Figures and data
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 and 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). Data are mean ± SEM; n = 3. **P < 0.01, ***P < 0.001. (E) Immunoblotting of the indicated proteins in HeLa cells from infected with HSV-1 (MOI = 1) for the indicated time points. (F) Immunoblotting of the indicated proteins in 3D4/21 cells from 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.
Viral infection triggers chromatin dysfunction and DDR activation via HDAC degradation.
(A, B and 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. Data are mean ± SEM; n = 100 cells/group. ***P < 0.001. (D and E) Comet assay assessing DNA damage in HeLa cells infected with HSV-1 (MOI = 1). Data are mean ± SEM; n = 40 cells/group. **P < 0.01, ***P < 0.001. (F and G) Comet assay assessing DNA damage in 3D4/21 cells infected with PRV (MOI = 1) at the indicated time points. Data are mean ± SEM; n = 40 cells/group. **P < 0.01, ***P < 0.001. (H and 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. ns, no significance. (L) Viral titers in HSV-1-infected HeLa cells (MOI = 2) treated with berzosertib (50 nM) or vehicle. Data are mean ± SEM; 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 (n = 12/group), ***P < 0.001.
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 FLAG-HDAC1 and FLAG-HDAC1 (K74R), and either 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 FLAG-HDAC1 and FLAG-HDAC1 (K74R), and either mock-infected or infected with HSV-1 (MOI = 1) at 24 hpi. Data are mean ± SEM; 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. Data are mean ± SEM; n = 3. **P < 0.01, ***P < 0.001.
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. Data are mean ± SEM; 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. Data are mean ± SEM; 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. Data are mean ± SEM; n = 3. **P < 0.01, ***P < 0.001.
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 and 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), Data are mean ± SEM; 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 and 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), Data are mean ± SEM; 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). Data are mean ± SEM; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 (K) Viral titer analysis in HeLa cells transfected with siHDAC1 transfected with FLAG-HDAC1,FLAG-HDAC1 (K74R) or FLAG-HDAC1 ΔNES, infected with HSV-1 (MOI = 1) at 24 hpi. Data are mean ± SEM; n = 3. **P < 0.01, ***P < 0.001.
A schematic model showing the targeted cytosolic degradation of class I histone deacetylases is essential for efficient alphaherpesvirus replication.
