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Prevention and Control in Lancang and Mekong Countries","award":["ZD2021CY001"],"award-info":[{"award-number":["ZD2021CY001"]}]},{"name":"project of Cooperation on Animal Biosecurity Prevention and Control in Lancang and Mekong Countries","award":["22N41900400"],"award-info":[{"award-number":["22N41900400"]}]},{"name":"project of Cooperation on Animal Biosecurity Prevention and Control in Lancang and Mekong Countries","award":["2022 YFD 1800100"],"award-info":[{"award-number":["2022 YFD 1800100"]}]},{"name":"project of Cooperation on Animal Biosecurity Prevention and Control in Lancang and Mekong Countries","award":["125161035"],"award-info":[{"award-number":["125161035"]}]},{"name":"project of Cooperation on Animal Biosecurity Prevention and Control in Lancang and Mekong Countries","award":["R01AI12820"],"award-info":[{"award-number":["R01AI12820"]}]},{"name":"NIH","award":["32273096"],"award-info":[{"award-number":["32273096"]}]},{"name":"NIH","award":["ZD2021CY001"],"award-info":[{"award-number":["ZD2021CY001"]}]},{"name":"NIH","award":["22N41900400"],"award-info":[{"award-number":["22N41900400"]}]},{"name":"NIH","award":["2022 YFD 1800100"],"award-info":[{"award-number":["2022 YFD 1800100"]}]},{"name":"NIH","award":["125161035"],"award-info":[{"award-number":["125161035"]}]},{"name":"NIH","award":["R01AI12820"],"award-info":[{"award-number":["R01AI12820"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Japanese encephalitis virus (JEV) NS2B-NS3 is a protein complex composed of NS3 proteases and an NS2B co-factor. 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 the 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 the 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 the 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 residues 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 the hydrophilic domain of NS2B co-contributed to the difference in NS2B(H)-NS3(pro) protease activities between GI and GIII. However, it will be crucial to explore these mutations in other in vivo and\/or in vitro models. Collectively, these observations will be useful for understanding the replication of JEV GI and GIII viruses.<\/jats:p>","DOI":"10.3390\/ijms252312680","type":"journal-article","created":{"date-parts":[[2024,11,26]],"date-time":"2024-11-26T05:06:53Z","timestamp":1732597613000},"page":"12680","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["NS2B-D55E and NS2B-E65D Variations Are Responsible for Differences in NS2B-NS3 Protease Activities Between Japanese Encephalitis Virus Genotype I and III in Fluorogenic Peptide Model"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1919-3653","authenticated-orcid":false,"given":"Abdul","family":"Wahaab","sequence":"first","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"},{"name":"The Department of Entomology, Center for Infectious Disease Dynamics, and the Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA 16802, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2216-5749","authenticated-orcid":false,"given":"Yan","family":"Zhang","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ke","family":"Liu","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4050-8429","authenticated-orcid":false,"given":"Jason L.","family":"Rasgon","sequence":"additional","affiliation":[{"name":"The Department of Entomology, Center for Infectious Disease Dynamics, and the Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA 16802, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lei","family":"Kang","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Muddassar","family":"Hameed","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chenxi","family":"Li","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4762-1635","authenticated-orcid":false,"given":"Muhammad Naveed","family":"Anwar","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-9564-3999","authenticated-orcid":false,"given":"Yanbing","family":"Zhang","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anam","family":"Shoaib","sequence":"additional","affiliation":[{"name":"School of Behavioral and Brain Sciences, University of Texas at Dallas, Richardson, TX 75080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4134-4024","authenticated-orcid":false,"given":"Beibei","family":"Li","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese 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China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,11,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Brecher, M., Li, Z., Liu, B., Zhang, J., Koetzner, C.A., Alifarag, A., Jones, S.A., Lin, Q., Kramer, L.D., and Li, H. 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