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

Simple item page
dc.contributor.authorWahaab, Abdulen
dc.contributor.authorZhang, Yanen
dc.contributor.authorLiu, Keen
dc.contributor.authorRasgon, Jason L.en
dc.contributor.authorKang, Leien
dc.contributor.authorHameed, Muddassaren
dc.contributor.authorLi, Chenxien
dc.contributor.authorAnwar, Muhammad Naveeden
dc.contributor.authorZhang, Yanbingen
dc.contributor.authorShoaib, Anamen
dc.contributor.authorLi, Beibeien
dc.contributor.authorQiu, Yafengen
dc.contributor.authorWei, Jianchaoen
dc.contributor.authorMa, Zhiyongen
dc.date.accessioned2024-12-13T17:45:44Zen
dc.date.available2024-12-13T17:45:44Zen
dc.date.issued2024-11-26en
dc.date.updated2024-12-13T16:21:00Zen
dc.description.abstractJapanese 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 <i>E. coli</i> 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.en
dc.description.versionPublished versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.citationWahaab, A.; Zhang, Y.; Liu, K.; Rasgon, J.L.; Kang, L.; Hameed, M.; Li, C.; Anwar, M.N.; Zhang, Y.; Shoaib, A.; Li, B.; Qiu, Y.; Wei, J.; Ma, Z. 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. Int. J. Mol. Sci. 2024, 25, 12680.en
dc.identifier.doihttps://doi.org/10.3390/ijms252312680en
dc.identifier.urihttps://hdl.handle.net/10919/123798en
dc.language.isoenen
dc.publisherMDPIen
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.titleNS2B-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 Modelen
dc.title.serialInternational Journal of Molecular Scienceen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten

Files

Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
ijms-25-12680.pdf
Size:
10.89 MB
Format:
Adobe Portable Document Format
Description:
Published version
Download
License bundle
Now showing 1 - 1 of 1
Name:
license.txt
Size:
1.5 KB
Format:
Item-specific license agreed upon to submission
Description:
Download

Collections

Journal Articles, Multidisciplinary Digital Publishing Institute (MDPI)
Scholarly Works, Biomedical Sciences and Pathobiology