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。

关键词:诺如病毒;RNA依赖性RNA聚合酶;GII.P12/GII.3;抑制剂

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