| Norovirus is the main foodborne pathogen of human acute gastroenteritis,which can lead to the spread and outbreak of acute gastroenteritis worldwide and make a serious threat to human health.Due to its rapid mutation and the unavailability of susceptible cell lines and animal models,the comprehensive understanding of norovirus has been seriously hindered.There are currently no approved effective vaccines and specific antiviral drugs for norovirus.Rich genetic diversity is the key factor for its continuous transmission,and the characteristics of rapid replication and high mutation promote the continuous emergence of the new mutants.However,the effect of the difference in replication ability of different genotypes of strains on the epidemic harm level of the virus is still unclear.The RNA-dependent RNA polymerase(RdRp)of norovirus is the key enzyme involved in the viral genome and subgenomic synthesis and is a major target for the development of direct-acting antivirals for norovirus.In this study,we took the polymerase of different strains of norovirus as the research object and studied the molecular mechanism leading to the epidemic difference of different strains of human norovirus mainly from the perspective of the replication level of RdRp.Besides,a simple,sensitive,rapid,and stable method for the determination of norovirus RdRp activity was established and used for high-throughput screening of antiviral probiotics.The main results were as follows:(1)Preparation and enzymatic characterization of the RdRp of norovirus epidemic differential strainsTo study the replication level of RdRp of epidemic differential strains of norovirus,the RdRp protein of different epidemic differential strains of human norovirus was highly expressed based on the E.coli expression system,and the high purity soluble RdRp was obtained by chromatography.The in vitro RNA synthesis experiments showed that the purified RdRp had biological activity,and the catalytic activity of different strains of RdRp was different.Under the same reaction conditions,the polymerase of the epidemic strain GII.P17/GII.17-L343 produced the most double-stranded RNA products from the substrate per unit time and showed the highest enzyme activity,while the non-epidemic strain GII.P8/GII.8-L601 had the lowest enzyme activity,the RdRp activity of the recombinant strain GII.P12/GII.3-L20 was between the GII.P17/GII.17-L343 and GII.P8/GII.8-L601.The Km values of RdRp of different strains also confirmed these results,and the Km values of different strains were also different,ranging from 0.013 to 0.151 mM.Compared with other strains,the Km value of epidemic strain GII.P17/GII.17-L343 was the lowest,which was 0.013mm,indicating that it has the greatest affinity with the substrate,and the maximum reaction rate can be achieved at a lower substrate concentration.The Km of GII.P8/GII.8-L601 was the highest,which was 0.151 mM,so the affinity with the substrate was the lowest.Therefore,at the same low substrate concentration,the production of double-stranded RNA catalyzed by GII.P17/GII.17-L343 RdRp was more and showed the highest activity.Besides,by systematically evaluating the effects of reaction temperature,template,substrate,and salt concentration on RdRp activity,the optimal reaction conditions for in vitro RdRp assays of different strains were determined.The optimum reaction temperature of different norovirus RdRp was between 15?and 30? and showed the best activity when the concentration of manganese ion was 1 mM.The utilization rates of templates and substrates were different but were in general agreement with the standard Michaelis-Menten model for enzyme kinetics.(2)Study on the molecular mechanism affecting the RdRp replication rate of norovirusThe results of amino acid sequence alignment of norovirus epidemic differential strain RdRp showed that the RdRp sequences of different strains were highly similar overall,however,a small amount of amino acid substitution was found in the conserved motifs of RdRp.Whether these amino acid variations would affect the replication level of RdRp,thus leading to the difference of epidemic ability of different strains.Given the significant difference in RdRp activity between the GII.17 and GII.8 strains,we chose the RdRp of epidemic strain GII.17 and non-epidemic strain GII.8 as the research object.Through rational design of RdRp mutants,we aim to screen the key amino acid sites affecting the polymerase activity in order to study the molecular mechanism leading to the difference in activity between the two strains.A series of GII.8 RdRp mutant proteins were prepared by prokaryotic expression system,and their enzymatic activities were determined based on RNA synthesis assays.The results of enzymatic activity showed that the change of polymerase amino acid sequence did affect RdRp catalytic activity,and the key amino acid site leading to the difference in enzyme activity between norovirus epidemic strain GII.17 and non-epidemic strain GII.8 was located at 160th position.The mutation of amino acid methionine to valine(M160V)at 160 positions increased the catalytic activity of GII.8 RdRp by 43%and the mutation site was in the conserved motif F1 of the polymerase.The F motif has been shown to be mainly involved in the transfer of nucleoside triphosphate and promote the synthesis of RNA,so it has an important effect on the replication rate of RdRp.The above results suggest that the changes in the conserved amino acid sites in the RNA polymerase of human norovirus significantly affect its replication kinetics.Moreover,the replication level of different norovirus strains was correlated with its prevalence.The impact of single point mutation of RdRp on virus replication and prevalence highlighted the dynamic interaction between replication rate and virus adaptability and prevalence.(3)Screening of norovirus RdRp inhibitors based on probioticsA simple,sensitive,rapid,and stable method for the determination of norovirus polymerase activity based on fluorescent dyes was established,which can be widely used in the rapid and high-throughput screening of virus polymerase inhibitors.The experimental data obtained by the new method are stable,the reaction cost is low,and the reaction process can be monitored in real-time.Based on the new method of RdRp activity assay,the inhibitory effect of compound inhibitors on norovirus RdRp was evaluated.It was found that nucleoside and non-nucleoside inhibitors ribavirin,favipiravir,and NF023 could effectively inhibit the ab initio synthesis of RdRp in the range of micromoles,and the half-maximum inhibitory concentrations(IC50 values)were 23 ?M,59?M and 11 ?M,respectively.Besides,based on the functional microbial resources accumulated by our team,the screening of antiviral probiotics was carried out.Multiple strains of microbial inhibitors with strong inhibitory effects on norovirus RdRp were obtained from 71 probiotics.Among them,fermented Lactobacillus PV22 and Lactobacillus Plantarum PV66 had a strong inhibitory effect on RdRp,with an inhibition rate of 88%and 90%,respectively.Based on the above studies,this study systematically characterized the biochemical characteristics of RNA polymerase of human norovirus strains with different epidemic characteristics,screened out the key amino acid sites of polymerase that have important effects on the replication rate of norovirus,and perfected the evolutionary molecular mechanism of the norovirus.Based on the in vitro RdRp assay,two probiotic strains with good inhibitory effect on norovirus RdRp were obtained.The above studies can provide important theoretical support for the formulation of prevention and control strategies and the development of antiviral drugs of norovirus. |
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