Effect of monkeypox virus B14R protein fragments binding to IL-1β on apoptosis in HeLa cells.

BACKGROUND

Interleukin-1 beta (IL-1β) is a key mediator of antiviral immunity, and B14R proteins encoded by monkeypox virus (MPV) can interfere with its signaling. This study investigates the differences in IL-1β-binding capacity and apoptotic regulatory effects between two B14R protein isoforms encoded by MPV clades: the truncated B14R-180 (West African clade) and the full-length B14R-326 (Central African clade).

METHODS

This study employed bioinformatics approaches to predict the binding affinity between B14R and IL-1β, and validated the findings through Enzyme-Linked Immunosorbent Assay (ELISA), flow cytometry, Quantitative RT-PCR (qRT-PCR), and Western blot analyses.

RESULTS

the research demonstrates that B14R-180 exhibits superior binding efficiency to IL-1β compared to B14R-326, attributed to more hydrogen bonds (9 vs. 7), lower binding free energy (ΔG = -12.4 vs. -2.0 kJ/mol), and optimized binding interfaces. ELISA confirms the significantly higher binding affinity of B14R-180 for IL-1β (P < 0.05), with stronger competitive inhibition by anti-IL-1β antibodies in vaccinia-immune sera. Flow cytometry reveals that the combination of B14R-180 and IL-1β significantly reduces cell apoptosis rates in HeLa cells after 48 h (h) (P < 0.05), whereas B14R-326 has no significant effect. Subsequent qRT-PCR indicates that the B14R-180/IL-1β co-treatment downregulates the transcriptional expression of caspase-3, caspase-8, caspase-9, and caspase-10 (P < 0.001). Notably, caspase-3 exhibited dual-level suppression, with significant reductions in both mRNA and protein abundance.

CONCLUSIONS

These results reveal distinct anti-apoptotic profiles of B14R isoforms and suggest that B14R-180 may enhance MPV immune evasion through high-affinity IL-1β binding. These findings provide critical mechanistic insights to guide the development of MPV vaccines and targeted therapeutic strategies.