Herpes simplex virus VP16, but not ICP0, is required to reduce histone occupancy and enhance histone acetylation on viral genomes in U2OS osteosarcoma cells

Abstract

The herpes simplex virus (HSV) genome rapidly becomes associated with histones after injection into the host cell nucleus. The viral proteins ICP0 and VP16 are required for efficient viral gene expression and have been implicated in reducing the levels of underacetylated histones on the viral genome, raising the possibility that high levels of underacetylated histones inhibit viral gene expression. The U2OS osteosarcoma cell line is permissive for replication of ICP0 and VP16 mutants and appears to lack an innate antiviral repression mechanism present in other cell types. We therefore used chromatin immunoprecipitation to determine whether U2OS cells are competent to load histones onto HSV DNA and, if so, whether ICP0 and/or VP16 are required to reduce histone occupancy and enhance acetylation in this cell type. High levels of underacetylated histone H3 accumulated at several locations on the viral genome in the absence of VP16 activation function; in contrast, an ICP0 mutant displayed markedly reduced histone levels and enhanced acetylation, similar to wild-type HSV. These results demonstrate that U2OS cells are competent to load underacetylated histones onto HSV DNA and uncover an unexpected role for VP16 in modulating chromatin structure at viral early and late loci. One interpretation of these findings is that ICP0 and VP16 affect viral chromatin structure through separate pathways, and the pathway targeted by ICP0 is defective in U2OS cells. We also show that HSV infection results in decreased histone levels on some actively transcribed genes within the cellular genome, demonstrating that viral infection alters cellular chromatin structure.

FIG. 1.

Gene expression profiles of KOS, n212, and V422 in HeLa and U2OS cells. HeLa and U2OS cells were infected with the indicated viruses in the presence of PAA as described in Materials and Methods. Total RNA was harvested after the indicated lengths of time and subjected to Northern blot analysis using probes for ICP27, TK, and VP16.

FIG. 2.

Histone H3 occupancy on KOS, n212, and V422 genomes in HeLa and U2OS cells. HeLa and U2OS cells were infected with the indicated viruses in the presence of PAA as described in Materials and Methods. Cell lysates were prepared at 1, 3, and 6 hpi. The amount of immunoprecipitated DNA from the ICP27 TSS (A), TK promoter (B), or VP16 promoter (C) was determined. Pooled data from three independent experiments are presented. Samples with mean values that differed significantly (P < 0.05, paired Student t test) are indicated (*).

FIG. 3.

Acetylated histone H3 on KOS, n212, and V422 genomes in HeLa and U2OS cells. HeLa and U2OS cells were infected with the viruses in the presence of PAA as described in Materials and Methods. Cell lysates were prepared at 6 and 9 hpi. The proportion of acetylated histone H3 associated with ICP27 TSS (A), TK promoter (B), and VP16 promoter (C) viral DNA was determined as the fraction of DNA associated with acetylated H3 normalized to the fraction of DNA associated with total histone H3. Pooled data from three independent experiments are presented. Samples with mean values that differed significantly (P < 0.05, paired Student t test) are indicated (*).

FIG. 4.

Effects of HSV infection on histone H3 occupancy at selected regions of the cellular genome. U2OS cells were infected with KOS, n212, or V422 or else mock infected in the presence of PAA as described in Materials and Methods. Cell lysates were prepared at 6 hpi. The amount of immunoprecipitated DNA associated with the GAPDH gene (A), U3 promoter (B), or Sat3 sequence (C) was determined. (D) Infections at 6 h were also performed in the presence of cycloheximide, and the amount of GAPDH DNA immunoprecipitated was determined. Samples with mean values that differed significantly (P < 0.05, paired Student t test) are indicated (*). The data displayed in panels A to C were pooled from three independent experiments performed over the course of approximately 1 week; the data displayed in panel D were pooled from a separate set of three independent experiments performed approximately 1 month later.