Ectromelia Virus Reprograms the Noncanonical NF-κB Signaling Pathway in HeLa Epithelial Cells

The noncanonical nuclear factor κB (NF-κB) pathway regulates adaptive immune responses and antiviral innate immunity. Orthopoxviruses, including the human pathogen variola virus and zoonotic viruses such as vaccinia virus and monkeypox virus, have evolved multiple strategies to suppress host antiviral signaling. Our previous work using in vitro immune cell models demonstrated that the mouse-specific orthopoxvirus ectromelia virus (ECTV) inhibits the noncanonical NF-κB signaling pathway. Here, we investigated the impact of ECTV on noncanonical NF-κB signaling in human epithelial HeLa cells. Tumor necrosis factor superfamily ligands were used to activate or modulate noncanonical NF-κB signaling. Using fluorescence microscopy and immunoblot analyses, we found that ECTV reduced LIGHT (TNFSF14)- and lymphotoxin α₁β₂ (LTα₁β₂)-induced nuclear accumulation of the NF-κB subunits RelB and p52. ECTV infection alone increased whole-cell RelB and NF-κB2/p100 levels but did not significantly alter nuclear RelB levels. In TNF-α-stimulated cells, ECTV reduced levels of cellular inhibitor of apoptosis protein 2 (cIAP2), whereas in LTα₁β₂-stimulated cells, ECTV reduced levels of TNF receptor-associated factor 3 (TRAF3). RT-qPCR analysis revealed that ECTV upregulated RELB, NFKB2, and CHUK expression. Viral infection also increased expression of TNFAIP3 and OTUD7B, genes encoding regulators of noncanonical NF-κB signaling. Moreover, infected cells exhibited increased expression of selected RelB-responsive genes, including FBRS, CCR10, and TNFRSF6B. Together, these findings indicate that ECTV modulates components associated with noncanonical NF-κB signaling in epithelial cells in vitro, without evidence of constitutive pathway activation.