Abstract
Understanding the proteolytic processing of polyprotein mediated by NS2B-NS3 protease contributes to the exploration of the mechanisms underlying infection of Japanese encephalitis virus (JEV), a zoonotic flavivirus. In this study, eukaryotic and prokaryotic cell models were employed to identify the cleavage sites mediated by viral NS2B-NS3 protease in JEV polyprotein. Artificial green fluorescent protein (GFP) substrates that contained the predicted cleavage site sequences of JEV polyprotein were expressed in swine testicle (ST) cells in the presence and absence of JEV infection, or co-expressed in E. coli with the recombinant NS2B-NS3 protease that was generated by fusing the N-terminal protease domain of NS3 to the central hydrophilic domain of NS2B. The cleavage of GFP substrates was examined by western blot. Among twelve artificial GFP substrates containing the cleavage site sequences predictively processed by host cell and/or NS2B-NS3 proteases, all sites were found to be cleaved by host cell proteases with different efficiencies. The sites at internal C, NS2A/NS2B, NS2B/NS3 and NS3/NS4A junctions, but not the sites at internal NS3, internal NS4A and NS4B/NS5 junctions were identified to be cleaved by JEV NS2B-NS3 protease. These data provide insight into the proteolytic processing of polyprotein, which is useful for understanding JEV replication and pathogenesis.
Highlights
Japanese encephalitis virus (JEV) is a zoonotic flavivirus belonging to the genus Flavivirus in the family Flaviviridae that comprises more than 70 species, such as dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV) and Zika virus
We identified the cleavage sites in JEV polyprotein using recombinant NS2B-NS3 protease and artificial green fluorescent protein (GFP) substrates containing the predicted cleavage site sequences in a prokaryotic cell model of E. coli
Itnhtehmis estcuhdayn,iwsme susuenddaeprlryoiknagrythoteicpcreoltlemoolydteicl opfrEo.cceoslsi ing of polyprotein to identifyatshewcelellavaasgJeEsViterseipnlitchaetipoonly. pIrnottehinisanstduodbys,ewrveedutsheadt tahepsrioteksaartyionttiecrncaelllCm, odel of E. coli to NS2A/NS2idB,eNntSi2fyB/NthSe3 calnedavNaSg3e/NsSit4eAs jiunncthtioenpsowlyerpercolteeaivnedanbdy NoSb2sBer-NveSd3 ptrhoatteatsheeosf ites at internal C, JEV
Summary
Japanese encephalitis virus (JEV) is a zoonotic flavivirus belonging to the genus Flavivirus in the family Flaviviridae that comprises more than 70 species, such as dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV) and Zika virus. It is endemic and the most substantial cause of human encephalitis in eastern and southern Asia with 30,000–50,000 cases annually [1,2]. According to the knowledge of polyprotein processing of other flaviviruses, JEV protease is speculated to cleave the polyprotein at intergenic junctions of NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 as well as at the internal sites within C, NS3 and NS4A [7,8]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
