Seminar Final.Key

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

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

Japanese Encephalitis Virus (JEV) Source : vietnammedicalpractice.com

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

Prevention and treatment of JEV disease 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

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

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

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

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

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. • 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 protease.

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

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 Measured the ﬂorescence Change

Result

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

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 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.

Discussion • 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 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.

Thank you for your attention.

Seminar Final.Key

by Chakard Chalayut

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