Herpes simplex virus 1 (HSV-1) genomes are associated with the repressive heterochromatic marks H3K9me2/me3 and H3K27me3 during latency. Previous studies have demonstrated that inhibitors of H3K9me2/me3 histone demethylases reduce the ability of HSV-1 to reactivate from latency. Here we demonstrate that GSK-J4, a specific inhibitor of the H3K27me3 histone demethylases UTX and JMJD3, inhibits HSV-1 reactivation from sensory neurons in vitro. These results indicate that removal of the H3K27me3 mark plays a key role in HSV-1 reactivation. Histone posttranslational modifications, both euchromatic and heterochromatic, are found in association with herpes simplex virus 1 (HSV-1) genes as the genome associates with nucleosomes during latency. The presence of permissive euchromatic marks (H3KAc, H9KAc, and H18K18Ac) in association with the latency-associated transcript (LAT) promoter and 5= exon during latency is consistent with its transcriptional activity during latency (1-4). Intrinsic to the latent state, however, are several repressive epigenetic marks that are also found in association with lytic genes of the latent HSV-1 genomes, notably, H3K9me2/me3 and H3K27me3, enriched along lytic genes. These marks have been implicated in helping to maintain lytic genes in a repressed state (3,5,6).Presumably, to reactivate from latency, the H3K9me2/me3 and H3K27me3 repressive marks need to be removed by chromatin remodeling enzymes. H3K27me3 is reversibly removed through the action of specific histone demethylases KDM6B/JMJD3 and KDM6A/ UTX (7). H3K9me3 can be removed by all members of the JMJD2 family (8), while H3K9me2/me3/1 can be removed by LSD1, KIAA1718, JHDM1F, and JMJD1A (9-12). In this study, we sought to determine whether blocking the removal of H3K27me3 would also block HSV-1 reactivation. This issue was addressed using GSK-J4, a specific inhibitor of both JMJD3 and UTX (13).Inhibition of JMJD3 and UTX blocks reactivation-induced H3K27me3 demethylation of HSV genomes. To understand the roles of UTX and JMJD3 in viral reactivation, we used an in vitro model of HSV-1 latency in primary adult murine trigeminal ganglion (TG) neurons (14), where reactivation is stimulated by nerve growth factor (NGF) depletion (15). TGs from 6-week-old outbred Swiss Webster mice were incubated in papain (Worthington, Lakewood, NJ) reconstituted with neurobasal A medium (Gibco; catalog no. 10888-022) followed by Hanks balanced salt solution containing dispase (4 mg/ml) and collagenase (4.7 mg/ml) (Sigma). Mechanically dissociated TGs were purified on an iodixinol gradient (OptiPrep; Sigma). Neuronal cells were counted and plated onto plates precoated with poly-D-lysine and laminin. Cells were maintained in neuralbasal media containing NGF, glial cell line-derived neurotrophic factor, neutrinin, L-glutamine, 2% (vol/vol) B-27 supplement minus AO (Gibco; catalog no. 10889-038), and 1% (vol/vol) penicillin-streptomycin. To suppress actively dividing nonneuronal cells, the media were supplemented with fluorodeoxyuridine for 72 h prior to i...

Herpes simplex virus (HSV) establishes a latent infection within sensory neurons of humans. Latency is characterized by the transcriptional repression of lytic genes by the condensation of lytic gene regions into heterochromatin. Recent data suggest that facultative heterochromatin predominates, and that cellular Polycomb proteins are involved in the establishment and maintenance of transcriptional repression during latency. This review summarizes these data and discusses the implication of viral and cellular factors in regulating heterochromatin composition.

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