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However, studying NSP4 within infectious virus has been hindered by essential RNA packaging signals at the 5\u2032 end of gene segment 10 (gs10) and an DLP-binding domain at its C-terminus, both of which constrain conventional mutagenesis. Using a plasmid-based reverse genetics system, we generated six recombinant rotaviruses (rRVs) encoding tagged or reporter-fused NSP4 via three strategies: expression from an alternative segment (gs7) in a bicistronic arrangement, N-terminal tagging of gs10 with translation shifted downstream of the native packaging sequence, and retention of the gs10 5\u2032 UTR and fusion of the first 20 amino acids fused to a reporter followed by bicistronic full-length NSP4 expression. All six rRVs were replication competent, with gs10-based constructs closely matching wild-type replication kinetics while gs7-based constructs showed modest attenuation and, in one case, genetic instability upon passage. Engineered NSP4 proteins were robustly expressed, correctly glycosylated, and capable of oligomerization. Live-cell imaging showed reporter fluorescence reliably tracked NSP4 synthesis and preceded NSP4-dependent intercellular calcium signals. Affinity purification of tagged NSP4 recovered viroplasm-associated and structural viral proteins along with candidate host interactors, including ANP32A, ANP32E, PPM1G, and H2AC1. These rRVs constitute a validated toolkit for dissecting NSP4 function during bona fide infection and establish a generalizable strategy for engineering constrained rotavirus gene segments.<\/jats:p>","DOI":"10.64898\/2026.09.04.749488","type":"posted-content","created":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T12:40:22Z","timestamp":1788871222000},"source":"Crossref","is-referenced-by-count":0,"title":["Development of fluorescent and affinity tagged variants of rotavirus nonstructural protein 4 using the reverse genetics system"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-2060-7501","authenticated-orcid":false,"given":"Ethan M.","family":"Huleatt","sequence":"first","affiliation":[{"name":"Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, TX, 77030, USA"},{"name":"Alkek Center for Metagenomics & Microbiome Research, Baylor College of Medicine, Houston, TX, 77030, 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Molecular Virology & Microbiology, Baylor College of Medicine, Houston, TX, 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4813-4249","authenticated-orcid":false,"given":"Mary K.","family":"Estes","sequence":"additional","affiliation":[{"name":"Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, TX, 77030, USA"},{"name":"Alkek Center for Metagenomics & Microbiome Research, Baylor College of Medicine, Houston, TX, 77030, USA"},{"name":"Department of Medicine, Baylor College of Medicine, Houston, TX, 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5249-3482","authenticated-orcid":false,"given":"Joseph M.","family":"Hyser","sequence":"additional","affiliation":[{"name":"Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, TX, 77030, USA"},{"name":"Alkek Center for Metagenomics & Microbiome Research, Baylor 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