Fig 1.
BET domain inhibition enhances HSV-1 and HSV-2 production.
(A) Titration of infectious virus production by plaque assay. Vero cells in triplicate were treated with a test compound or with 0.1% DMSO (Mock) at 2 hr prior to inoculation. HSV-1 or HSV-2 at 1 pfu/cell (MOI = 1) was used. The compounds were left throughout the infection. The samples were harvested at 24 hr PI and used to titrate infectious virus by plaque assay. The concentrations are as the following: JQ1 at 300 nM, PFI-1 at 500 nM, I-BET-762 (I-BET) at 1 μM, TG101348 (TG) at 3 μM, and TSA at 150 nM.
(B) Representative compounds JQ1 and PFI-1 on plaque sizes. Vero cells in 6-well plates were infected in the presence or absence of a test compound with approximately 50–100 pfu/well of HSV-1 or HSV-2. Wells from plaque assay were photographed and the sizes of 30 randomly selected plaques were measured using ImageJ. The relative sizes the plaques were plotted and presented as mean ± SD. Data were analyzed using paired T test for statistical significance. * denotes p<0.05, and ** as p<0.01.
Table 1.
Summary of names, PubChem IDs, and the minimal concentrations defined to enhance HSV-2 infection*.
The epigenetics compound library contains 129 small molecule compounds that are classified into inhibitors of histone deacetylases (HDACi), lysine demethylases, histone acetyltransferases (HATs), DNA methyltransferases (Dnmts), and the epigenetic reader domain inhibitors, to name a few. Thirteen compounds showed enhancement effect on infection, while no compounds showed inhibitory effect. To quantitatively describe their effect on HSV-2 infection, we determined the concentration that would cause 20% changes in cell viability from HSV-2-infected controls. In this regard, the compounds were series diluted and tested on Vero cells in triplicated samples. An MOI of 1 was used for infections. HSV infection causes cytolytic effect that was determined at 48–60 hr PI by an MTT assay.
Fig 2.
Bromodomain inhibition promotes HSV-1 and HSV-2 infection.
(A) Effect of BD1 and BD2 inhibition on HSV-1 infection. Vero cells were treated with BD1 inhibitor JQ1 (300 nM), BD2 inhibitor RVX-208 (RVX, 500 nM), or HDAC inhibitor TSA (150 nM). (-)JQ1, an inactive enantiomer of JQ1, at 300 nM was also included as a control. The cells were infected with HSV-1 or HSV-2 (1 MOI) at 2 hr post treatment. The cells were harvested at 24 hr PI and used for plaque assay. The data are presented as mean ± SEM of triplicate samples. (B and C) Dose effect of JQ1 on HSV infection. Vero cells were treated with JQ1 at concentrations as indicated. HSV-1 or HSV-2 at 1 MOI were used. The samples were harvested at 24 hr PI for virus titration by plaque assay (B) or used for protein expression analysis by western blot (C). The bar graph represents quantitative measurement of viral protein expression (relative to infected but untreated controls). The experiment was performed independently 3 times. Data are presented as mean ± SEM of two independent measurements. (D) Time effect of JQ1 addition on HSV infection. HeLa cells were treated with JQ1 at 300 nM at time points as indicated. The time of inoculation was counted as 0, and -2 hr refers to 2 hr prior to infection. In parallel experiments, the virus was incubated with 300 nM JQ1 at RT for 1 hr and then used to infect the cells after dilution (Tx-V). The cells were infected with HSV-1 at 0.5 MOI for 36 hr and virus production was determined by plaque assay. (E) JQ1 accelerates HSV-1 infection. HeLa cells were mock-treated or treated with 300 nM JQ1 prior to HSV-1 infection (MOI = 0.3). The samples were harvested at times as indicated and virus production was titrated.
Fig 3.
Dependency of BRD4 expression for HSV-1 infection and JQ1 activity.
HeLa cells were transfected with a control siRNA (siCtrl) or with siRNA (si-1, -2, -3) targeting BRD4. The cells were then used for infection (HSV-1, 1 MOI) at 48 hr post transfection. Virus production was determined by plaque assay, while viral protein synthesis was determined at 24 hr PI by immunoblotting. (A) siRNA treatment on BRD4 expression and on HSV-1 infection determined by immunoblotting for viral ICP0 and cellular BRD4 expression. Bar graph representative measurement of BRD4 and ICP0 expression. ** denotes p≤0.01. The experiment was performed independently 3 times. (B) Knockdown of BRD4 expression on infectious virus production. Data are presented as mean ± SEM of duplicated samples. (C, D, E) Suppression of BRD4, but not BRD2 or BRD3, inhibits HSV infection. The effect of individual siRNAs on BRD2, BRD3, and BRD4 on corresponding gene expression in HeLa cells was validated by real-time PCR. Gene expression was normalized to GAPDH and expressed as relative levels to siCtrl (C). Suppression of BRD4, but not BRD2 or BRD3 inhibited virus infection, determined by titration study (D) and by immunoblotting for HSV-1 proteins (E). F. Suppression of BRD4 expression ablates JQ1 enhancement effect on HSV-1 infection. Bar graphs at the right represent quantitative measurement of BRD4 and ICP0 protein bands. JQ1 was tested at 300 nM.
Fig 4.
Overexpression of BRD4 mutants on HSV-1 infection.
(A) Schematic drawing of BRD4 constructs. The DNA was subcloned into p3xFLAG-CMV-24 vector for mammalian cell expression. (B) BRD4 wt and its mutants were detected in the nuclei of transfected 293T cells. (C) Transient transfection of BRD4 wt had no effect on HSV-1 infection, determined by ICP0 and ICP4 expression. 293T cells in 6-well plates were transfected with p3xFLAG empty vector or with p3xFLAG-BRD4 wt at 0.3 and 1.0 μg for 24 hr. The cells were then infected with HSV-1 at 0.5 MOI for 24 hr. (D) Transient transfection of BD1, BD1/2 or ΔBD1 increases HSV-1 ICP0 and ICP4 expression. 293T cells were transfected and used as in (C). (E) BRD4 mutants promote HSV-1 and HSV-2 infection as were determined by plaque forming assay.
Fig 5.
HSV-1 infection induces protein complex formation and the association with viral gene promoters.
HeLa cells were infected with HSV-1 at 10 MOI for times as indicated. The samples were then processed for detection of protein association by immunoprecipitation assay, for protein co-localization by immunostaining, for protein-DNA interactions by a modified ChIP assay, respectively. (A) HSV-1 infection promotes protein association involving BRD4, CDK9, and RNAP II. Proteins in the lysates were used as control for input. The bar graph represents quantitative measurement of protein band intensities in anti-BRD4 immunocomplexes. The data are presented as mean ± SEM of 2 independent measurement. (B and C) Co-localization of BRD4 with CDK9 (B) and Rpb-1/RNAP II (C). The nuclei were stained using DAPI (blue). (D to G) Viral gene promoters in the BRD4 or Rbp-1/RNAP II immunocomplexes were determined by PCR (D, F) and real-time PCR (E, G). The GAPDH and IFNβ gene promoters in the complexes were included as controls for validation of ChIP assays.
Fig 6.
JQ1 increases BRD4 complex interaction with viral gene promoters.
HeLa cell were infected with HSV-1 at 10 MOI in the absence or presence of 500 nM JQ1 for 6 hr. Protein association and their interaction with viral gene promoters were determined by immunoprecipitation, by immunostaining, and by ChIP assays, respectively. (A) JQ1 promotes protein complex formation involving BRD4, CDK9, and Rbp-1/RNAP II. Right panel bar graphs represent quantitative measurement of protein band intensities from immunoprecipitation assay. (B) JQ1 promotes BRD4 co-localization with CDK9. The enlarged areas, labeled as a, b, and c, are presented underneath the images. DAPI (blue) staining shows the nuclei. (C, D) BRD4 association with viral and host gene promoters by qPCR measurement (C) and PCR (D). (E) JQ1 promotes viral DNA synthesis and induces BRD4 redistribution. Serum-starved HeLa cells were infected with HSV-1 (10 MOI) for 4 hr in the absence or presence of 500 nM JQ1. The cells were then labeled with EdU for another 4 hr. BRD4 (Red) was visualized by staining with anti-BRD4 antibody. Green: newly synthesized DNA.
Fig 7.
Requirement of CDK9 kinase activity for HSV-1 infection.
(A) JQ1 treatment promotes HSV-1 infection and increases the phosphorylation of Rbp-1/RNAP II. HeLa cells were infected with HSV-1 (1 MOI) in the absence or presence of 300 nM JQ1 for 24 hr. Protein expression and modification was determined using specific antibodies by immunoblotting. Lower panel bar graph represents quantitative measurement of ICP0 and phosphorylated Rpb-1 band intensities relative to GAPDH. (B) CDK9 inhibitor LDC000067 (LDC) dose-dependently blocks Rbp-1 phosphorylation (upper panels) and viral gene expression, determined by western blotting and qPCR, respectively. (C) Knock down of CDK9 expression blocks Rbp-1 phosphorylation and viral gene expression, determined by western blotting and qPCR, respectively. (D) CDK9 inhibitor LDC000067 (LDC) abolishes JQ1 effect on Rbp-1 phosphorylation and HSV-1 gene expression. (E) Knock down of CDK9 expression diminishes JQ1 effect on Rbp-1 phosphorylation and viral gene expression.
Fig 8.
Model of BRD4 regulation of herpes simplex virus replication.
BRD4 participates in epigenetics regulation by recognition of acetyl lysine residues with its bromodomains (BD1, BD2). BRD4 also regulates the transcription of cellular and viral genes by recruitment of the positive transcription elongation factor (P-TEFb) and transcriptional factors. P-TEFb is itself under a stringent control by the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP) complex. Infection by HSV-1 or HSV-2 causes BRD4 relocation and rededicates BRD4 and the protein complex to viral gene transcription. JQ1 (red dots) or BD1 domain proteins functions by dislodging BRD4 from chromatin association.