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Flaviviruses are known to cause a variety of diseases in humans in different parts of the world. There are very limited numbers of antivirals to combat flavivirus infection, and therefore new drug targets must be explored. The flavivirus NS2B-NS3 proteases are responsible for the cleavage of the flavivirus polyprotein, which is necessary for productive viral infection and for causing clinical infections; therefore, they are a promising drug target for devising novel drugs against different flaviviruses. This review highlights the structural details of the NS2B-NS3 proteases of different flaviviruses, and also describes potential antiviral drugs that can interfere with the viral protease activity, as determined by various studies. Moreover, optimized in vitro reaction conditions for studying the NS2B-NS3 proteases of different flaviviruses may vary and have been incorporated in this review. The increasing availability of the in silico and crystallographic/structural details of flavivirus NS2B-NS3 proteases in free and drug-bound states can pave the path for the development of promising antiflavivirus drugs to be used in clinics. However, there is a paucity of information available on using animal cells and models for studying flavivirus NS2B-NS3 proteases, as well as on the testing of the antiviral drug efficacy against NS2B-NS3 proteases. Therefore, on the basis of recent studies, an effort has also been made to propose potential cellular and animal models for the study of flavivirus NS2B-NS3 proteases for the purposes of exploring flavivirus pathogenesis and for testing the efficacy of possible drugs targets, in vitro and in vivo. Published version

Japanese Encephalitis Virus (JEV) NS2B-NS3 is a protein complex composed of NS3 proteases and a NS2B cofactor. The N-terminal protease domain (180 residues) of NS3 (NS3(pro)) interacts directly with a central 40-amino acid hydrophilic domain of NS2B (NS2B(H)) to form an active serine protease. In this study, the recombinant NS2B(H)-NS3(pro) proteases were prepared in E. coli and used to compare the enzymatic activity between genotype I (GI) and III (GIII) NS2B-NS3 proteases. The GI NS2B(H)-NS3(pro) was able to cleave the sites at internal C, NS2A/NS2B, NS2B/NS3 and NS3/NS4A junctions that were identical to the sites proteolytically processed by GIII NS2B(H)-NS3(pro). Analysis of the enzymatic activity of recombinant NS2B(H)-NS3(pro) proteases using a model of fluorogenic peptide substrate revealed that the proteolytical processing activity of GIII NS2B(H)-NS3(pro) was significantly higher than that of GI NS2B(H)-NS3(pro). There were eight amino acid variations between GI and GIII NS2B(H)-NS3(pro), which may be responsible for the difference in enzymatic activities between GI and GIII proteases. Therefore, recombinant mutants were generated by exchanging NS2B(H) and NS3(pro) domains between GI and GIII NS2B(H)-NS3(pro) and subjected to protease activity analysis. Substitution of NS2B(H) significantly altered the protease activities, as compared to the parental NS2B(H)-NS3(pro), suggesting that NS2B(H) played an essential role in regulation of NS3(pro) protease activity. To further identify the amino acids responsible for the difference in protease activities, multiple substitution mutants including the individual and combined mutations at the variant residue 55 and 65 of NS2B(H) were generated and subjected to protease activity analysis. Replacement of NS2B-55 and NS2B-65 of GI to GIII significantly increased the enzymatic activity of GI NS2B(H)-NS3(pro) protease, whereas mutation of NS2B-55 and NS2B-65 of GIII to GI remarkably reduced the enzymatic activity of GIII NS2B(H)-NS3(pro) protease. Overall, these data demonstrated that NS2B-55 and NS2B-65 variations in hydrophilic domain of NS2B co-contributed to the difference in NS2B(H)-NS3(pro) protease activities between GI and GIII. These observations gain an insight into the role of NS2B in regulation of NS3 protease activities, which is useful for understanding the replication of JEV GI and GIII viruses., Competing Interests: Conflict of Interest The authors declare no conflict of interest.