Moreover, the disease is not restricted to Asia and cases also occur sporadically in northern Australia and western Pacific [6]. which are human pathogens, including West Nile computer virus (WNV), Dengue computer virus (DEN), Yellow fever computer virus (YFV), Murray Valley Encephalitis Computer virus (MVEV), Kunjin Computer virus (KUNV) and Tick-Borne Encephalitis Computer virus (TBEV) [2]. Japanese encephalitis (JE) is among the most important viral encephalitides in Asia [3]C[5]. Moreover, the disease is usually not restricted to Asia and cases also occur sporadically in northern Australia and western Pacific [6]. Of about 68,000 estimated annual cases, approximately 20C30% are fatal, and 30C50% of survivors have significant neurologic sequelae [7], [8]. Since the zoonosis is usually endemic in large parts of Asia, it is not likely to ever be extinguished. Currently there is no antiviral therapy for JEV or any other flaviviral infection, and so far the main strategy to control the incidence is usually by preventive methods such as vaccination and preventing mosquito bites [9]C[11]. Even though improvements in JEV vaccination protection has reduced the JE incidence, about 55,000 (81%) out of the total annual cases still occur in areas with well established or developing JE vaccination programs [12]. Effective antiviral therapy is usually thus urgently needed, especially for those cases where the contamination has become prolonged. One approach to develop anti-JEV therapy is usually to interfere with the life cycle of the computer virus, and exploit the molecular targets such as envelope glycoprotein, NS3 protease, NS3 helicase, NS5 methyltransferase and NS5 RNA- dependent RNA polymerase [13]. Unlike several other flaviviruses such as DEN, WNV and MVEV [14]C[16] whose protease enzymes are extensively characterised as potential drug R406 besylate targets, the JEV protease is usually comparatively less analyzed with a view to structure-activity relations. The JEV two-component protease NS2B/NS3 is responsible for processing the viral polyprotein precursor to the mature viral proteins involved in viral pathogenesis, and therefore considered an important drug target in JEV [17], [18]. The N-terminal one-third (180 residues) of NS3 represents the protease domain name NS3(pro) that works in coordination with the C-terminal two-third portion RNA helicase during viral propagation [19], [20]. The proteolytic domain name contains a classical catalytic triad of H51, D75 and S135, and autocatalytic proteolytic cleavage at the NS2B/NS3 polyprotein junction prospects to the formation of a non-covalent complex of NS2B and NS3 [21]. Earlier studies have revealed that a 35C48 amino acid residues long central hydrophilic region NS2B(H) of NS2B interacts directly with the NS3(pro) and promotes folding of NS3(pro) into a catalytically qualified conformation [22]C[25]. Currently, there is no X-ray crystallographic structure available for the JEV protease, but crystal structures of the comparable proteases from DEN and WNV have provided insight into the mechanism of cofactor-dependent activation and revealed an induced fit mechanism of catalysis [26], [27]. By analysis of chimeric viral proteases of DEN2 and YFV, it was shown that this YFV polyprotein cleavage sites were efficiently cleaved R406 besylate by the chimeric protease made up of the YFV or DEN2 NS3 protease domain name, while the DEN2 polyprotein sites were not cleaved by the YFV chimeric protease made up of YFV NS3(pro), suggesting that cleavage requires specific local interactions between substrates and the binding CREB4 pocket site of the enzyme [28]. The substrate recognition sequence is highly conserved in all flaviviruses and consists of two basic residues in P2 and P1 followed by a small unbranched amino acid in P1 [22], [43]. Substrate profiling studies found that the WNV protease was highly selective for the cleavage site sequence motif (K/R)GG, whereas DEN protease also tolerated the presence of bulky residues such as Phe, Trp, or Tyr at either the P1 or the P2 site, provided that the other position was occupied by Gly [21], [28], [29]. The aim of this study was to develop a fast and easy methodology for cloning, expression and purification of the active JEV NS2B(H)-NS3 serine protease and to obtain numerical data for kinetic constants by using ?uorogenic model peptide substrates for serine proteases. In addition, we also characterized inhibition of the protease by conventional serine protease inhibitors. To the best of.Similar to the protein from DEN, the 29.8 kDa (His)6NS2B(H)-NS3pro protein of JEV exhibits anomalous migration in SDS-PAGE gels. including West Nile virus (WNV), Dengue virus (DEN), Yellow fever virus (YFV), Murray Valley Encephalitis Virus (MVEV), Kunjin Virus (KUNV) and Tick-Borne Encephalitis Virus (TBEV) [2]. Japanese encephalitis (JE) is among the most important viral encephalitides in Asia [3]C[5]. Moreover, the disease is not restricted to Asia and cases also occur sporadically in northern Australia and western Pacific [6]. Of about 68,000 estimated annual cases, approximately 20C30% are fatal, and 30C50% of survivors have significant neurologic sequelae [7], [8]. Since the zoonosis is endemic in large parts of Asia, it is not likely to ever be extinguished. Currently there is no antiviral therapy for JEV or any other flaviviral infection, and so far the main strategy to control the incidence is by preventive methods such as vaccination and preventing mosquito bites [9]C[11]. Although the improvements in JEV vaccination coverage has reduced the JE incidence, about 55,000 (81%) out of the total annual cases still occur in areas with well established or developing JE vaccination programs [12]. Effective antiviral therapy is thus urgently needed, especially for those cases where the infection has become persistent. One approach to develop anti-JEV therapy is to interfere with the life cycle of the virus, and exploit the molecular targets such as envelope glycoprotein, NS3 protease, NS3 helicase, NS5 methyltransferase and NS5 RNA- dependent RNA polymerase [13]. Unlike several other flaviviruses such as DEN, WNV and MVEV [14]C[16] whose protease enzymes are extensively characterised as potential drug targets, the JEV protease is comparatively less studied with a view to structure-activity relations. The JEV two-component protease NS2B/NS3 is responsible for processing the viral polyprotein precursor to the mature viral proteins involved in viral pathogenesis, and therefore considered an important drug target in JEV [17], [18]. The N-terminal one-third (180 residues) of NS3 represents the protease domain NS3(pro) that works in coordination with the C-terminal two-third portion RNA helicase during viral propagation [19], [20]. The proteolytic domain contains a classical catalytic triad of H51, D75 and S135, and autocatalytic proteolytic cleavage at the NS2B/NS3 polyprotein junction leads to the formation of a non-covalent complex of NS2B and NS3 [21]. Earlier studies have revealed that a 35C48 amino acid residues long central hydrophilic region NS2B(H) of NS2B interacts directly with the NS3(pro) and promotes folding of NS3(pro) into a catalytically competent conformation [22]C[25]. Currently, there is no X-ray crystallographic structure available for the JEV protease, but crystal structures of the similar proteases from DEN and WNV have provided insight into the mechanism of cofactor-dependent activation and revealed an induced fit mechanism of catalysis [26], [27]. By analysis of chimeric viral proteases of DEN2 and YFV, it was shown that the YFV polyprotein cleavage sites were efficiently cleaved by the chimeric protease containing the YFV or DEN2 NS3 R406 besylate protease domain, while the DEN2 polyprotein sites were not cleaved by the YFV chimeric protease containing YFV NS3(pro), suggesting that cleavage requires specific local interactions between substrates and the binding pocket site of the enzyme [28]. The substrate recognition sequence is highly conserved in all flaviviruses and consists of two basic residues in P2 and P1 followed by a small unbranched amino acid in P1 [22], [43]. Substrate profiling studies found that the WNV protease was highly selective for the cleavage site sequence motif (K/R)GG, whereas DEN protease also tolerated the presence of bulky residues such as Phe, Trp, or Tyr at either the P1 or the P2 site, provided that the other position was occupied by Gly [21], [28], [29]. The aim of this study was to develop a fast and easy.