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
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Japanese Encephalitis Virus
Flaviviridae family
Dengue (Den)
West nile virus (WNV)
Yellow fever virus (YFV)
Japanese Encephalitis Virus (JEV)
ETC...
2

Japanese Encephalitis Virus
Flaviviridae family
Dengue (Den)
West nile virus (WNV)
Yellow fever virus (YFV)
Japanese Encephalitis Virus (JEV)
ETC...
2

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
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Japanese Encephalitis Virus
http://www.fehd.gov.hk
3

Japanese Encephalitis Virus
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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
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Japanese Encephalitis Virus
http://www.cdc.gov
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Japanese Encephalitis Virus
50,000 cases/year
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Japanese Encephalitis Virus
10,000 Death Cases/year
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Japanese Encephalitis Virus
30% fatality rate
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Prevention and treatment of
JEV disease
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Prevention and treatment of
JEV disease
Drug
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Prevention and treatment of
JEV disease
Drug No drug exist
Vaccine development
5

Prevention and treatment of
JEV disease
Drug No drug exist
Vaccine development Available vaccine
Mosquitoes control
5

Prevention and treatment of
JEV disease
Drug No drug exist
Vaccine development Available vaccine
Mosquitoes control Elimination of
mosquitoes
breeding places
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Molecular biology of
Japanese Encephalitis Virus
www. molecular-virology.uni-hd.de
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Molecular biology of
Japanese Encephalitis Virus
www. molecular-virology.uni-hd.de
6

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

Molecular biology of
Japanese Encephalitis Virus
www. molecular-virology.uni-hd.de
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The NS2B
130 aa
activating domain central hydrophilic region
(Falgout et al, 1993)
3 membrane spanning parts
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The NS2B
130 aa
activating domain central hydrophilic region
(Falgout et al, 1993)
3 membrane spanning parts Hypothetical model
NS2B-NS3 complex
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The NS2B
130 aa
activating domain central hydrophilic region
(Falgout et al, 1993)
3 membrane spanning parts Hypothetical model
NS2B-NS3 complex
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The NS2B
Hypothetical model
NS2B-NS3 complex
Brinkworth et al, 1999
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The NS2B
Hypothetical model
NS2B-NS3 complex
58 VSGKATDMWLERAADISWEMDAAITGSSRRLDVKLDDDGDFHLIDDPGVP 101
Brinkworth et al, 1999
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The NS3
Theoretical model from PDB
2I84
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The NS3
Protease
Theoretical model from PDB
2I84
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The NS3
Protease
NTPase
Theoretical model from PDB
2I84
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The NS3
Protease
NTPase
RNA Helicase
Theoretical model from PDB
2I84
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The NS3
Chymotrypsin-like fold
2-β barrel domains
Inactive alone
Enzyme’s pocket is small
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The NS3 protease
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The NS3 protease
Complexation with NS2B cofactor
conformational change
alteration of the enzyme
pocket additional substrate
binding site
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The NS3 protease
NS3 serine protease
domain 20 kDa
catalytic residues His51,
Asp75, Ser135
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Lin. C W et al,2007
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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
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Compare to the structure
JEV WNV
JEV homology model from Den Den 2
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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)
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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?
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