Function of NS2B for NS3
   protease activation of
Japanese encephalitis virus

               Mr.Chakard Chalayut
     Ad...
Japanese Encephalitis Virus
          (JEV)
  Culex tritaeniorhynchus.




Source: fehd.gov.hk          Source: news.bbc.c...
Japanese Encephalitis Virus
          (JEV)




   Source : vietnammedicalpractice.com
Japanese Encephalitis Virus
           (JEV)

  50,000 Cases
  10,000
30% fatality rate
 Source: medwork84.com



        ...
Prevention and treatment of
          JEV disease


       Drug                No drug exist


Vaccine development       A...
Molecular biology of Japanese Encephalitis Virus




               Source : molecular-virology.uni-hd.de
The NS2B
  • 130 aa                         Hypothetical model
  • activating domain              NS2B-NS3 complex
      c...
The NS3

                                 Protease

                                  NTPase


                           ...
The NS3 protease
       Complexation with NS2B cofactor

        •NS3 serine protease
        domain 20 of thechange pocke...
• The result shown the slightly differences inby
  Some physico-chemical properties shared
  all the proteases.
  biochemi...
Homology Modelling of
   NS3 protease
Homology Modelling of
   NS3 protease



WNV      JEV       DEN
• The NS2B-NS3 can adopt two distinctfit mechanism.
  NS2B-NS3 WNV shown the induced conformation.
• Two component of protease has a substrate
  specificity.
• Two component of protease has a unique
  activation mechanism....
• To investigate the function determination in
  the JEV NS2B for the activation of NS3
  protease.
Method 1.and mutation forms
Expression and purification of deleted
               of NS2B-NS3 protease

        Full length...
Method 1.
Result




Lane 1- 5 = Washing Fraction with 100 mM Imidazole
Lane 6 -10 = Eluted Fraction with 400 mM Imidazole
Method II
Trans-cleavage of fluorogenie peptide substrate


           NS2B-NS3 protease

        Incubate with GKR-AMC


 ...
Result
Method III
   Molecular Modelling of a JEV NS2B-NS3 complex

         JEV NS2B and NS3 protease protein

      Based on WN...
Result
Result




Lane 1- 5 = Washing Fraction with 100 mM Imidazole
Lane 6 -10 = Eluted Fraction with 400 mM Imidazole
Discussion
• Ser46 and Ile60 are essential region of JEV
  NS2B for activation of NS3 protease.
• Alanine substitution dem...
Discussion
• Residues Lys78 to Leu87 of JEV NS2B may
  involved in the formation of the active site in
  the NS2B-NS3 prot...
• Due to the substrate specificity of the
  NS2B-NS3 protease.
  • Can we make a very specific anti-viral drug
    to Flaviv...
Thank you for your attention.
Seminar Final.Key
Seminar Final.Key
Seminar Final.Key
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Seminar Final.Key

  1. 1. Function of NS2B for NS3 protease activation of Japanese encephalitis virus Mr.Chakard Chalayut Advisor: Assist.Prof Gerd Katzenmeier Laboratory of Molecular virology Institute of Molecular Biology & Genetics
  2. 2. Japanese Encephalitis Virus (JEV) Culex tritaeniorhynchus. Source: fehd.gov.hk Source: news.bbc.co.uk
  3. 3. Japanese Encephalitis Virus (JEV) Source : vietnammedicalpractice.com
  4. 4. Japanese Encephalitis Virus (JEV) 50,000 Cases 10,000 30% fatality rate Source: medwork84.com Source: cdc.gov
  5. 5. Prevention and treatment of JEV disease Drug No drug exist Vaccine development Available vaccine Elimination of mosquitoes Mosquitoes control breeding places
  6. 6. Molecular biology of Japanese Encephalitis Virus Source : molecular-virology.uni-hd.de
  7. 7. The NS2B • 130 aa Hypothetical model • activating domain NS2B-NS3 complex central hydrophilic region (Falgout et al, 1993) • 3 membrane spanning parts hydrophobicity plot ฺBrinkworth et al, 1999 51 DMWLERAADISWEMDAAITGSSRRLDVKLDDDGDFHLIDDPGVP 95
  8. 8. The NS3 Protease NTPase •Chymotrypsin-like fold 2-β barrel Helicase RNA domains •Inactive alone Theoretical model from PDB •Enzyme’s pocket is small 2I84
  9. 9. The NS3 protease Complexation with NS2B cofactor •NS3 serine protease domain 20 of thechange pocket kDa enzyme conformational alteration •catalytic residues binding site additional substrate His51, Asp75, Ser135
  10. 10. • The result shown the slightly differences inby Some physico-chemical properties shared all the proteases. biochemical properties.
  11. 11. Homology Modelling of NS3 protease
  12. 12. Homology Modelling of NS3 protease WNV JEV DEN
  13. 13. • The NS2B-NS3 can adopt two distinctfit mechanism. NS2B-NS3 WNV shown the induced conformation.
  14. 14. • Two component of protease has a substrate specificity. • Two component of protease has a unique activation mechanism. • The NS3 protease share the similar structure in the Flavivirus group but are different in cofactor binding.
  15. 15. • To investigate the function determination in the JEV NS2B for the activation of NS3 protease.
  16. 16. Method 1.and mutation forms Expression and purification of deleted of NS2B-NS3 protease Full length NS2B and NS3 protease PCR to amply deleted and mutation forms of NS2B Clone into vector Expression and Purification SDS-PAGE and Western Blot
  17. 17. Method 1.
  18. 18. Result Lane 1- 5 = Washing Fraction with 100 mM Imidazole Lane 6 -10 = Eluted Fraction with 400 mM Imidazole
  19. 19. Method II Trans-cleavage of fluorogenie peptide substrate NS2B-NS3 protease Incubate with GKR-AMC Measured the florescence Change
  20. 20. Result
  21. 21. Method III Molecular Modelling of a JEV NS2B-NS3 complex JEV NS2B and NS3 protease protein Based on WNV crystallographic structure Perform the structural modelling by using Swiss modeling workstation & MEDock program
  22. 22. Result
  23. 23. Result Lane 1- 5 = Washing Fraction with 100 mM Imidazole Lane 6 -10 = Eluted Fraction with 400 mM Imidazole
  24. 24. Discussion • Ser46 and Ile60 are essential region of JEV NS2B for activation of NS3 protease. • Alanine substitution demonstrate the functional conformation of Trp53, Glu55 and Arg56 in JEV NS2B for the cleavage ability of NS2B-NS3 protease. • Residues Ala67 to Asp76 of JEV NS2B suggested to provide the additional β- strands to stabilize the fold of NS3 protease.
  25. 25. Discussion • Residues Lys78 to Leu87 of JEV NS2B may involved in the formation of the active site in the NS2B-NS3 protease.
  26. 26. • Due to the substrate specificity of the NS2B-NS3 protease. • Can we make a very specific anti-viral drug to Flavivirus ? The pan-Flavivirus NS3 protease drugs may be developed for Flaviviral diseases.
  27. 27. Thank you for your attention.

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