Epigenetic Modulation of Gene Expression from Quiescent Herpes Simplex Virus Genomes

Abstract

The ability of herpes simplex virus to persist in cells depends on the extent of viral-gene expression, which may be controlled by epigenetic mechanisms. We used quiescent infection with the viral mutants d109 and d106 to explore the effects of cell type and the presence of the viral protein ICP0 on the expression and chromatin structure of the human cytomegalovirus (HCMV) tk and gC promoters on the viral genome. Expression from the HCMV promoter on the d109 genome decreased with time and was considerably less in HEL cells than in Vero cells. Expression from the HCMV promoter in d106 was considerably more abundant than in d109, and this increased with time in both cell types. The same pattern of expression was seen on the tk and gC genes on the viral genomes, although the levels of tk and gC RNA were approximately 10 2 -and 10 5 -fold lower than those of wild-type virus in d106 and d109, respectively. In micrococcal-nuclease digestion experiments, nucleosomes were evident on the d109 genome, and the amount of total H3 as determined by chromatin immunoprecipitation was considerably greater on d109 than d106 genomes. The acetylation of histone H3 on the d106 genomes was evident at early and late times postinfection in Vero cells, but only at late times in HEL cells. The same pattern was observed for H3 acetylated on lysine 9. Trimethylation of H3K9 on d109 genomes was evident only at late times postinfection in Vero cells, while it was observed both early and late in HEL cells. Heterochromatin protein 1␥ (HP1␥) was generally present only on d109 genomes at late times postinfection of HEL cells. The observations of chromatin structure correlate with the expression patterns of the three analyzed genes on the quiescent genomes. Therefore, several mechanisms generally affect the expression and contribute to the silencing of persisting genomes. These are the abundance of nucleosomes, the acetylation state of the histones, and heterochromatin. The extents to which these different mechanisms contribute to repression vary in different cell types and are counteracted by the presence of ICP0.

Activities of ICP0 Involved in the Reversal of Silencing of Quiescent Herpes Simplex Virus 1

“…The primers used for ChIP and cDNA quantification and their locations relative to the transcription start site of the gene to be analyzed are as previously published (33). d106 DNA was also included in each plate, in a standard curve of 1:10 dilutions from 250,000 to 25 copies per well, which covers the threshold cycle values for the ChIP DNA samples tested.…”

“…Chromatin immunoprecipitation (ChIP) was carried out as previously described (33,76), with a few modifications. A total of 5 ϫ 10 6 MRC-5 cells were seeded into 100-mm dishes (ϳ10 5 cells/cm 2 ) and infected with d109 at a multiplicity of infection (MOI) of 10 PFU per cell at room temperature for 1 h. After adsorption, the inoculum was removed and 37°C 5% fetal bovine serum (FBS) in Dulbecco's modified Eagle's medium was added.…”

“…As in latency in animal models, viral DNA was shown to be in a chromatin structure in a cell culture model of quiescence where incoming viral DNA is chromatinized in the absence of viral immediate-early (IE) proteins (33). Specifically, it was seen that in HEL cells, histones are deposited on the incoming genome by 4 h postinfection (hpi), and these histones begin to acquire trimethylation of histone H3K9.…”

“…Previously, we have shown that ICP0 expression is able to prevent the accumulation of histones, repressive histone modifications, and higher-order chromatin structure on the incoming viral genome, as well as cause the hyperacetylation of the few histones that do bind (33). We have also shown that provision of ICP0 can result in the removal of heterochromatin and histones H3 and H4 and result in the acetylation of remaining histones on genomes that have been repressed in relatively long-term quiescence (33a).…”

Herpes Simplex Virus 1 ICP0 Phosphorylation Site Mutants Are Attenuated for Viral Replication and Impaired for Explant-Induced Reactivation

“…Specifically, ICP0's E3 ubiquitin ligase activity promotes the disruption of ND10 by mediating the proteolysis (directly or indirectly) of SUMO-conjugated isoforms of PML and Sp100 (12,20,50) or by dissociating the ND10 components hDaxx and ATRX (45), stimulating viral transcription. In addition to affecting viral transcription, ICP0 assists HSV-1 in counteracting other intrinsic and innate host defenses that include chromatinization or repression of the viral genome (13,14,23,24,28) and activation and establishment of the interferon response (35,40,48,49,54).…”