Function of NS2B for NS3 protease activation of

Japanese encephalitis virus

Mr.Chakard ChalayutAdvisor: Assist.Prof Gerd Katzenmeier

Laboratory of Molecular virologyInstitute of Molecular Biology & Genetics

Japanese Encephalitis Virus(JEV)

Source: fehd.gov.hk Source: news.bbc.co.uk

Culex tritaeniorhynchus.

Japanese Encephalitis Virus(JEV)

Source : vietnammedicalpractice.com

Japanese Encephalitis Virus(JEV)

Source: medwork84.com

Source: cdc.gov

30% fatality rate50,000 Cases10,000 Cases

Prevention and treatment of JEV disease

Drug No drug exist

Vaccine development

Mosquitoes control Elimination of mosquitoes breeding places

Available vaccine

Molecular biology of Japanese Encephalitis Virus

Source : molecular-virology.uni-hd.de

The NS2BHypothetical model NS2B-NS3 complex

hydrophobicity plot

51 DMWLERAADISWEMDAAITGSSRRLDVKLDDDGDFHLIDDPGVP 95

Brinkworth et al, 1999

• 130 aa• activating domain central hydrophilic region (Falgout et al, 1993)• 3 membrane spanning parts

The NS3

•Chymotrypsin-like fold2-β barrel domains •Inactive alone•Enzyme’s pocket is small

NTPase

Protease

RNA Helicase

Theoretical model from PDB 2I84

The NS3 protease

conformational change alteration of the enzyme pocket additional substrate binding site

•NS3 serine protease domain 20 kDa•catalytic residues His51, Asp75, Ser135

Complexation with NS2B cofactor

• The result shown the slightly differences in biochemical properties.

• Some physico-chemical properties shared by all the proteases.

Homology Modelling of NS3 protease

Homology Modelling of NS3 protease

JEVWNV DEN

• NS2B-NS3 WNV can adopt two distinct conformation.• The NS2B-NS3 shown the induced fit mechanism.

• 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.Expression and purification of deleted and mutation forms

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 ImidazoleLane 6 -10 = Eluted Fraction with 400 mM Imidazole

Method II

NS2B-NS3 protease

Incubate with GKR-AMC

Measured the florescence Change

Trans-cleavage of fluorogenie peptide substrate

Result

Method III

JEV NS2B and NS3 protease protein

Based on WNV crystallographic structure

Perform the structural modelling by using Swiss modeling workstation & MEDock program

Molecular Modelling of a JEV NS2B-NS3 complex

Result

Result

Lane 1- 5 = Washing Fraction with 100 mM ImidazoleLane 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.