Cloning, Expression, Purification and
Enzymological Characterization of
NS2B(H)/NS3 protease of
Japanese Encephalitis Virus...
Japanese Encephalitis Virus
             Flaviviridae family
Dengue (Den)

West nile virus (WNV)
Yellow fever virus (YFV)
...
Japanese Encephalitis Virus
             Flaviviridae family
Dengue (Den)

West nile virus (WNV)
Yellow fever virus (YFV)
...
Japanese Encephalitis Virus
Mosquito-borne neurotropic flavivirus
  causes severe central nerve system diseases
Divided int...
Japanese Encephalitis Virus




       http://www.fehd.gov.hk

                                3
Japanese Encephalitis Virus




                              4
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
...
Japanese Encephalitis Virus




        http://www.cdc.gov

                              4
Japanese Encephalitis Virus



 50,000 cases/year



                              4
Japanese Encephalitis Virus



10,000 Death Cases/year



                              4
Japanese Encephalitis Virus



  30% fatality rate


                              4
Prevention and treatment of
JEV disease




                              5
Prevention and treatment of
JEV disease
     Drug




                              5
Prevention and treatment of
JEV disease
       Drug           No drug exist

Vaccine development




                     ...
Prevention and treatment of
JEV disease
       Drug            No drug exist

Vaccine development   Available vaccine

 Mo...
Prevention and treatment of
JEV disease
       Drug            No drug exist

Vaccine development   Available vaccine

 Mo...
Molecular biology of
Japanese Encephalitis Virus




                 www. molecular-virology.uni-hd.de
                  ...
Molecular biology of
Japanese Encephalitis Virus




                 www. molecular-virology.uni-hd.de
                  ...
Molecular biology of
Japanese Encephalitis Virus




                 www. molecular-virology.uni-hd.de
                  ...
Molecular biology of
Japanese Encephalitis Virus




                 www. molecular-virology.uni-hd.de
                  ...
The NS2B
 130 aa
  activating domain central hydrophilic region
 (Falgout et al, 1993)
 3 membrane spanning parts




    ...
The NS2B
 130 aa
  activating domain central hydrophilic region
 (Falgout et al, 1993)
 3 membrane spanning parts        H...
The NS2B
 130 aa
  activating domain central hydrophilic region
 (Falgout et al, 1993)
 3 membrane spanning parts        H...
The NS2B


                         Hypothetical model
                         NS2B-NS3 complex




Brinkworth et al, 199...
The NS2B


                                    Hypothetical model
                                    NS2B-NS3 complex



...
The NS3




 Theoretical model from PDB
             2I84
                              8
The NS3
                              Protease




 Theoretical model from PDB
             2I84
                         ...
The NS3
                              Protease

                                NTPase




 Theoretical model from PDB
   ...
The NS3
                              Protease

                                 NTPase




                              ...
The NS3




       Chymotrypsin-like fold
        2-β barrel domains
          Inactive alone
      Enzyme’s pocket is sma...
The NS3 protease




                   9
The NS3 protease
    Complexation with NS2B cofactor




                   conformational change
                   alter...
The NS3 protease

            NS3 serine protease
            domain 20 kDa
            catalytic residues His51,
        ...
Lin. C W et al,2007
                      10
Ser46 to Ile60 were essential region required for NS3
protease activity.
Ala substition of Trp50, Glu55, and Arg56 in NS2B...
Compare to the structure



JEV                                   WNV




JEV homology model from Den   Den 2
            ...
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 ...
From the different on NS2B(H)-NS3 protease complex
structure and cofactor specificity.Can we do the NS2B
cofactor analog as...
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Flavivirus Group meeting

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Flavivirus Group meeting

  1. 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. 2. Japanese Encephalitis Virus Flaviviridae family Dengue (Den) West nile virus (WNV) Yellow fever virus (YFV) Japanese Encephalitis Virus (JEV) ETC... 2
  3. 3. Japanese Encephalitis Virus Flaviviridae family Dengue (Den) West nile virus (WNV) Yellow fever virus (YFV) Japanese Encephalitis Virus (JEV) ETC... 2
  4. 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. 5. Japanese Encephalitis Virus http://www.fehd.gov.hk 3
  6. 6. Japanese Encephalitis Virus 4
  7. 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. 8. Japanese Encephalitis Virus http://www.cdc.gov 4
  9. 9. Japanese Encephalitis Virus 50,000 cases/year 4
  10. 10. Japanese Encephalitis Virus 10,000 Death Cases/year 4
  11. 11. Japanese Encephalitis Virus 30% fatality rate 4
  12. 12. Prevention and treatment of JEV disease 5
  13. 13. Prevention and treatment of JEV disease Drug 5
  14. 14. Prevention and treatment of JEV disease Drug No drug exist Vaccine development 5
  15. 15. Prevention and treatment of JEV disease Drug No drug exist Vaccine development Available vaccine Mosquitoes control 5
  16. 16. Prevention and treatment of JEV disease Drug No drug exist Vaccine development Available vaccine Mosquitoes control Elimination of mosquitoes breeding places 5
  17. 17. Molecular biology of Japanese Encephalitis Virus www. molecular-virology.uni-hd.de 6
  18. 18. Molecular biology of Japanese Encephalitis Virus www. molecular-virology.uni-hd.de 6
  19. 19. Molecular biology of Japanese Encephalitis Virus www. molecular-virology.uni-hd.de 6
  20. 20. Molecular biology of Japanese Encephalitis Virus www. molecular-virology.uni-hd.de 6
  21. 21. The NS2B 130 aa activating domain central hydrophilic region (Falgout et al, 1993) 3 membrane spanning parts 7
  22. 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. 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. 24. The NS2B Hypothetical model NS2B-NS3 complex Brinkworth et al, 1999 7
  25. 25. The NS2B Hypothetical model NS2B-NS3 complex 58 VSGKATDMWLERAADISWEMDAAITGSSRRLDVKLDDDGDFHLIDDPGVP 101 Brinkworth et al, 1999 7
  26. 26. The NS3 Theoretical model from PDB 2I84 8
  27. 27. The NS3 Protease Theoretical model from PDB 2I84 8
  28. 28. The NS3 Protease NTPase Theoretical model from PDB 2I84 8
  29. 29. The NS3 Protease NTPase RNA Helicase Theoretical model from PDB 2I84 8
  30. 30. The NS3 Chymotrypsin-like fold 2-β barrel domains Inactive alone Enzyme’s pocket is small 8
  31. 31. The NS3 protease 9
  32. 32. The NS3 protease Complexation with NS2B cofactor conformational change alteration of the enzyme pocket additional substrate binding site 9
  33. 33. The NS3 protease NS3 serine protease domain 20 kDa catalytic residues His51, Asp75, Ser135 9
  34. 34. Lin. C W et al,2007 10
  35. 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. 36. Compare to the structure JEV WNV JEV homology model from Den Den 2 11
  37. 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. 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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