Crystal Structure Of RNA Helicase From Saint Louis Encephalitis Virus And Screening Of Its Inhibitors

Objective:St.Louis encephalitis virus(SLEV),a member of flaviviridae family,has been widely transmitted through arthropods in the Americas since the first patient appeared in the 20 th century.The symptoms of infection may include low fever,memory loss,and meningoencephalitis,etc.It is therefore extremely urgent to find treatments for SLEV.The nonstructural protein NS3 of SLEV plays an essential role in virus replication,like that of other flaviviruses.The c-terminal domain of NS3 are involved in helicase function as well as nucleoside triphosphatase and RNA triphosphatase activities.In addition,RNA helicase is required for replication of SLEV to open the hydrogen bond between double stranded RNA.This makes NS3 helicase an attractive drug target for treating viral infections.Methods:The SLEV helicase gene was cloned into pET-32M-3C vector and transformed into E.coli for protein expression.The target protein was purified through three chromatography steps: affinity chromatography,ion exchange chromatography,and gel filtration chromatography.The SLEV helicase crystals were obtained via the hanging-drop vapor diffusion method.The crystal structures were determined using X-ray diffraction.The virtual screening method was used to find the potential inhibitors of SLEV helicase.Results:In addition to expressing and purifying the SLEV helicase protein,the crystal of the helicase was also obtained.SLEV helicase structural information was obtained by X-ray diffraction based on the crystal structure.The structure and sequence were compared with that of other flavivirus helicases and the critical amino acids of ATP binding sites were known.Inhibitors specifically binding to SLEV helicase were successfully identified through virtual screening,which can competitively bind the ATP binding sites,inhibiting helicase function and viral gene replication as well.Conclusion:In summary,we solved the crystal structure of SLEV helicase,obtained 26 SLEV inhibitors targeting SLEV helicase from 11061 compounds in the Apex Bio compound library through virtual screening technology,which may provide new theoretical guidance for the development of anti-flavivirus drugs.