Cellular SNF2H chromatin-remodeling factor promotes herpes simplex virus 1 immediate-early gene expression and replication

Abstract

Like other DNA viruses that replicate in the nucleus, herpes simplex virus 1 (HSV-1) regulates the association of histones with its genome to promote viral replication and gene expression. We previously demonstrated that SNF2H, a member of the ISWI family of chromatin-remodeling factors, is concentrated in HSV-1 replication compartments in the nuclei of infected cells, suggesting that this cellular enzyme plays a role in viral replication. We show here that small interfering RNA (siRNA)-mediated knockdown of SNF2H in HEp-2 cells resulted in an approximately 20-fold decrease in HSV-1 replication, arguing that SNF2H promotes efficient HSV-1 replication. Decreases in HSV-1 replication were observed with multiple SNF2H-specific siRNAs, and the extent of the replication decrease correlated with the amount of SNF2H knockdown, indicating that the phenotype resulted from decreased SNF2H levels rather than off-target effects of the siRNAs. We also observed a decrease in the accumulation of immediate-early (IE) gene products in HSV-1-infected cells in which SNF2H was knocked down. Histone H3 occupancy on viral promoters was increased in HSV-1-infected cells that were transfected with SNF2H-specific siRNAs, suggesting that SNF2H promotes removal of histones from viral promoters during infection. Furthermore, chromatin immunoprecipitation (ChIP) studies showed that SNF2H associated with the HSV-1 genome during infection, which suggests that SNF2H may directly remodel viral chromatin. We hypothesize that SNF2H is recruited to viral promoters during HSV-1 infection, where it can remodel the chromatin state of the viral genome, facilitate the transcription of immediate-early genes, and enhance viral replication.

FIG 1

Decreased SNF2H levels result in reduced HSV-1 replication. (A) Immunoblot measuring levels of SNF2H following transfection of HEp-2 cells with either SNF2H-specific siRNA or nontarget control siRNA. siRNA-transfected cells were either mock infected or infected with HSV-1 strain KOS at an MOI of 10 for the time indicated (hours postinfection [hpi]). The cellular GAPDH gene product serves as a recovery and loading control. (B) SNF2H knockdown following transfection of SNF2H-specific siRNA resulted in a decrease in HSV-1 replication relative to viral replication in cells transfected with nontarget control siRNA. siRNA-transfected cells were infected with HSV-1 at an MOI of 10, and at the times indicated, samples were harvested, and viral yield (PFU/ml) was determined by plaque assay on Vero cells. Results are averages of at least four independent experiments, and the error bars represent the standard errors of the means.

FIG 2

The extent of SNF2H knockdown correlates with reduction of HSV-1 replication. (A) Immunoblot evaluating the efficiency of SNF2H knockdown following transfection of HEp-2 cells with different siRNAs targeting SNF2H or a control nontargeting siRNA. The cellular GAPDH protein serves as a recovery and loading control. (B) The efficiency of SNF2H knockdown correlates with the extent of the observed decrease in HSV-1 replication. siRNA-transfected HEp-2 cells were infected with HSV-1 at an MOI of 10. At 24 hours postinfection, cultures were harvested, and viral yield was determined by plaque assay on Vero cells. Data are presented as the fold decreases in viral replication relative to the level of viral replication observed in HEp-2 cells transfected with nontarget control siRNA. Results are the averages of three independent experiments, and the error bars represent the standard errors of the means.

FIG 3

Reducing SNF2H levels results in decreased ICP0 accumulation. (A) Immunoblot measuring the levels of HSV-1 immediate-early proteins in SNF2H-specific siRNA-transfected HEp2 cells relative to nontarget control siRNA-transfected cells. The cells were either mock infected or infected with HSV-1 at an MOI of 10 for the time indicated. (B) Quantification of the immunoblot in panel A, as described in Materials and Methods. a.u. arbitrary units.

FIG 4

The extent of SNF2H knockdown correlates with the reduction of ICP0 levels. Immunoblot measuring HSV-1 ICP0 protein levels in cells transfected with SNF2H-specific or -nonspecific siRNAs. HEp-2 cells were transfected with either one of the four individual siRNAs targeting SNF2H, or the nontargeting negative-control siRNA and were then either mock infected or infected with HSV-1 at an MOI of 10 for the time indicated. The cellular GAPDH protein serves as a recovery and loading control.

FIG 5

SNF2H associates with the HSV-1 genome. Chromatin immunoprecipitation assay measuring SNF2H levels on viral promoters in HeLa cells that were transfected with a plasmid expressing SNF2H. Cells were infected with HSV-1 at an MOI of 20 for the time indicated and fixed with formaldehyde. Chromatin was prepared and subjected to immunoprecipitation using either an antibody specific for SNF2H (SNF2H Ab) or control rabbit IgG. Immunoprecipitated DNA was measured by real-time PCR. (A) ICP0 gene promoter; (B) ICP4 gene promoter; (C) ICP27 gene promoter; (D) NucE region of the cellular pS2 promoter. Data are presented as the percentages of DNA immunoprecipitated, and the values are the averages of three independent experiments. Error bars represent the standard errors of the means.

FIG 6

Histone H3 levels on viral promoters increase when SNF2H levels are reduced. Chromatin immunoprecipitation assay measuring histone H3 levels on viral promoters in HEp-2 cells that were transfected with SNF2H-specific or nontarget control siRNA. The cells were infected with HSV-1 at an MOI of 10 for the time indicated, and then the cells were fixed. Chromatin was prepared and subjected to immunoprecipitation using either an antibody specific for histone H3 or normal rabbit IgG. Immunoprecipitated DNA was measured by real-time PCR. (A) ICP0 gene promoter; (B) ICP4 gene promoter; (C) ICP27 gene promoter; (D) GAPDH pseudogene. Data are presented as the percentages of the DNA immunoprecipitated with background signal from the normal rabbit IgG subtracted. The values are averages of at least five independent experiments. Error bars represent the standard errors of the means.