Effect of monkeypox virus B14R protein fragments binding to IL-1β on apoptosis in HeLa cells
Xiaojuan Zhu1, Yunfeng Shan1, Qiao Qiao1
1NHC Key Laboratory of Enteric Pathogenic Microbiology, Jiangsu Provincial Medical Key Laboratory of Pathogenic Microbiology in Emerging Major Infectious Diseases, Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, 210009, China.
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.
Insights
Monkeypox virus B14R-180 protein binds Interleukin-1 beta (IL-1β) more effectively than B14R-326, reducing cell apoptosis. This suggests B14R-180 enhances monkeypox virus immune evasion.
Area of Science:
- Virology
- Immunology
- Structural Biology
Background:
- Interleukin-1 beta (IL-1β) is crucial for antiviral immunity.
- Monkeypox virus (MPV) B14R proteins can inhibit IL-1β signaling.
- Two MPV B14R isoforms, B14R-180 and B14R-326, exhibit distinct structures and origins.
Purpose of the Study:
- To compare the IL-1β binding capacity of MPV B14R-180 and B14R-326.
- To investigate the differential effects of these isoforms on apoptosis.
- To elucidate the mechanisms underlying MPV immune evasion strategies.
Main Methods:
- Bioinformatics analysis to predict binding affinity.
- Enzyme-Linked Immunosorbent Assay (ELISA) to validate binding.
- Flow cytometry to assess apoptosis rates.
- Quantitative RT-PCR (qRT-PCR) and Western blot to analyze caspase expression.
Main Results:
- B14R-180 demonstrated significantly higher IL-1β binding affinity than B14R-326.
- B14R-180, in combination with IL-1β, significantly reduced apoptosis in HeLa cells.
- B14R-180/IL-1β co-treatment downregulated key caspases (caspase-3, -8, -9, -10) at both transcriptional and protein levels.
Conclusions:
- MPV B14R isoforms possess distinct anti-apoptotic activities.
- High-affinity IL-1β binding by B14R-180 likely contributes to MPV immune evasion.
- Findings offer insights for developing MPV vaccines and therapeutics.
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