The targeted cytosolic degradation of class I histone deacetylases is essential for efficient alphaherpesvirus replication
- College of Veterinary Medicine, Henan Agricultural University, China
- Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs, China
- Key Laboratory of Animal Growth and Development of Henan Province, Henan Agricultural University, China
- State Key Laboratory of Membrane Biology, School of Pharmaceutical Sciences, Tsinghua University, China
- Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, China
- International Joint Research Center of National Animal Immunology, Henan Agricultural University, China
Figures
Figure 1
Viral infection induces degradation of class I HDACs and hyperacetylation of histones H3 and H4.
(A) Immunoblotting analysis of the indicated HDACs in HeLa cells infected with HSV-1 (MOI = 1) for the indicated time points. (B) Immunoblotting analysis of the indicated HDACs in 3D4/21 cells infected with PRV-QXX (MOI = 1) for the indicated time points. (C, D) qRT-PCR analysis of HDAC1 and HDAC2 mRNA levels, normalized to β-actin expression, in HeLa cells infected with HSV-1 (C) or 3D4/21 cells infected with PRV-QXX (D) (MOI = 1). Statistical significance of the different time points in comparison with the control (0 h) is shown in the table (mean ± SD; n=3; **p<0.01, ***p<0.001). (E) Immunoblotting of the indicated proteins in HeLa cells infected with HSV-1 (MOI = 1) for the indicated time points. (F) Immunoblotting of the indicated proteins in 3D4/21 cells infected with PRV-QXX (MOI = 1) for the indicated time points. (G) Immunoblotting analysis of the indicated proteins in liver tissues from mice mock-infected or infected with HSV-1 (1×10⁶ pfu per mouse) at 5 days post-infection (n=3). (H) Immunoblotting of the indicated proteins in HeLa cells transfected with siControl or siHDAC1/2. Note: The numerical values beneath each lane in the western blot images represent the relative abundance of the corresponding protein bands, as quantified by densitometric analysis and normalized to the signal intensity of the first lane, which serves as the control group.
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Figure 1—source data 1
PDF file containing original western blots for Figure 1A, B, E–H, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/110309/elife-110309-fig1-data1-v1.zip
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Figure 1—source data 2
Original files for western blot analysis displayed in Figure 1A, B, E–H.
- https://cdn.elifesciences.org/articles/110309/elife-110309-fig1-data2-v1.zip
Figure 2
Viral infection triggers chromatin dysfunction and DDR activation via HDAC degradation.
(A–C) Immunofluorescence staining of γ-H2AX (red) and H2AX (green) in HSV-1-infected HeLa cells (A) or PRV-infected 3D4/21 cells (B) (MOI = 1). Panel (C) presents the quantitative analysis of γ-H2AX foci intensity or nuclear fluorescence signal derived from panels (A) and (B). Scale bars: 10 μm. Statistical significance of the different time points in comparison with the control (0 h) is shown in the table (mean ± SD; n=100 cells/group; ***p<0.001). (D, E) Comet assay assessing DNA damage in HeLa cells infected with HSV-1 (MOI = 1). Scale bars: 10 μm. Statistical significance of the different time points in comparison with the control (0 h) is shown in the table (mean ± SD; n=40 cells/group; **p<0.01, ***p<0.001). (F, G) Comet assay assessing DNA damage in 3D4/21 cells infected with PRV (MOI = 1) at the indicated time points. Statistical significance of the different time points in comparison with the control (0 h) is shown in the table (mean ± SD; n=40 cells/group; **p<0.01, ***p<0.001). (H, I) Immunoblotting analysis of DDR markers (p-ATM, p-ATR, p-Chk1, p-Chk2, γ-H2AX) and viral ICP4/EP0 in HSV-1-infected HeLa cells (G) or PRV-QXX-infected 3D4/21 cells (H) (MOI = 1). (J) Immunoblotting analysis of DDR markers (p-ATM, p-ATR, γ-H2AX) and HDAC1/HDAC2 in HeLa cells transfected with siHDAC1/2. (K) HeLa cells were treated with vehicle and berzosertib (0–60 μM) for 0–48 h. Cell proliferation was analyzed by CCK-8 assay. Statistical significance of the different time points in comparison with the control (0 μM) is shown in the table (mean ± SD; ns, no significance). (L) Viral titers in HSV-1-infected HeLa cells (MOI = 2) treated with berzosertib (50 nM) or vehicle. t=0 h defined as time of medium replacement post-adsorption. Statistical significance of the different time points in comparison with the control (vehicle) is shown in the table (mean ± SD; n=3. *p<0.05, **p<0.01, ***p<0.001). (M) Survival curves of HSV-1-infected mice (1×10⁶ PFU/mouse) treated with berzosertib (20 mg/kg) or vehicle. Statistical significance of berzosertib treatment (10-day regimen) versus vehicle control in HSV-1-infected groups is summarized in the table (mean ± SD; n=12/group, ***p<0.001). Note: The numerical values beneath each lane in the western blot images represent the relative abundance of the corresponding protein bands, as quantified by densitometric analysis and normalized to the signal intensity of the first lane, which serves as the control group.
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Figure 2—source data 1
PDF file containing original western blots for Figure 2H–J, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/110309/elife-110309-fig2-data1-v1.zip
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Figure 2—source data 2
Original files for western blot analysis displayed in Figure 2H–J.
- https://cdn.elifesciences.org/articles/110309/elife-110309-fig2-data2-v1.zip
Figure 3
Viral infection induces K63-linked ubiquitination of HDAC1/2.
(A) Immunoblotting analysis of the indicated proteins in HeLa cells either mock-infected or infected with HSV-1 (MOI = 1), treated with vehicle, 3-MA (10 μM), MG-132 (10 μM), or chloroquine (10 μM) for the indicated times. (B) Ubiquitination assay of HDAC1 in HeLa cells either mock-infected or infected with HSV-1 (MOI = 1) at 24 hpi. (C) Ubiquitination assay of FLAG-HDAC1 in HeLa cells transfected with HA-ubiquitin variants (WT, K48, K63), and either mock-infected or infected with HSV-1 (MOI = 1) at 24 hpi. (D) Schematic representation of HDAC1 deletion mutants. (E–I) Ubiquitination assays of FLAG-HDAC1 mutant variants in HeLa cells either mock-infected or infected with HSV-1 (MOI = 1) at 24 hpi. (J) Ubiquitination assays of FLAG-HDAC2 variants (WT and K75R) in HeLa cells either mock-infected or infected with HSV-1 (MOI = 1) at 24 hpi. (K) Immunoblotting analysis of the indicated proteins in HeLa cells transfected with empty vector, FLAG-HDAC1 (wild-type), or FLAG-HDAC1 (K74R), followed by mock-infected or infected with HSV-1 (MOI = 1) at 24 hpi. (L) qRT-PCR analysis of HSV-1 ICP0, ICP8, and gB mRNA levels, normalized to β-actin expression, in HeLa cells transfected with empty vector, FLAG-HDAC1 (wild-type), or FLAG-HDAC1 (K74R), followed by mock-infected or infected with HSV-1 (MOI = 1) at 24 hpi. Statistical significance was determined: (i) relative to the empty-vector control group, and (ii) between cells transfected with FLAG-HDAC1 versus those transfected with FLAG-HDAC1 (K74R), as summarized in the table (mean ± SD; n=3; ns >0.05, *p<0.05, **p<0.01, ***p<0.001). (M) Viral titer analysis in HeLa cells transfected with FLAG-HDAC1 or FLAG-HDAC1 (K74R), and either mock-infected or infected with HSV-1 (MOI = 1) at 24 hpi. Statistical significance was determined: (i) relative to the empty-vector control group and (ii) between cells transfected with FLAG-HDAC1 versus those transfected with FLAG-HDAC1 (K74R), as summarized in the table (mean ± SD; n=3; **p<0.01, ***p<0.001). Note: The numerical values beneath each lane in the western blot images represent the relative abundance of the corresponding protein bands, as quantified by densitometric analysis and normalized to the signal intensity of the first lane, which serves as the control group.
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Figure 3—source data 1
PDF file containing original western blots for Figure 3A–C, E–K, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/110309/elife-110309-fig3-data1-v1.zip
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Figure 3—source data 2
Original files for western blot analysis displayed in Figure 3A–C, E–K.
- https://cdn.elifesciences.org/articles/110309/elife-110309-fig3-data2-v1.zip
Figure 4
MDM2 mediates viral-induced HDAC1/2 degradation via K74/K75 ubiquitination.
(A) qRT-PCR analysis validation of E3 ligase knockdown efficiency (shPIRH2, shKCTD11, shMDM2, shCHFR, shUHRF1, shTRIM46) in HeLa cells. Statistical significance of shRNA-mediated knockdown relative to the scramble control was assessed, and the results are summarized in the table (mean ± SD; n=3; ***p<0.001). (B) Immunoblotting analysis of HDAC1/2 stability in E3 ligase-knockdown HeLa cells infected with HSV-1 (MOI = 1). (C) Co-IP assay of endogenous HDAC1-MDM2 interaction in HSV-1-infected HeLa cells (MOI = 1; 24 hpi). (D) Co-IP assay of FLAG-HDAC1 with EGFP-MDM2 (WT or ΔRING) in HSV-1-infected HeLa cells (MOI = 1; 24 hpi). (E) Co-IP assay of FLAG-HDAC1 (WT or K74R) with EGFP-MDM2 in HSV-1-infected HeLa cells (MOI = 1; 24 hpi). (F) Ubiquitination assays of FLAG-HDAC1 (WT or K74R) co-expressed with EGFP-MDM2 (WT or ΔRING) in HSV-1-infected HeLa cells (MOI = 1; 24 hpi). (G–I) qRT-PCR analysis of HSV-1 ICP0 (H), ICP8 (I), and gB (J) mRNA levels, normalized to β-actin expression, in Scramble and shMDM2 HeLa cells, and either mock-infected or infected with HSV-1 (MOI = 1) at 24 hpi. Statistical significance of shMDM2-mediated knockdown relative to the scramble control was assessed at each time point and is summarized in the table (mean ± SD; n=3; ns >0.05, *p<0.05, **p<0.01, ***p<0.001). (J) Viral titer analysis in Scramble and shMDM2 HeLa cells, either mock-infected or infected with HSV-1 (MOI = 2) at 24 hpi. t=0 h defined as time of medium replacement post-adsorption. Statistical significance of shMDM2-mediated knockdown relative to the scramble control was assessed at each time point and is summarized in the table (mean ± SD; n=3; **p<0.01, ***p<0.001). Note: The numerical values beneath each lane in the western blot images represent the relative abundance of the corresponding protein bands, as quantified by densitometric analysis and normalized to the signal intensity of the first lane, which serves as the control group.
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Figure 4—source data 1
PDF file containing original western blots for Figure 4B–F, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/110309/elife-110309-fig4-data1-v1.zip
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Figure 4—source data 2
Original files for western blot analysis displayed in Figure 4B–F.
- https://cdn.elifesciences.org/articles/110309/elife-110309-fig4-data2-v1.zip
Figure 5
Cytoplasmic translocation enables MDM2-mediated HDAC1 degradation.
(A) HeLa cells were treated with vehicle and LMB (0–60 μM) for 0–48 h. Cell proliferation was analyzed by CCK-8 assay. ns, no significance. (B, C) Immunofluorescence staining of HDAC1 (red) and viral ICP4 (green) in HSV-1-infected HeLa cells (MOI = 1)±leptomycin B (LMB; 10 nM; 24 h). Scale bar: 10 μm. (C) shows the quantitative analysis of (B), Statistical significance was determined for: (i) HSV-1 infection versus no infection in the absence of LMB; and (ii) HSV-1 infection with LMB treatment versus vehicle control, as summarized in the table (mean ± SD; n=100; ***p<0.001). (D) CHX chase assay measuring HDAC1/2 degradation kinetics in HSV-1-infected HeLa cells (MOI = 1)±LMB (10 μM). (E) Ubiquitination assay of HDAC1 in HSV-1-infected HeLa cells (MOI = 1)±LMB (10 μM; 24 h). (F) Immunoblotting analysis of ubiquitination in HDAC1 from cytosolic and nuclear fractions of HeLa cells infected with HSV-1. (G, H) Immunofluorescence analysis of MDM2 (red) and ICP4 (green) in HSV-1-infected HeLa cells (MOI = 1)±LMB (10 μM; 24 h). Scale bar: 10 μm. (H) shows the quantitative analysis of (G), Statistical significance was determined for: (i) HSV-1 infection versus no infection in the absence of LMB; and (ii) HSV-1 infection with LMB treatment versus vehicle control, as summarized in the table (mean ± SD; n=100; ***p<0.001). (I) Immunoblotting analysis of DDR markers (γ-H2AX) and HDAC1/2 in HSV-1-infected HeLa cells (MOI = 1)±LMB (10 μM). (J) Viral titers in HSV-1-infected HeLa cells (MOI = 2)±LMB (10 μM). t=0 h defined as time of medium replacement post-adsorption. Statistical significance of LMB treatment versus vehicle control in HSV-1-infected groups is summarized in the table (mean ± SD; n=3; *p<0.05, **p<0.01, ***p<0.001). (K) Viral titer analysis in HeLa cells transfected with siHDAC1 transfected with empty vector, FLAG-HDAC1, FLAG-HDAC1 (K74R), or FLAG-HDAC1 ∆NES, infected with HSV-1 (MOI = 1) at 24 hpi. Statistical significance was determined: (i) relative to the empty-vector control group; and (ii) among cells transfected with FLAG-HDAC1, FLAG-HDAC1 (K74R), and FLAG-HDAC1 ∆NES, as summarized in the table (mean ± SD; n=3; **p<0.01, ***p<0.001). Note: The numerical values beneath each lane in the western blot images represent the relative abundance of the corresponding protein bands, as quantified by densitometric analysis and normalized to the signal intensity of the first lane, which serves as the control group.
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Figure 5—source data 1
PDF file containing original western blots for Figure 5D–F, I, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/110309/elife-110309-fig5-data1-v1.zip
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Figure 5—source data 2
Original files for western blot analysis displayed in Figure 5D–F, I.
- https://cdn.elifesciences.org/articles/110309/elife-110309-fig5-data2-v1.zip
Figure 6
A schematic model showing the targeted cytosolic degradation of class I histone deacetylases is essential for efficient alphaherpesvirus replication.
Author response image 1
Author response image 2
Author response image 3
Additional files
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Supplementary file 1
List of shRNAs and primers used in this study.
- https://cdn.elifesciences.org/articles/110309/elife-110309-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/110309/elife-110309-mdarchecklist1-v1.docx
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The targeted cytosolic degradation of class I histone deacetylases is essential for efficient alphaherpesvirus replication
eLife 15:RP110309.
https://doi.org/10.7554/eLife.110309.4
