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Flavivirus Group meeting

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Flavivirus Group meeting at KU

Flavivirus Group meeting at KU

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  • 1. Cloning, Expression, Purification and Enzymological Characterization of NS2B(H)/NS3 protease of Japanese Encephalitis Virus. Chakard Chalayut Advisor: Assit. Prof. Gerd Katzenmier Laboratory of Molecular Virology Institute of Molecular Biology & Genetics 1
  • 2. Japanese Encephalitis Virus Flaviviridae family Dengue (Den) West nile virus (WNV) Yellow fever virus (YFV) Japanese Encephalitis Virus (JEV) ETC... 2
  • 3. Japanese Encephalitis Virus Flaviviridae family Dengue (Den) West nile virus (WNV) Yellow fever virus (YFV) Japanese Encephalitis Virus (JEV) ETC... 2
  • 4. Japanese Encephalitis Virus Mosquito-borne neurotropic flavivirus causes severe central nerve system diseases Divided into 4 Genotypes. For unknown reason genotypes 3 is the most outbreak. Culex tritaeniorhynchus is the important vector. http://www.fehd.gov.hk 3
  • 5. Japanese Encephalitis Virus http://www.fehd.gov.hk 3
  • 6. Japanese Encephalitis Virus 4
  • 7. Japanese Encephalitis Virus J E V c a u s e s s e v e re central nerve system diseases such as poliomyelitis- like acute flaccid paralysis, aseptic m e n i n g i t i s a n d http://www.wonder.cdc.gov encephalitis 4
  • 8. Japanese Encephalitis Virus http://www.cdc.gov 4
  • 9. Japanese Encephalitis Virus 50,000 cases/year 4
  • 10. Japanese Encephalitis Virus 10,000 Death Cases/year 4
  • 11. Japanese Encephalitis Virus 30% fatality rate 4
  • 12. Prevention and treatment of JEV disease 5
  • 13. Prevention and treatment of JEV disease Drug 5
  • 14. Prevention and treatment of JEV disease Drug No drug exist Vaccine development 5
  • 15. Prevention and treatment of JEV disease Drug No drug exist Vaccine development Available vaccine Mosquitoes control 5
  • 16. Prevention and treatment of JEV disease Drug No drug exist Vaccine development Available vaccine Mosquitoes control Elimination of mosquitoes breeding places 5
  • 17. Molecular biology of Japanese Encephalitis Virus www. molecular-virology.uni-hd.de 6
  • 18. Molecular biology of Japanese Encephalitis Virus www. molecular-virology.uni-hd.de 6
  • 19. Molecular biology of Japanese Encephalitis Virus www. molecular-virology.uni-hd.de 6
  • 20. Molecular biology of Japanese Encephalitis Virus www. molecular-virology.uni-hd.de 6
  • 21. The NS2B 130 aa activating domain central hydrophilic region (Falgout et al, 1993) 3 membrane spanning parts 7
  • 22. The NS2B 130 aa activating domain central hydrophilic region (Falgout et al, 1993) 3 membrane spanning parts Hypothetical model NS2B-NS3 complex 7
  • 23. The NS2B 130 aa activating domain central hydrophilic region (Falgout et al, 1993) 3 membrane spanning parts Hypothetical model NS2B-NS3 complex 7
  • 24. The NS2B Hypothetical model NS2B-NS3 complex Brinkworth et al, 1999 7
  • 25. The NS2B Hypothetical model NS2B-NS3 complex 58 VSGKATDMWLERAADISWEMDAAITGSSRRLDVKLDDDGDFHLIDDPGVP 101 Brinkworth et al, 1999 7
  • 26. The NS3 Theoretical model from PDB 2I84 8
  • 27. The NS3 Protease Theoretical model from PDB 2I84 8
  • 28. The NS3 Protease NTPase Theoretical model from PDB 2I84 8
  • 29. The NS3 Protease NTPase RNA Helicase Theoretical model from PDB 2I84 8
  • 30. The NS3 Chymotrypsin-like fold 2-β barrel domains Inactive alone Enzyme’s pocket is small 8
  • 31. The NS3 protease 9
  • 32. The NS3 protease Complexation with NS2B cofactor conformational change alteration of the enzyme pocket additional substrate binding site 9
  • 33. The NS3 protease NS3 serine protease domain 20 kDa catalytic residues His51, Asp75, Ser135 9
  • 34. Lin. C W et al,2007 10
  • 35. Ser46 to Ile60 were essential region required for NS3 protease activity. Ala substition of Trp50, Glu55, and Arg56 in NS2B shown significantly reduced NS3 protease activity. Lin. C W et al,2007 10
  • 36. Compare to the structure JEV WNV JEV homology model from Den Den 2 11
  • 37. Report From Novartis Den 2 protease can be activated by Den1,2,3 ,WNV and YFV NS2B(H) From Jan L.R. et al 1995 Den 4 protease can’t be activated by JEV NS2B(H) but JEV protease can activated by Den 4 NS2B(H) 12
  • 38. From the different on NS2B(H)-NS3 protease complex structure and cofactor specificity.Can we do the NS2B cofactor analog as an universal drug for flavivirus? 13

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