Function of NS2B for NS3
protease activation of
Japanese encephalitis virus
Advisor: Assist.Prof Gerd Katzenmeier
Laboratory of Molecular virology
Institute of Molecular Biology & Genetics
Prevention and treatment of
Drug No drug exist
Vaccine development Available vaccine
Elimination of mosquitoes
Mosquitoes control breeding places
Molecular biology of Japanese Encephalitis Virus
Source : molecular-virology.uni-hd.de
• 130 aa Hypothetical model
• activating domain NS2B-NS3 complex
central hydrophilic region
(Falgout et al, 1993)
• 3 membrane spanning parts
ฺBrinkworth et al, 1999
51 DMWLERAADISWEMDAAITGSSRRLDVKLDDDGDFHLIDDPGVP 95
2-β barrel Helicase
Theoretical model from PDB
•Enzyme’s pocket is small
The NS3 protease
Complexation with NS2B cofactor
•NS3 serine protease
domain 20 of thechange pocket
•catalytic residues binding site
additional substrate His51,
• The result shown the slightly differences inby
Some physico-chemical properties shared
all the proteases.
Homology Modelling of
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
• Two component of protease has a unique
• The NS3 protease share the similar
structure in the Flavivirus group but are
different in cofactor binding.
• To investigate the function determination in
the JEV NS2B for the activation of NS3
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
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
Lane 1- 5 = Washing Fraction with 100 mM Imidazole
Lane 6 -10 = Eluted Fraction with 400 mM Imidazole
• 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.
• Residues Lys78 to Leu87 of JEV NS2B may
involved in the formation of the active site in
the NS2B-NS3 protease.
• Due to the substrate speciﬁcity of the
• Can we make a very speciﬁc anti-viral drug
to Flavivirus ?
The pan-Flavivirus NS3 protease drugs may
be developed for Flaviviral diseases.