{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T16:31:10Z","timestamp":1790267470069,"version":"4.1.0"},"reference-count":51,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2021,1,31]],"date-time":"2021-01-31T00:00:00Z","timestamp":1612051200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Centers of Biomedical Research Excellence","award":["P30GM114737"],"award-info":[{"award-number":["P30GM114737"]}]},{"DOI":"10.13039\/100001159","name":"Hawaii Community Foundation","doi-asserted-by":"publisher","award":["15ADVC-75878"],"award-info":[{"award-number":["15ADVC-75878"]}],"id":[{"id":"10.13039\/100001159","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>West Nile virus (WNV) nonstructural protein 3 (NS3) harbors the viral triphosphatase and helicase for viral RNA synthesis and, together with NS2B, constitutes the protease responsible for polyprotein processing. NS3 is a soluble protein, but it is localized to specialized compartments at the rough endoplasmic reticulum (RER), where its enzymatic functions are essential for virus replication. However, the mechanistic details behind the recruitment of NS3 from the cytoplasm to the RER have not yet been fully elucidated. In this study, we employed immunofluorescence and biochemical assays to demonstrate that NS3, when expressed individually and when cleaved from the viral polyprotein, is localized exclusively to the cytoplasm. Furthermore, NS3 appeared to be peripherally recruited to the RER and proteolytically active when NS2B was provided in trans. Thus, we provide evidence for a potential additional role for NS2B in not only serving as the cofactor for the NS3 protease, but also in recruiting NS3 from the cytoplasm to the RER for proper enzymatic activity. Results from our study suggest that targeting the interaction between NS2B and NS3 in disrupting the NS3 ER localization may be an attractive avenue for antiviral drug discovery.<\/jats:p>","DOI":"10.3390\/v13020216","type":"journal-article","created":{"date-parts":[[2021,1,31]],"date-time":"2021-01-31T04:53:00Z","timestamp":1612068780000},"page":"216","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Potential Dual Role of West Nile Virus NS2B in Orchestrating NS3 Enzymatic Activity in Viral Replication"],"prefix":"10.3390","volume":"13","author":[{"given":"Alanna C.","family":"Tseng","sequence":"first","affiliation":[{"name":"Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96813, USA"},{"name":"Molecular Biosciences and Bioengineering Graduate Program, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, Honolulu, HI 96822, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2044-2784","authenticated-orcid":false,"given":"Vivek R.","family":"Nerurkar","sequence":"additional","affiliation":[{"name":"Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96813, USA"},{"name":"Molecular Biosciences and Bioengineering Graduate Program, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, Honolulu, HI 96822, USA"},{"name":"Pacific Center for Emerging Infectious Diseases Research, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96813, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kabi R.","family":"Neupane","sequence":"additional","affiliation":[{"name":"Division of Math and Sciences, Leeward Community College, University of Hawaii, Pearl City, HI 96782, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Helmut","family":"Kae","sequence":"additional","affiliation":[{"name":"Division of Math and Sciences, Leeward Community College, University of Hawaii, Pearl City, HI 96782, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pakieli H.","family":"Kaufusi","sequence":"additional","affiliation":[{"name":"Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96813, USA"},{"name":"Molecular Biosciences and Bioengineering Graduate Program, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, Honolulu, HI 96822, USA"},{"name":"Pacific Center for Emerging Infectious Diseases Research, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96813, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,1,31]]},"reference":[{"key":"ref_1","unstructured":"van Regenmortel, M.H.V., Fauquet, C.M., Bishop, D.H.L., Carstens, E.B., Estes, M.K., Lemon, S.M., Maniloff, J., Mayo, M.A., McGeoch, D.J., and Pringle, C.R. (2000). Flaviviridae. Virus taxonomy: Classification and nomenclature of viruses. 7th Report of the International Committee for the Taxonomy of Viruses, Academic Press."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"511","DOI":"10.1001\/jama.290.4.511","article-title":"Neurologic manifestations and outcome of West Nile virus infection","volume":"290","author":"Sejvar","year":"2003","journal-title":"JAMA"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1016\/0042-6822(89)90162-1","article-title":"Analyses of the terminal sequences of West Nile virus structural proteins and of the in vitro translation of these proteins allow the proposal of a complete scheme of the proteolytic cleavages involved in their synthesis","volume":"169","author":"Nowak","year":"1989","journal-title":"Virology"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/0042-6822(86)90082-6","article-title":"Primary structure of the West Nile flavivirus genome region coding for all nonstructural proteins","volume":"149","author":"Castle","year":"1986","journal-title":"Virology"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1099\/vir.0.80411-0","article-title":"Inhibition of West Nile virus entry by using a recombinant domain III from the envelope glycoprotein","volume":"86","author":"Chu","year":"2005","journal-title":"J. Gen. Virol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"8632","DOI":"10.1128\/JVI.01253-15","article-title":"Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly","volume":"89","author":"VanBlargan","year":"2015","journal-title":"J. Virol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"10787","DOI":"10.1128\/JVI.75.22.10787-10799.2001","article-title":"Assembly and maturation of the flavivirus Kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively","volume":"75","author":"Mackenzie","year":"2001","journal-title":"J. Virol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"6650","DOI":"10.1128\/jvi.71.9.6650-6661.1997","article-title":"Ultrastructure of Kunjin virus-infected cells: Colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures","volume":"71","author":"Westaway","year":"1997","journal-title":"J. Virol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"10438","DOI":"10.1128\/JVI.00986-10","article-title":"The endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex","volume":"84","author":"Gillespie","year":"2010","journal-title":"J. Virol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1016\/j.chom.2009.03.007","article-title":"Composition and three-dimensional architecture of the dengue virus replication and assembly sites","volume":"5","author":"Welsch","year":"2009","journal-title":"Cell Host Microbe"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1852","DOI":"10.1128\/jvi.63.5.1852-1860.1989","article-title":"Proper processing of dengue virus nonstructural glycoprotein NS1 requires the N-terminal hydrophobic signal sequence and the downstream nonstructural protein NS2a","volume":"63","author":"Falgout","year":"1989","journal-title":"J. Virol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"9608","DOI":"10.1128\/jvi.71.12.9608-9617.1997","article-title":"trans-Complementation of yellow fever virus NS1 reveals a role in early RNA replication","volume":"71","author":"Lindenbach","year":"1997","journal-title":"J. Virol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"4609","DOI":"10.1128\/JVI.02424-12","article-title":"Membrane topology and function of dengue virus NS2A protein","volume":"87","author":"Xie","year":"2013","journal-title":"J. Virol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"30715","DOI":"10.1074\/jbc.272.49.30715","article-title":"Cotranslational membrane insertion of the serine proteinase precursor NS2B-NS3(Pro) of dengue virus type 2 is required for efficient in vitro processing and is mediated through the hydrophobic regions of NS2B","volume":"272","author":"Clum","year":"1997","journal-title":"J. Biol. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1006\/viro.1998.9156","article-title":"Subcellular localization and some biochemical properties of the flavivirus Kunjin nonstructural proteins NS2A and NS4A","volume":"245","author":"Mackenzie","year":"1998","journal-title":"Virology"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"8854","DOI":"10.1074\/jbc.M512697200","article-title":"Subcellular localization and membrane topology of the Dengue virus type 2 Non-structural protein 4B","volume":"281","author":"Miller","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"19100","DOI":"10.1074\/jbc.270.32.19100","article-title":"Association between NS3 and NS5 proteins of dengue virus type 2 in the putative RNA replicase is linked to differential phosphorylation of NS5","volume":"270","author":"Kapoor","year":"1995","journal-title":"J. Biol. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3108","DOI":"10.1128\/JVI.73.4.3108-3116.1999","article-title":"The serine protease and RNA-stimulated nucleoside triphosphatase and RNA helicase functional domains of dengue virus type 2 NS3 converge within a region of 20 amino acids","volume":"73","author":"Li","year":"1999","journal-title":"J. Virol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2757","DOI":"10.1093\/emboj\/21.11.2757","article-title":"An RNA cap (nucleoside-2\u2019-O-)-methyltransferase in the flavivirus RNA polymerase NS5: Crystal structure and functional characterization","volume":"21","author":"Egloff","year":"2002","journal-title":"EMBO J."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"12141","DOI":"10.1074\/jbc.M310839200","article-title":"Requirements for West Nile virus (-)- and (+)-strand subgenomic RNA synthesis in vitro by the viral RNA-dependent RNA polymerase expressed in Escherichia coli","volume":"279","author":"Nomaguchi","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"707","DOI":"10.1016\/0042-6822(91)90440-M","article-title":"The carboxy-terminal part of the NS 3 protein of the West Nile flavivirus can be isolated as a soluble protein after proteolytic cleavage and represents an RNA-stimulated NTPase","volume":"184","author":"Wengler","year":"1991","journal-title":"Virology"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2467","DOI":"10.1128\/jvi.65.5.2467-2475.1991","article-title":"Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins","volume":"65","author":"Falgout","year":"1991","journal-title":"J. Virol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"12883","DOI":"10.1074\/jbc.M611318200","article-title":"Functional characterization of cis and trans activity of the Flavivirus NS2B-NS3 protease","volume":"282","author":"Bera","year":"2007","journal-title":"J. Biol. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"17270","DOI":"10.1074\/jbc.M801719200","article-title":"The two-component NS2B-NS3 proteinase represses DNA unwinding activity of the West Nile virus NS3 helicase","volume":"283","author":"Chernov","year":"2008","journal-title":"J. Biol. Chem."},{"key":"ref_25","unstructured":"Knipe, D.M., and Howley, P.M. (2013). Fields Virology, Wolters Kluwer\/Lippencott Williams & Wilkins. [6th ed.]."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4640","DOI":"10.3390\/v7082837","article-title":"Flaviviral Replication Complex: Coordination between RNA Synthesis and 5\u2019-RNA Capping","volume":"7","author":"Klema","year":"2015","journal-title":"Viruses"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"691","DOI":"10.1016\/j.febslet.2009.01.008","article-title":"Analysis of the nucleoside triphosphatase, RNA triphosphatase, and unwinding activities of the helicase domain of dengue virus NS3 protein","volume":"583","author":"Wang","year":"2009","journal-title":"FEBS Lett."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2244","DOI":"10.1016\/j.bbamem.2015.06.010","article-title":"Membrane topology of NS2B of dengue virus revealed by NMR spectroscopy","volume":"1848","author":"Li","year":"2015","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"8873","DOI":"10.1074\/jbc.M609919200","article-title":"The non-structural protein 4A of dengue virus is an integral membrane protein inducing membrane alterations in a 2K-regulated manner","volume":"282","author":"Miller","year":"2007","journal-title":"J. Biol. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"372","DOI":"10.1038\/nsmb1073","article-title":"Structural basis for the activation of flaviviral NS3 proteases from dengue and West Nile virus","volume":"13","author":"Erbel","year":"2006","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2081","DOI":"10.1099\/vir.0.012864-0","article-title":"NS4A regulates the ATPase activity of the NS3 helicase: A novel cofactor role of the non-structural protein NS4A from West Nile virus","volume":"90","author":"Shiryaev","year":"2009","journal-title":"J. Gen. Virol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2605","DOI":"10.1099\/vir.0.81844-0","article-title":"Dengue virus NS4B interacts with NS3 and dissociates it from single-stranded RNA","volume":"87","author":"Umareddy","year":"2006","journal-title":"J. Gen. Virol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1016\/j.virol.2013.08.006","article-title":"Protein-protein interactions among West Nile non-structural proteins and transmembrane complex formation in mammalian cells","volume":"446","author":"Yu","year":"2013","journal-title":"Virology"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2034","DOI":"10.1128\/jvi.67.4.2034-2042.1993","article-title":"Deletion analysis of dengue virus type 4 nonstructural protein NS2B: Identification of a domain required for NS2B-NS3 protease activity","volume":"67","author":"Falgout","year":"1993","journal-title":"J. Virol."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Su, X.C., Ozawa, K., Qi, R., Vasudevan, S.G., Lim, S.P., and Otting, G. (2009). NMR analysis of the dynamic exchange of the NS2B cofactor between open and closed conformations of the West Nile virus NS2B-NS3 protease. PLoS Negl. Trop. Dis., 3.","DOI":"10.1371\/journal.pntd.0000561"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"6042","DOI":"10.1128\/jvi.65.11.6042-6050.1991","article-title":"Processing of the yellow fever virus nonstructural polyprotein: A catalytically active NS3 proteinase domain and NS2B are required for cleavages at dibasic sites","volume":"65","author":"Chambers","year":"1991","journal-title":"J. Virol."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Kaufusi, P.H., Tseng, A.C., Kelley, J.F., and Nerurkar, V.R. (2020). Selective Reactivity of Anti-Japanese Encephalitis Virus NS4B Antibody Towards Different Flaviviruses. Viruses, 12.","DOI":"10.3390\/v12020212"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"425","DOI":"10.1016\/j.virol.2008.11.047","article-title":"West Nile virus infection modulates human brain microvascular endothelial cells tight junction proteins and cell adhesion molecules: Transmigration across the in vitro blood-brain barrier","volume":"385","author":"Verma","year":"2009","journal-title":"Virology"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1007\/978-1-4939-3670-0_5","article-title":"Functional Analysis of West Nile Virus Proteins in Human Cells","volume":"1435","author":"Kaufusi","year":"2016","journal-title":"Methods Mol. Biol."},{"key":"ref_40","unstructured":"Sambrook, J., and Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1038\/ni921","article-title":"IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway","volume":"4","author":"Fitzgerald","year":"2003","journal-title":"Nat. Immunol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: An open-source platform for biological-image analysis","volume":"9","author":"Schindelin","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"3993","DOI":"10.1529\/biophysj.103.038422","article-title":"Automatic and quantitative measurement of protein-protein colocalization in live cells","volume":"86","author":"Costes","year":"2004","journal-title":"Biophys. J."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Kaufusi, P.H., Kelley, J.F., Yanagihara, R., and Nerurkar, V.R. (2014). Induction of endoplasmic reticulum-derived replication-competent membrane structures by West Nile virus non-structural protein 4B. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0084040"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"12895","DOI":"10.1128\/JVI.00942-09","article-title":"Crystal structure of a novel conformational state of the flavivirus NS3 protein: Implications for polyprotein processing and viral replication","volume":"83","author":"Assenberg","year":"2009","journal-title":"J. Virol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"2379","DOI":"10.1074\/jbc.M114.607341","article-title":"The C-terminal 50 amino acid residues of dengue NS3 protein are important for NS3-NS5 interaction and viral replication","volume":"290","author":"Tay","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"3471","DOI":"10.1128\/JVI.03454-14","article-title":"Mapping the Interactions between the NS4B and NS3 proteins of dengue virus","volume":"89","author":"Zou","year":"2015","journal-title":"J. Virol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/j.virusres.2004.01.025","article-title":"The non-structural 3 (NS3) protein of dengue virus type 2 interacts with human nuclear receptor binding protein and is associated with alterations in membrane structure","volume":"102","author":"Chua","year":"2004","journal-title":"Virus Res."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"5451","DOI":"10.1128\/JVI.01982-05","article-title":"Nuclear localization of flavivirus RNA synthesis in infected cells","volume":"80","author":"Uchil","year":"2006","journal-title":"J. Virol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1128\/CVI.00500-13","article-title":"Dengue virus subverts the interferon induction pathway via NS2B\/3 protease-IkappaB kinase epsilon interaction","volume":"21","author":"Pantoja","year":"2014","journal-title":"Clin. Vaccine Immunol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"393","DOI":"10.1016\/j.bbrc.2013.09.078","article-title":"A small compound targeting the interaction between nonstructural proteins 2B and 3 inhibits dengue virus replication","volume":"440","author":"Pambudi","year":"2013","journal-title":"Biochem. Biophys. Res. Commun."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/2\/216\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:17:47Z","timestamp":1760159867000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/2\/216"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,1,31]]},"references-count":51,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2021,2]]}},"alternative-id":["v13020216"],"URL":"https:\/\/doi.org\/10.3390\/v13020216","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,1,31]]}}}