Fig 1: HSV-1 US3 phosphorylates HDAC6 to enhance its deacetylase activity and cytosolic interactions with TRIM56.(A) HDAC6 enzymatic activity assay was conducted by measuring AFC fluorescence intensity following infection with HSV-1 (MOI = 5) or treatment with tubacin (1 μM) in indicated time. Data were analyzed using the unpaired t test, which are shown as mean ± SD (n = 4 biological replicates). (B) Representative immunofluorescent images of HDAC6 (green) and nucleus (blue) in N2a cells infected with HSV-1 (MOI = 5) for the indicated time. Scale bars, 10 µm. The enlarged insets show the localization of HDAC6. Scale bars, 2 µm. (C) Western blot analysis of HDAC6 in cytosol and nucleus in N2a cells infected with HSV-1 (MOI = 5) for the indicated time, and histograms show quantified data (below). Data were analyzed using the unpaired t test, which are shown as mean ± SD (n = 3 biological replicates). (D) Co-IP analysis the TRIM56-HDAC6 interaction in cells infected with HSV-1 (MOI = 5) for the indicated times. (E) Western blot analysis of IRF3 and TBK1 in N2a cells transfected with HDAC6 WT or ∆NLS for 72 h followed by infection with HSV-1 (MOI = 3) for 12 h. (F) Co-IP and RT-qPCR assay were performed to detect the cGAS-bound viral DNA in N2a cells transfected with HDAC6 WT or ∆NLS for 72 h followed by infection with HSV-1 (MOI = 3) for 12 h. Data were analyzed using the unpaired t-test, which are shown as mean ± SD (n = 3 biological replicates). (G) Viral plaque assays in N2a cells transfected with HDAC6 WT or ∆NLS for 72 h followed by infection with HSV-1 (MOI = 3) for 12 h. Data were analyzed using the unpaired t test, which are shown as mean ± SD (n = 3 biological replicates). (H) HDAC6 and its phosphoserine were analyzed through Co-IP assay upon HSV-1 (MOI = 10) infection at different times. (I) Co-IP assay analysis of the US3-HDAC6 interaction and phosphoserine in N2a cells transfected with Flag-US3 for 48 h. (J) N2a cells were transfected with shRNA HDAC6 for 72 h and later transfected with Flag-US3 for 48 h. Western blotting was used to assess the levels of indicated proteins in N2a cells infected with HSV-1 (MOI = 3) for 12 h. Histograms show the quantification of P-TBK1 or P-IRF3 levels relative to that of TBK1 or IRF3. Data were analyzed using the unpaired t test, which are shown as mean ± SD (n = 3 biological replicates). Source data are available online for this figure.
Fig 2: HDAC6 interacts with and deacetylates TRIM56 at K110.(A) HDAC6-based immunoprecipitation and LC‒MS/MS (HDAC6 Interactome) to identify the interacting proteins of HDAC6 in N2a cells. (B) The Reactome enrichment analysis of HDAC6-interacting proteins. (C) Acetyl-lysine immunoprecipitation and LC‒MS/MS (Acetylome) on N2a cells treated with or without tubacin (1 μM) for 2 h. (D) Volcano plot displays the differential acetylation of amino acid sites, with red representing up-regulation and blue indicating downregulation (P < 0.05, log2 fold change ≥0.263). (E) Network diagram shows 83 HDAC6-inteacting proteins regulated by tubacin. Grey-bordered rectangles represent HDAC6-interacting proteins targeted by tubacin (TubA). The color changes in rectangles without grey borders signify the differences in acetylated site values following tubacin treatment. The purple rectangles depict antiviral-associated proteins that are targeted by tubacin and interact with HDAC6 proteins. (F) The Reactome enrichment analysis of acetylated proteins in the turquoise module. (G) The top 10 essential sites of the turquoise module were identified using the MCC algorithm. The importance of the sites in the network is indicated by the color saturation. (H) Co-IP analysis of the interaction between HDAC6 and TRIM56 in N2a cells infected with HSV-1 (MOI = 3) for 12 h. (I) Co-IP analysis of the interaction between HDAC6 and TRIM56 in 293T cells co-transfected with HA-HDAC6 and Flag-TRIM56. (J–L) Co-IP analysis of the acetylated TRIM56 in N2a cells treated with tubacin (1 μM) for 12 h (J), transfected with HDAC6 siRNA (K), transfected with HDAC6 WT or HD1/2 m (L). The black arrow indicates the protein band of acetylated TRIM56. (M) N2a cells were transfected with Flag-TRIM56 WT or K110R for 72 h followed by infection with HSV-1 (MOI = 3) for 12 h. Co-IP analysis of the acetylated Flag-TRIM56 in cell lysates. Source data are available online for this figure.
Fig 3: HDAC6 promotes HSV-1 infection.(A, B) RT-qPCR analysis of the mRNA expression of HDAC6 in N2a cells transfected with HDAC6 siRNA and infected with HSV-1 (MOI = 3) for 12 h. Data were analyzed using the unpaired t test, which are shown as mean ± SD (n = 3 biological replicates). (B, C) The DNA copy number of viral genes in BV2 (B) and HMC3 cells (C) infected with HSV-1 (MOI = 3) in the presence or absence of tubacin (1 μM) for 12 h. Data were analyzed using the unpaired t test, which are shown as mean ± SD (n = 3 biological replicates). (D, E) Viral plaque assays of BV2 (D) and HMC3 cells (E) treated with or without HSV-1 (MOI = 3) in the presence or absence of tubacin (1 μM) for 12 h. The number of plaques was calculated. Data were analyzed using the unpaired t test, which are shown as mean ± SD (n = 3 biological replicates). (F, G) Western blot assay was performed to assess the levels of ICP0 and gD in N2a cells infected with HSV-1 (MOI = 3) for 12 h followed by treatment with tubacin (1 μM) in the indicated times of HSV-1 infection. Source data are available online for this figure.
Fig 4: Acetylation of K110 within the TRIM56 B-box1 domain interacts with the HDAC6 and regulates IFN-β production.(A) Schematic illustration of HDAC6 and TRIM56 structure. FL, full length; CD1/2, histone deacetylase 1 and histone deacetylase 2 domain; B-box1, B box-type 1 domain. (B) N2a cells were infected with HSV-1 (MOI = 3) for 12 h after overexpression of HDAC6-FL, TRIM56-FL, HDAC6-CD1/2 or TRIM56 B-box1, and ELISA assay was conducted to detect IFN-β production. Data were analyzed using the unpaired t-test, which are shown as mean ± SD (n = 3 biological replicates). (C–E), N2a cells were transfected with ASO-TRIM56 B-box1. (C) RT-qPCR assay of TRIM56 mRNA expression. Data were analyzed using the unpaired t-test, which are shown as mean ± SD (n = 5 biological replicates). (D) Western blotting was used to assess the levels of indicated proteins in N2a cells infected with HSV-1 (MOI = 3) for 12 h. (E) ELISA was used to assess the levels of IFN-β in the culture supernatants. Data were analyzed using the unpaired t test, which are shown as mean ± SD (n = 3 biological replicates). (F, G), N2a cells transfected with TRIM56-FL or TRIM56 B-box1 for 72 h followed by infection with HSV-1 (MOI = 3) for 12 h. (F) ELISA was used to assess the levels of IFN-β in the culture supernatants. Data were analyzed using the unpaired t test, which are shown as mean ± SD (n = 3 biological replicates). (G) Co-IP assay of cGAS monoubiquitination. The black arrow indicates the protein band of cGAS monoubiquitination. (H–J) N2a cells were infected with HSV-1 (MOI = 3) for 12 h after overexpression of TRIM56 WT or K110A, and the effects on the cGAS-STING signaling pathway (H), IFN-β production (I), and viral plaque formation (J) were analyzed. Data were analyzed using the unpaired t test, which are shown as mean ± SD (n = 3 biological replicates). Source data are available online for this figure.
Fig 5: HDAC6 promotes HSV-1 infection.(A, B) N2a cells were transfected with negative control (NC) siRNA or HDAC6 siRNA for 72 h followed by infection with HSV-1 (MOI = 3) for 12 h. The formation of viral plaques (A) and viral titers (B) were measured. Data were analyzed using the unpaired t test, which are shown as mean ± SD (n = 3 biological replicates). (C, D) N2a cells were transfected with vector, HDAC6 WT or HDAC6 deacetylase activity mutation (HD1/2 m) for 72 h followed by infection with HSV-1 (MOI = 3) for 12 h. The formation of viral plaques was measured. Data were analyzed using the unpaired t test, which are shown as mean ± SD (n = 3 biological replicates). (E, F) Effect of tubacin (1 μM) on the DNA copy number of viral genes (E) and viral plaque formation (F). Data were analyzed using unpaired t test, which are shown as mean ± SD (n = 3 biological replicates). (G) Effect of tubacin (1 μM) on EGFP-HSV-1 (MOI = 1) infection for 24 h, scale bars, 200 μm. Source data are available online for this figure.
Supplier Page from Abcam for HDAC6 Activity Assay Kit (Fluorometric)