Epigenetic repression of herpes simplex virus infection by the nucleosome remodeler CHD3

Abstract

Upon infection, the genome of herpes simplex virus is rapidly incorporated into nucleosomes displaying histone modifications characteristic of heterochromatic structures. The initiation of infection requires complex viral-cellular interactions that ultimately circumvent this repression by utilizing host cell enzymes to remove repressive histone marks and install those that promote viral gene expression. The reversion of repression and activation of viral gene expression is mediated by the cellular coactivator HCF-1 in association with histone demethylases and methyltransferases. However, the mechanisms and the components that are involved in the initial repression remain unclear. In this study, the chromatin remodeler chromodomain helicase DNA binding (CHD3) protein is identified as an important component of the initial repression of the herpesvirus genome. CHD3 localizes to early viral foci and suppresses viral gene expression. Depletion of CHD3 results in enhanced viral immediate early gene expression and an increase in the number of transcriptionally active viral genomes in the cell. Importantly, CHD3 can recognize the repressive histone marks that have been detected in the chromatin associated with the viral genome and this remodeler is important for ultimately reducing the levels of accessible viral genomes. A model is presented in which CHD3 represses viral infection in opposition to the actions of the HCF-1 coactivator complex. This dynamic, at least in part, determines the initiation of viral infection.

Importance: Chromatin modulation of herpesvirus infection is a dynamic process involving regulatory components that mediate suppression and those that promote viral gene expression and the progression of infection. The mechanisms by which the host cell employs the assembly and modulation of chromatin as an antiviral defense strategy against an invading herpesvirus remain unclear. This study defines a critical cellular component that mediates the initial repression of infecting HSV genomes and contributes to understanding the dynamics of this complex interplay between host cell and viral pathogen.

FIG 1

Repression and activation of the HSV genome through opposing chromatin modulation activities. Upon infection, the HSV genome is subject to impacts of cellular chromatin modulation machinery that suppresses the virus by the assembly of the genome into heterochromatic structures. In opposition to this, the HCF-1 coactivator complex contains histone modification activities that circumvent the accumulation of repressive marks and promote the installation of positive marks.

FIG 2

Depletion of chromodomain helicase DNA binding (CHD) proteins identifies CHD3 as a repressor of HSV-1 IE gene expression. (A) Phylogenetic tree of human CHD proteins generated by the Clustal W method. (B) HeLa cells were depleted of CHD proteins using siRNA pools and infected with HSV-1 (1.0 PFU/cell) for 2 h. The levels of viral IE ICP27 mRNA are expressed as ratios to the levels in control siRNA (siCntrl)-treated cells. *, significant difference relative to siRNA control (2-tailed t test, n = 4). (C and D) HeLa cells were depleted of CHD3 or CHD4, followed by infection with HSV-1 (1.0 PFU/cell) for 1.5 h. The levels of viral IE (ICP0, ICP4, ICP22, ICP27) and E (UL29, UL30, UL52) mRNAs are expressed as ratios to the levels in siCntrl-treated cells. (E to F) HeLa cells were depleted of CHD3 and CHD4 proteins using siRNA pools (p) or individual siRNAs (si-1, si-2) and infected with HSV-1 (1.0 PFU/cell) for 1.5 h. The levels of viral (ICP27) and cellular (TBP) control mRNAs are expressed as ratios to the levels in control siRNA-treated cells. (G) Western blot of IE proteins (ICP0, ICP4) in CHD3- or CHD4-depleted cells. The ratios to levels in control siRNA-treated cells are shown and are normalized to the actin-loading control.

FIG 3

CHD3 localizes to early viral foci during initiation of infection. (A) MRC-5 cells were depleted of CHD3, CHD4, or CHD6 and infected with HSV-1 (1.0 PFU/cell) for the indicated times. The levels of viral IE (ICP27, ICP22, ICP4) mRNAs are shown relative to siRNA control-treated cells. *, significant difference relative to siRNA control (multiple t test, n = 3). (B and C) Confocal images of MRC-5 cells infected with HSV-1 (10.0 PFU/cell) for 1.5 h. Cells were stained for DAPI, CHD3, and ICP4. The IE protein ICP4 was used as a marker for early viral foci. (C) 3-D volume of an infected cell illustrating the colocalization of CHD3 (green) with ICP4 (red). DAPI, blue.

FIG 4

A large percentage of infecting HSV genomes are repressed by CHD3. (A) HFF cells were infected with HSV-1 (0.001 PFU/cell) for 24 h followed by CSK treatment as described in Materials and Methods. Cells were stained for DAPI, CHD3, and ICP4. (B) MRC-5 cells were depleted of CHD3 or CHD4 and infected with HSV-1 (10.0 PFU/cell) for 2 h. The numbers and sizes (small, 0.4 to 1.0 µM; medium, 1.0 to 2.5 µM; large, >2.5 µM) of ICP4 foci are expressed as ratios to those in siRNA control-treated cells (siCntrl) (n > 270 cells). *, significant relative to siRNA control (ANOVA with Dunnett’s post hoc multiple comparisons test, n = 270).

FIG 5

Depletion of CHD3 compensates for TCP-mediated repression of IE gene expression. Control and CHD3-depleted HeLa cells were treated with DMSO or TCP and subsequently infected with HSV-1 (1.0 PFU/cell) for 1.5 h (A) or 2 and 4 h (B). The levels of viral (ICP27, ICP4, ICP0) and cellular control (TBP, LSD1, actin) mRNAs (A) and proteins (B) are expressed as ratios to the levels in siRNA control-treated cells.

FIG 6

CHD3 promotes FAIRE insolubility of the HSV-1 genome. FAIRE insolubility of the HSV genome is enhanced by TCP treatment and reduced by depletion of CHD3. HeLa cells were infected with HSV-1 (1.0 PFU/cell) for 4 h. The levels of HSV-1 genomes (ICP0) were determined in FAIRE (soluble) and insoluble chromatin (compacted) fractions. (A) Cells were treated with TCP (2.0 mM) for 3 h; (B) cells were transfected with control or CHD3 siRNAs; (C) control or CHD3 siRNA transfected cells were treated with DMSO or TCP. The results are shown relative to the total extracted DNAs.