Expression and subcellular localization of zebrafish histone H2A. (A) The expressions of histone H2A and SVCV-N in zebrafish larvae in response to SVCV infection. Total RNA was extracted from zebrafish larvae collected at 6, 12, 24, 48 and 72 hpi. Data represented means ± SEM (n = 3), and were tested for statistical significance. *p < 0.05; **p < 0.01; ns, not significant. (B) Subcellular localization of zebrafish histone H2A by immunofluorescence analysis in the mock-infected and SVCV-infected ZF4 cells. Scale bar: 10 μm. (C) Cytoplasmic fractions from the mock-infected and SVCV-infected ZF4 cells. (D) Nuclear fractions from the mock-infected and SVCV-infected ZF4 cells. For (C, D), ZF4 cells cultured in 6-well plates overnight were infected with SVCV at an MOI of 0.1, 1, 5 and 10. At 24 hpi, the cells were harvested and used for preparation of nuclear and cytoplasmic extracts. (E) Cytoplasmic fractions from SVCV-infected, poly I:C-stimulated and untreated ZF4 cells. (F) Nuclear fractions from SVCV-infected, poly I:C-stimulated and untreated ZF4 cells. For (E, F), ZF4 cells passed in 6-well plates overnight were infected with SVCV at an MOI of 1 or stimulated with poly I:C (10 μg/mL) or left untreated. At 6, 24 and 48 hpi, the cells were harvested and used for preparation of nuclear and cytoplasmic extracts.

Zebrafish histone H2A negatively regulates RLR signaling. (A) Effect of zebrafish histone H2A on the IFN1 promoter activity mediated by poly I:C stimulation. (B) Effect of zebrafish histone H2A on the IFN1 promoter activity mediated by RLR signaling pathway. (C) Effect of zebrafish histone H2A on the IFN3 promoter activity mediated by poly I:C stimulation. (D) Effect of zebrafish histone H2A on the IFN3 promoter activity mediated by RLR signaling pathway. For (A, C), EPC cells transfected with various indicated plasmids and DNA concentration were stimulation with poly I:C. Another 24 h later, the cells were lysed and used for luciferase activity assay. For (B, D), EPC cells were transfected with the indicated combination. At 48 h post-transfection, the cells were used for luciferase activity assay. (E) Effect of zebrafish histone H2A on the larvae survival. (F) The confirmation of zebrafish histone H2A expression in the histone H2A-overexpressed larvae with SVCV infection. (G) Effect of zebrafish histone H2A on the expression of SVCV-N gene. For (E–G), zebrafish larvae microinjected with p3×FLAG or H2A-FLAG were infected with 2×10⁶ PFU/mL SVCV for 24 h. For (E), larvae were monitored for 6 days. For (F, G), larvae were collected at 24 and 48 hpi, and used for qRT-PCR. (H) Effect of zebrafish histone H2A on the expression of antiviral genes involved in RLR signaling pathway. Zebrafish larvae microinjected with p3×FLAG or H2A-FLAG were collected at 1, 3 and 7 dpf, and used for qRT-PCR. For (A–D, F–H), data represented means ± SEM (n = 3), and were tested for statistical significance. *p < 0.05; **p < 0.01; ns, not significant.

Zebrafish histone H2A interacts with TBK1 and IRF3 to disrupt the formation of TBK1-IRF3 complex. (A) Zebrafish histone H2A interacted with TBK1 and IRF3. (B) Zebrafish histone H2A colocalized with TBK1 and IRF3. (C) Zebrafish histone H2A interacted with TBK1 to inhibit the formation of TBK1-IRF3 complex. For (A, C), co-IP was performed with anti-FLAG-conjugated agarose beads in EPC cells. The cell lysates and bound proteins were analyzed by immunoblotting with the indicated Abs. All experiments were repeated for at least three times with similar results. The expression ratio for IRF3 protein was quantified by Quantity One. For (B), immunofluorescence analysis was performed in EPC cells transfected with H2A-FLAG and TBK1-GFP or IRF3-GFP. Scale bar: 25 μm.

Zebrafish histone H2A degrades TBK1 and IRF3 via the lysosomal pathway. (A) Effect of zebrafish histone H2A on the protein expression of TBK1. (B) Effect of zebrafish histone H2A on the protein expression of IRF3. For (A, B), EPC cells seeded in six-well plates were transfected with various indicated plasmids and DNA concentration. After 48 h post-transfection, cell lysates were analyzed by Western blotting using the indicated Abs. (C) Effect of MG132 on the histone H2A-mediated protein degradation of TBK1. (D) Effect of MG132 on the histone H2A-mediated protein degradation of IRF3. (E) Effect of 3-MA on the histone H2A-mediated protein degradation of TBK1. (F) Effect of 3-MA on the histone H2A-mediated protein degradation of IRF3. (G) Effect of CQ on the histone H2A-mediated protein degradation of TBK1. (H) Effect of CQ on the histone H2A-mediated protein degradation of IRF3. (I) Effect of NH₄Cl on the histone H2A-mediated protein degradation of TBK1. (J) Effect of NH₄Cl on the histone H2A-mediated protein degradation of IRF3. For (C–J), EPC cells seeded in six-well plates were transfected with various indicated plasmids and DNA concentration. After 48 h post-transfection, cells were treated with DMSO, MG132, 3-MA, CQ or NH₄Cl with indicated concentration for 6 h. Following this, cell lysates were analyzed by Western blotting using the indicated Abs. The expression ratio for TBK1 or IRF3 protein was quantified by Quantity One. All experiments were repeated for at least three times with similar results.

Zebrafish histone H2A inhibits the effects mediated by TBK1. (A) Effect of zebrafish histone H2A on the TBK1-mediated antiviral activity. (B) The confirmation of zebrafish histone H2A expression in the histone H2A-overexpressed ZF4 cells with SVCV infection. (C) Effect of zebrafish histone H2A on the expression of SVCV-N gene mediated by TBK1. (D) Effect of zebrafish histone H2A on the expression of SVCV-G gene mediated by TBK1. (E) Effect of zebrafish histone H2A on the expression of IFN1 mediated by TBK1. (F) Effect of zebrafish histone H2A on the expression of IFN3 mediated by TBK1. (G) Effect of zebrafish histone H2A on the expression of PKZ mediated by TBK1. (H) Effect of zebrafish histone H2A on the expression of RSAD2 mediated by TBK1. (I) Effect of zebrafish histone H2A on the expression of MxA mediated by TBK1. (J) Effect of zebrafish histone H2A on the expression of MxB mediated by TBK1. (K) Effect of zebrafish histone H2A on the expression of MxC mediated by TBK1. (L) Effect of zebrafish histone H2A on the expression of MxE mediated by TBK1. For (A–L) ZF4 cells were transfected with the indicated plasmids. At 36 h post-transfection, these cells were used for SVCV infection at an MOI of 1. At 48 hpi, the supernatants of transfected cells were collected for the determination of virus titers. The cell pellets of transfected cells were collected and used for qRT-PCR. Data represented means ± SEM (n = 3), and were tested for statistical significance. *p < 0.05; **p < 0.01. The asterisk above the error bars indicates statistical significance using the group microinjected with empty plasmid as the control group. The asterisk above the bracket indicates statistical significance between the two groups connected by the bracket.

Zebrafish histone H2A impairs the protein expression of cytoplasmic (A) and nuclear IRF3 protein (B). For (A, B), EPC cells seeded in 6-well plates were transfected with various indicated plasmids. After 36 h post-transfection, the cells were infected with SVCV. After 12 h, the cells were harvested and used for preparation of nuclear and cytoplasmic extracts. The expression ratio for IRF3 protein was quantified by Quantity One.

Histone H2A nuclear/cytoplasmic trafficking is essential for the negative regulation of histone H2A. (A) Effect of the inhibition of histone H2A nuclear/cytoplasmic trafficking on the protein degradation of cytoplasmic TBK1. (B) Effect of the inhibition of histone H2A nuclear/cytoplasmic trafficking on the protein degradation of nuclear TBK1. (C) Effect of the inhibition of histone H2A nuclear/cytoplasmic trafficking on the protein degradation of cytoplasmic IRF3. (D) Effect of the inhibition of histone H2A nuclear/cytoplasmic trafficking on the protein degradation of nuclear IRF3. For A–D), EPC cells seeded in 6-well plates were transfected with various indicated plasmids. After 36 h post-transfection, the cells were treated with LMB at the indicated concentration or left untreated. After 12 h, the cells were harvested and used for preparation of nuclear and cytoplasmic extracts. The expression ratio for TBK1 or IRF3 protein was quantified by Quantity One. (E) Effect of the inhibition of histone H2A nuclear/cytoplasmic trafficking on the histone H2A-mediated SVCV replication. (F) Effect of the inhibition of histone H2A nuclear/cytoplasmic trafficking on the expression of SVCV-N mediated by histone H2A. (G) Effect of the inhibition of histone H2A nuclear/cytoplasmic trafficking on the expression of SVCV-P mediated by histone H2A. (H) Effect of the inhibition of histone H2A nuclear/cytoplasmic trafficking on the expression of SVCV-G mediated by histone H2A. For (E–H), EPC cells seeded in 24-well plates were transfected with p3×FLAG or H2A-FLAG, then treated with LMB or left untreated. After 48 h posttransfection, the cells were infected with SVCV. Another 24 h later, the supernatants of infected cells were collected for the determination of virus titers. The cell pellets of infected cells were collected for qRT-PCR. Data represented means ± SEM (n = 3), and were tested for statistical significance. **p < 0.01; ns, not significant. The asterisk above the error bars indicates statistical significance using the group microinjected with empty plasmid as the control group. The asterisk above the bracket indicates statistical significance between the two groups connected by the bracket.

Proposed model illustrating how histone H2A is utilized by SVCV to evade host immune response. Histone H2A is nuclear-localized in the absence of pathogen infection. SVCV infection promotes histone H2A nuclear/cytoplasmic trafficking. In the cytoplasm, histone H2A degrades TBK1 and IRF3, which lead to the impaired formation of TBK1-IRF3 functional complex and the decreased expression of nuclear IRF3 protein. In the cell nucleus, the impaired expressions of IRF3 and/or histone H2A inhibit transcriptional regulation of immune genes, and impede the production of type I IFNs and ISGs in response to SVCV infection.