Study On The Crystal Structure And Enzyme Function Of Ilheus Virus Helicase And Antiviral Drug Screening

Objective:The Ilheus virus（ILHV）is an encephalitis associated arthropod-borne flavivirus.It was first identified in Ilheus City in the northeast Brazil and has since spread around the world.No specific vaccines or drugs are currently available for the treatment of ILHV infections.The ILHV helicase,like other flavivirus helicases,possesses 5’-triphosphatase activity.This allows it to perform ATP hydrolysis to generate energy and to sustain double-stranded RNA’s unwinding during ILHV genome replication.Thus,ILHV helicase is an ideal target for antiviral drug designing.This study aimed to resolute the crystal structure of ILHV helicase and to analyze the enzyme function,and based on these studies,to screen inhibitors of the helicase.The study may provide a theoretical basis for the designing of anti-Ilheus virus drugs.Methods:In this study,we resolved the crystal structure of ILHV helicase protein using structural biology related methods.By molecular docking of ATP and Mn²⁺with ILHV helicase,the amino acid residues that are crucial to the ATP hydrolysis process of helicase were identified,and their important roles in the maintenance of the helicase activity were confirmed by site-directed mutagenesis and ATPase activity assay.By targeting these key amino acid sites,we coupled two small molecules to these sites by molecular docking,and found these two molecules could bind to the active pocket of the helicase.Results:We resolved the crystal structure of the ILHV helicase at a 1.75-（?）resolution.We then conducted molecular docking of ATP-Mn²⁺to the ILHV helicase.Comparisons with related flavivirus helicases indicated that both the NTP and the RNA-ILHV helicase binding sites were conserved across intra-genus species,suggesting that ILHV helicase adopts an identical mode in recognizing ATP/Mn²⁺.However,the P-loop in the active site showed a distinctive conformation,reflecting a different local structural rearrangement.The enzyme activity of ILHV helicase was also characterized,which showed a relatively lower activity than that of the DENV,ZIKV,MVEV,and ALSV helicases.Structure-guided mutagenesis revealed that R26A,E110A,and Q280A greatly reduced the ATPase activity of the helicase.Moreover,we docked two small molecule inhibitors of DENV helicase（ST-610 and suramin）to the ILHV helicase,and found that these two molecules had the potential to inhibit the activity of ILHV helicase as well.Conclusion:The study resolved the crystal structure of the ILHV helicase at a high-resolution,identified three key amino acids responsible for the ATPase activity of the helicase,and by molecular docking,screened out two potential inhibitors（ST-610 and suramin）of the helicase.The study may provide useful information to the future development of antiviral drugs targeting ILHV.