The Human Herpesviruses persist in the form of a latent infection in specialized cell types. During latency, the herpesvirus genomes associate with cellular histone proteins and the viral lytic genes assemble into transcriptionally repressive heterochromatin. Although there is divergence in the nature of heterochromatin on latent herpesvirus genomes, in general, the genomes assemble into forms of heterochromatin that can convert to euchromatin to permit gene expression and therefore reactivation. This reversible form of heterochromatin is known as facultative heterochromatin and is most commonly characterized by polycomb silencing. Polycomb silencing is prevalent on the cellular genome and plays a role in developmentally regulated and imprinted genes, as well as X chromosome inactivation. As herpesviruses initially enter the cell in an un-chromatinized state, they provide an optimal system to study how de novo facultative heterochromatin is targeted to regions of DNA and how it contributes to silencing. Here, we describe how polycomb-mediated silencing potentially assembles onto herpesvirus genomes, synergizing what is known about herpesvirus latency with facultative heterochromatin targeting to the cellular genome. A greater understanding of polycomb silencing of herpesviruses will inform on the mechanism of persistence and reactivation of these pathogenic human viruses and provide clues regarding how de novo facultative heterochromatin forms on the cellular genome.

Herpes Simplex Virus (HSV) establishes a latent infection in neurons, in which viral transcription is restricted and viral promoters are associated with heterochromatin. In response to certain stimuli, the virus reactivates to permit transmission. The exact physiological triggers of reactivation, the cell signaling pathways involved, and how signals act on heterochromatin-associated lytic promoters, are not understood. Previously, we identified a role for a neural stress pathway involving DLK and JNK activity in HSV reactivation, triggered by nerve growth factor (NGF) deprivation. Reactivation was associated with a JNK-dependent histone phospho/methyl switch on lytic gene promoters. Because the same histone phospho/methyl switch occurs in cortical neurons following hyperexcitability (triggered by forskolin), we examined whether HSV reactivation was linked to hyperexcitability, and the contribution of JNK activity and histone phosphorylation. Using our primary neuronal model of HSV reactivation, we found that forskolin triggered DLK/JNK-dependent reactivation via a pathway that was distinct from NGF deprivation. The initial burst of HSV lytic gene expression in response to forskolin occurred independently of histone demethylase activity, and was accompanied by a histone phospho/methyl switch. To determine whether forskolin-mediated reactivation was linked to neuronal activity, we investigated the contribution of ion channel activity. Inhibition of voltage-gated potassium and sodium channels, or hyperpolarization-activated cyclic nucleotide-gated channels, prevented forskolin-mediated reactivation. In addition, hyperexcitability, resulting from the removal of a tetrodotoxin block, triggered HSV reactivation in a DLK/JNK-dependent manner. We next investigated whether physiological triggers induce HSV reactivation via hyperexcitability. IL-1 induced DNA damage associated with hyperexcitability in adult neurons. IL-1 also triggered DLK/JNK-dependent HSV reactivation, which was dependent on ion channel activity. Therefore, these data indicate that neuronal hyperexcitability in response to physiological stimuli, such as inflammation, trigger HSV reactivation, and mark out the activation of DLK/JNK and a histone phospho/methyl switch as key events in the hyperexcitability-mediated reactivation.