底物和盐浓度对RdRp功能的影响。【结果】GII.P12/GII.3诺如病毒的RdRp在30 ℃、1 mmol/L氯化锰条件下酶活性最佳。酶动力学实验表明底物GTP和模板polyC的K_m值分别为79.0µmol/L和10.6 µg/mL。体外抑制试验表明，RdRp在微摩尔范围内可被利巴韦林、法维拉韦和NF023有效抑制，半数最大抑制浓度分别为23、59和11 µmol/L。 【结论】首次表征了诺如病毒易重组基因型GII.P12/GII.3毒株RdRp的酶学特性。基于荧光的酶活性测试表明，RdRp在体外具有催化活性，这为诺如病毒RdRp抑制剂的筛选及诺如病毒感染的治疗提供了良好的技术和理论支持。

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.