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Our previous work using\n                    <jats:italic>in vitro<\/jats:italic>\n                    immune cell models demonstrated that the mouse-specific orthopoxvirus ectromelia virus (ECTV) inhibits the noncanonical NF-\u03baB signaling pathway. Here, we investigated the impact of ECTV on noncanonical NF-\u03baB signaling in human epithelial HeLa cells. Tumor necrosis factor superfamily ligands were used to activate or modulate noncanonical NF-\u03baB signaling. Using fluorescence microscopy and immunoblot analyses, we found that ECTV reduced LIGHT (TNFSF14)- and lymphotoxin \u03b1\n                    <jats:sub>1<\/jats:sub>\n                    \u03b2\n                    <jats:sub>2<\/jats:sub>\n                    (LT\u03b1\n                    <jats:sub>1<\/jats:sub>\n                    \u03b2\n                    <jats:sub>2<\/jats:sub>\n                    )-induced nuclear accumulation of the NF-\u03baB subunits RelB and p52. ECTV infection alone increased whole-cell RelB and NF-\u03baB2\/p100 levels but did not significantly alter nuclear RelB levels. In TNF-\u03b1-stimulated cells, ECTV reduced levels of cellular inhibitor of apoptosis protein 2 (cIAP2), whereas in LT\u03b1\n                    <jats:sub>1<\/jats:sub>\n                    \u03b2\n                    <jats:sub>2<\/jats:sub>\n                    -stimulated cells, ECTV reduced levels of TNF receptor-associated factor 3 (TRAF3). RT-qPCR analysis revealed that ECTV upregulated\n                    <jats:italic>RELB<\/jats:italic>\n                    ,\n                    <jats:italic>NFKB2<\/jats:italic>\n                    , and\n                    <jats:italic>CHUK<\/jats:italic>\n                    expression. Viral infection also increased expression of\n                    <jats:italic>TNFAIP3<\/jats:italic>\n                    and\n                    <jats:italic>OTUD7B<\/jats:italic>\n                    , genes encoding regulators of noncanonical NF-\u03baB signaling. Moreover, infected cells exhibited increased expression of selected RelB-responsive genes, including\n                    <jats:italic>FBRS<\/jats:italic>\n                    ,\n                    <jats:italic>CCR10<\/jats:italic>\n                    , and\n                    <jats:italic>TNFRSF6B<\/jats:italic>\n                    . Together, these findings indicate that ECTV modulates components associated with noncanonical NF-\u03baB signaling in epithelial cells\n                    <jats:italic>in vitro<\/jats:italic>\n                    , without evidence of constitutive pathway activation.\n                  <\/jats:p>","DOI":"10.2478\/aite-2026-0025","type":"journal-article","created":{"date-parts":[[2026,10,17]],"date-time":"2026-10-17T14:57:13Z","timestamp":1792249033000},"source":"Crossref","is-referenced-by-count":0,"title":["Ectromelia Virus Reprograms the Noncanonical NF-\u03baB Signaling Pathway in HeLa Epithelial Cells"],"prefix":"10.2478","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9718-7657","authenticated-orcid":false,"given":"Justyna","family":"Struzik","sequence":"first","affiliation":[{"name":"Department of Preclinical Sciences, Faculty of Veterinary Medicine , Warsaw University of Life Sciences\u2013SGGW , Warsaw , Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5436-6556","authenticated-orcid":false,"given":"Lidia","family":"Szulc-D\u0105browska","sequence":"additional","affiliation":[{"name":"Department of Preclinical Sciences, Faculty of Veterinary Medicine , Warsaw University of Life Sciences\u2013SGGW , Warsaw , Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4824-1797","authenticated-orcid":false,"given":"Micha\u0142","family":"Koper","sequence":"additional","affiliation":[{"name":"Institute of Genetics and Biotechnology, Faculty of Biology , University of Warsaw , Warsaw , Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7865-400X","authenticated-orcid":false,"given":"Ma\u0142gorzata","family":"Giery\u0144ska","sequence":"additional","affiliation":[{"name":"Department of Preclinical Sciences, Faculty of Veterinary Medicine , Warsaw University of Life Sciences\u2013SGGW , Warsaw , Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"374","published-online":{"date-parts":[[2026,10,17]]},"reference":[{"key":"2026101714570883118_j_aite-2026-0025_ref_001","doi-asserted-by":"crossref","unstructured":"Alejo A, Ruiz-Arg\u00fcello MB, Pontejo SM et al. 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