In neurons, various stimuli, such as growth factor deprivation or the severing of axons, activate JNK by dual leucine zipper kinase (DLK) and JNK-interacting protein 3 (JIP3), which initiates a cellular stress response. To test whether this JNK pathway is involved in herpes simplex virus reactivation, the authors generated latently infected cells by infecting primary neurons isolated from mouse sympathetic ganglia and treating them with interferons and acyclovir to inhibit lytic infection. Adding the corticosteroid dexamethasone or inhibitors of growth factor signalling pathways to these latently infected cells can then induce virus reactivation. Using this latency model, the authors were able to show that the production of viral mRNA and virus reactivation could be blocked with JNK inhibitors or shRNA-mediated depletion of DLK or JIP3. JNK inhibition also reduced virus reactivation in neurons that were explanted from mice infected with herpes simplex virus, which confirmed the central role of the JNK pathway in herpes simplex virus reactivation. As inhibiting JNK or depleting DLK or JIP3 had no effect on virus replication in de novo infected neurons, this process seems to be specific for reactivation.
The promoters of latent herpes simplex virus are associated with extensive histone modifications, particularly the presence of repressive methylation marks in lysine residues. Interestingly, blocking demethylation and thus removal of these repressive histone modifications had no effect on herpes simplex virus reactivation in the in vitro latency model. Cliffe et al. therefore speculated that a methyl/phospho switch (by which the phosphorylation of serine residues neighbouring the methylated lysine residues initiates gene expression, overriding the repressive marks) mediates reactivation. Indeed, virus reactivation was accompanied by histone phosphorylation of serines next to the methylated lysines, and JNK inhibition blocked this phosphorylation. Furthermore, chromatin immunoprecipitation showed an enrichment of JNK on viral lytic promoters, suggesting that JNK mediates the methyl/phospho switch, which activates expression of herpes simplex virus genes.
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