{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,6]],"date-time":"2026-02-06T21:07:31Z","timestamp":1770412051926,"version":"3.49.0"},"reference-count":52,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,1,24]],"date-time":"2026-01-24T00:00:00Z","timestamp":1769212800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan","award":["AP23488393"],"award-info":[{"award-number":["AP23488393"]}]},{"name":"Ministry of Healthcare of the Republic of Kazakhstan","award":["BR25293293"],"award-info":[{"award-number":["BR25293293"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>The flavivirus NS1 protein is a component of the viral replication complex and plays diverse, yet poorly understood, roles in the viral life cycle. To enable real-time visualization of the developing replication organelle and biochemical analysis of tagged NS1 and its interacting partners, we engineered a replication-competent yellow fever virus (YFV) replicon encoding a C-terminal fusion of NS1 with green fluorescent protein (NS1-GFP). The initial variant was non-viable in the absence of trans-complementation with wild-type NS1; however, viability was partially restored through the introduction of co-adaptive mutations in GFP (Q204R\/A206V) and NS4A (M108L). Subsequent cell culture adaptation generated a 17-nucleotide frameshift within the NS1-GFP linker, resulting in a more flexible and less hydrophobic linker sequence. The optimized genome, in the form of a replicon, replicates in packaging cells that produce YFV structural proteins, as well as in naive BHK-21 cells. In the packaging cells, the adapted NS1-GFP replicon produces titers of infectious particles of approximately 106 FFU\/mL and is genetically stable over five passages. The expressed NS1-GFP fusion protein localizes to the endoplasmic reticulum and co-fractionates with detergent-resistant heavy membranes, a hallmark of flavivirus replication organelles. This NS1-GFP replicon provides a novel platform for studying NS1 functions and can be further adapted for proximity-labeling strategies aimed at identifying the still-unknown protease responsible for NS1-NS2A cleavage.<\/jats:p>","DOI":"10.3390\/biology15030220","type":"journal-article","created":{"date-parts":[[2026,1,26]],"date-time":"2026-01-26T10:24:36Z","timestamp":1769423076000},"page":"220","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Replication-Competent Flavivirus Genome with a Stable GFP Insertion at the NS1-NS2A Junction"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2075-307X","authenticated-orcid":false,"given":"Pavel","family":"Tarlykov","sequence":"first","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin Hwy. 13\/5, Astana 010000, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6915-8207","authenticated-orcid":false,"given":"Bakytkali","family":"Ingirbay","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin Hwy. 13\/5, Astana 010000, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5719-6118","authenticated-orcid":false,"given":"Dana","family":"Auganova","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin Hwy. 13\/5, Astana 010000, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-5885-8963","authenticated-orcid":false,"given":"Tolganay","family":"Kulatay","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin Hwy. 13\/5, Astana 010000, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8885-2387","authenticated-orcid":false,"given":"Viktoriya","family":"Keyer","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin Hwy. 13\/5, Astana 010000, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7565-9454","authenticated-orcid":false,"given":"Sabina","family":"Atavliyeva","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin Hwy. 13\/5, Astana 010000, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-9532-6232","authenticated-orcid":false,"given":"Maral","family":"Zhumabekova","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin Hwy. 13\/5, Astana 010000, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-3594-425X","authenticated-orcid":false,"given":"Arman","family":"Abeev","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin Hwy. 13\/5, Astana 010000, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9880-9382","authenticated-orcid":false,"given":"Alexandr V.","family":"Shustov","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin Hwy. 13\/5, Astana 010000, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1007\/s00705-023-05835-1","article-title":"Renaming of the genus Flavivirus to Orthoflavivirus and extension of binomial species names within the family Flaviviridae","volume":"168","author":"Postler","year":"2023","journal-title":"Arch. 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