{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T17:07:40Z","timestamp":1785431260504,"version":"3.56.0"},"posted":{"date-parts":[[2026,2,7]]},"group-title":"Biochemistry","reference-count":64,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,2,7]],"date-time":"2026-02-07T00:00:00Z","timestamp":1770422400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["R01AI045818"],"award-info":[{"award-number":["R01AI045818"]}],"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,2,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  The NS3 helicases from the\n                  <jats:italic>Flaviviridae<\/jats:italic>\n                  family of viruses exhibit nucleotide-hydrolysis-dependent, nucleic-acid-unwinding activity. The RNA unwinding activity for NS3 helicases from the\n                  <jats:italic>Ortho<\/jats:italic>\n                  f\n                  <jats:italic>lavivirus<\/jats:italic>\n                  genus has not been fully explored and contrasts with NS3 helicase from Hepatitis C virus (HCV) of the\n                  <jats:italic>Hepacivirus<\/jats:italic>\n                  genus, which has thus far served as the prototypical model enzyme from this family of viruses. To begin to understand the functional differences between flavivirus NS3 helicases, we first developed an expression and purification system for full-length untagged NS3 protein from West Nile virus (WNV) and Zika virus (ZIKV). Both enzymes exhibit RNA-stimulated ATPase activity and are dependent on the nucleoside triphosphatase active site of the enzyme. Unlike HCV NS3, orthoflavivirus NS3s do not efficiently pre-assemble on a 3\u2019-ssRNA-tailed dsRNA substrate in the absence of ATP-Mg which is a prerequisite for formation of a productive HCV NS3-RNA complex that can exhibit a rapid burst of RNA unwinding. Instead, to observe RNA unwinding by WNV and ZIKV NS3s, low Mg-ATP concentrations are required at a time coincident when NS3 encounters the RNA substrate. In addition, we find that orthoflavivirus NS3s require translocation beyond the displaced strand to completely unwind a dsRNA substrate. Last, we find that orthoflavivirus NS5 stimulates the ability of NS3 to unwind dsRNA. These results suggest that functional differences exist between the flavivirus NS3 helicases and illuminate that orthoflavivirus NS3s require a functional interaction with the NS5 protein for coordination of its activity, as it is believed these two proteins constitute the viral replicase.\n                <\/jats:p>","DOI":"10.64898\/2026.02.06.704446","type":"posted-content","created":{"date-parts":[[2026,2,8]],"date-time":"2026-02-08T01:40:11Z","timestamp":1770514811000},"source":"Crossref","is-referenced-by-count":1,"title":["Unwinding of an RNA duplex by the Orthoflavivirus NS3 helicase requires translocation beyond the displaced strand and is stimulated by the NS5 RdRp"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2345-9776","authenticated-orcid":false,"given":"Jamie J.","family":"Arnold","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA"},{"name":"Department of Cell Biology and Physiology, University of North Carolina School of Medicine, Chapel Hill, NC 27599"},{"name":"Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC 27599"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shubeena","family":"Chib","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA"},{"name":"OHSU Technology Transfer (OHSU Innovates), Oregon Health & Science University (OHSU), 3181 SW Sam Jackson Park Road, Portland, OR 97239"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7564-5642","authenticated-orcid":false,"given":"Craig E.","family":"Cameron","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA"},{"name":"Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC 27599"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026021102003174000_2026.02.06.704446v1.1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.micro.112408.134012"},{"key":"2026021102003174000_2026.02.06.704446v1.2","doi-asserted-by":"publisher","DOI":"10.5501\/wjv.v2.i2.32"},{"key":"2026021102003174000_2026.02.06.704446v1.3","doi-asserted-by":"publisher","DOI":"10.3390\/v6072826"},{"key":"2026021102003174000_2026.02.06.704446v1.4","doi-asserted-by":"crossref","unstructured":"Morita, E. , and Suzuki, Y. 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