The histone variant H3.3 regulates gene expression during lytic infection with herpes simplex virus type 1

Abstract

It has been proposed that incorporation of the histone variant H3.3 within actively transcribed regions of a genome helps to facilitate transcription. In this report we use lytic infection by herpes simplex virus type 1 (HSV-1) as a model to examine the temporal profile of histone H3 incorporation and to determine whether the variant histone H3.3 has a direct effect on transcription. We find that canonical H3.1 and variant H3.3 exhibit distinct temporal associations with the genome in cell lines expressing equal amounts of epitope-tagged H3 variants. At the earliest times examined after infection, the HSV-1 genome is incorporated into chromatin that predominantly contains the variant H3.3, whereas incorporation of canonical H3.1 occurs later in infection and is dependent on replication of the HSV-1 genome. Further, inhibition of H3.3 association, via reduced expression of the H3.3 chaperone HIRA, significantly reduces the levels of HSV-1 mRNA. These findings show that incorporation of H3.3 facilitates transcription, and they provide new evidence for a regulatory role of chromatin composition during HSV-1 acute infection.

FIG. 1.

HeLa cells stably expressing human Flag-tagged H3.1 and H3.3. Cell extracts were collected; inputs and Flag-immunoprecipitated samples were probed with monoclonal antibody to the Flag epitope. β-Actin was used as a loading control. All further experiments were carried out using clone 13 (Flag-H3.1) and clone 1 (Flag-H3.3). IP, immunoprecipitation; Western, Western blotting.

FIG. 2.

Flag-tagged H3.1 and H3.3 are differentially incorporated into the HSV genome during acute infection. In all cases cells were infected at an MOI of 1 with HSV-1 and harvested at the indicated time postinfection. Data for the promoter and transcribed regions of the ICP0 (A), tK (B), and VP16 (C) genes are presented. The data are presented as percentages of input signals. All data represent the average of three independent experiments, and error bars represent 1 standard deviation of the data.

FIG. 3.

Effect of PAA treatment on H3.1 and H3.3 incorporation. (A) The genome copy numbers for untreated and PAA-treated cells are shown. Genome copy number was quantified by real-time PCR and normalized to the number of cells. (B) The effect of PAA treatment on H3.1 incorporation in control cells and Flag-H3.1 cells. (C) PAA effect on H3.3 incorporation in control cells and Flag-H3.3 cells. All data represent the average of three independent experiments, and error bars represent 1 standard deviation of the data.

FIG. 4.

siRNA directed to the human HIRA transcript reduces the level of HIRA mRNA and protein. (A) Effect of siRNA treatment on mRNA levels for HIRA. Cells were transfected twice with siRNA at 48 and 24 h prior to infection. mRNA was isolated at 1, 3, 6, and 10 h after infection at an MOI of 1 with HSV-1. Relative mRNA levels were determined by quantitative PCR and normalized to 28S rRNA. All data represent the average of three independent experiments, and error bars represent 1 standard deviation of the data. (B) Western blot analysis was performed with HIRA-specific antibody; β-actin was used as a loading control. RT-PCR, reverse transcription-PCR.

FIG. 5.

HIRA knockdown lowers the incorporation of Flag-H3.3 within the HSV-1 genome. Flag-H3.3 incorporation was compared in control siRNA- and HIRA siRNA-treated cells stably expressing Flag-H3.3. Promoter and transcribed regions of ICP0 (A), tK (B), and VP16 (C) genes were examined by ChIP. Values are expressed as a percentage of input, and all data represent the average of three independent experiments; error bars represent 1 standard deviation of the data.

FIG. 6.

Lowered H3.3 at HSV-1 genes leads to a decrease in gene expression and genome copy number. (A) HeLa cells were treated with control siRNA or HIRA siRNA. Cells were transfected twice with siRNAs at 48 and 24 h prior to infection. mRNA was isolated at 1, 3, 6, and 10 h after infection at an MOI of 1 with HSV-1. Relative mRNA levels for ICP0, tK, and VP16 (A) and cyclin-G-associated kinase (B) were determined by quantitative PCR and normalized to 28S rRNA. (C) Genome copy number was quantified by real-time PCR and normalized to the number of cells. All data represent the average of three independent experiments, and error bars represent 1 standard deviation of the data. RT-PCR, reverse transcription-PCR.