my seminar 1 slide

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. Japanese Encephalitis Virus
(JEV)
Culex tritaeniorhynchus.
Source: fehd.gov.hk Source: news.bbc.co.uk

3. Japanese Encephalitis Virus
(JEV)
Source : vietnammedicalpractice.com

4. Japanese Encephalitis Virus
(JEV)
50,000 Cases
10,000
30% fatality rate
Source: medwork84.com
Source: cdc.gov

5. Prevention and treatment of
JEV disease
Drug No drug exist
Vaccine development Available vaccine
Elimination of mosquitoes
Mosquitoes control breeding places

6. Molecular biology of Japanese Encephalitis Virus
Source : molecular-virology.uni-hd.de

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. 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. 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

11. • The result shown the slightly differences inby
Some physico-chemical properties shared
all the proteases.
biochemical properties.

12. Homology Modelling of
NS3 protease

13. Homology Modelling of
NS3 protease
WNV JEV DEN

16. • The NS2B-NS3 can adopt two distinctfit mechanism.
NS2B-NS3 WNV shown the induced conformation.

17. • 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.

18. • To investigate the function determination in
the JEV NS2B for the activation of NS3
protease.

19. 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

20. Method 1.

21. Result
Lane 1- 5 = Washing Fraction with 100 mM Imidazole
Lane 6 -10 = Eluted Fraction with 400 mM Imidazole

22. Method II
Trans-cleavage of fluorogenie peptide substrate
NS2B-NS3 protease
Incubate with GKR-AMC
Measured the florescence Change

23. Result

24. 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

25. Result

26. Result
Lane 1- 5 = Washing Fraction with 100 mM Imidazole
Lane 6 -10 = Eluted Fraction with 400 mM Imidazole

27. 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.

28. Discussion
• Residues Lys78 to Leu87 of JEV NS2B may
involved in the formation of the active site in
the NS2B-NS3 protease.

29. • 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.

30. Thank you for your attention.