Stress Triggers Expression of Bovine Herpesvirus 1 Infected Cell Protein 4 (bICP4) RNA during Early Stages of Reactivation from Latency in Pharyngeal Tonsil

BoHV-1, a neurotropic herpesvirus, establishes, maintains, and reactivates from latency in neurons. BoHV-1 DNA is also detected in pharyngeal tonsil (PT) from latently infected calves. Bovine herpesvirus 1 (BoHV-1), an important pathogen of cattle, establishes lifelong latency in sensory neurons within trigeminal ganglia (TG) after acute infection. The BoHV-1 latency-reactivation cycle, like other alphaherpesvirinae subfamily members, is essential for viral persistence and transmission. Notably, cells within pharyngeal tonsil (PT) also support a quiescent or latent BoHV-1 infection. The synthetic corticosteroid dexamethasone, which mimics the effects of stress, consistently induces BoHV-1 reactivation from latency allowing early stages of viral reactivation to be examined in the natural host. Based on previous studies, we hypothesized that stress-induced cellular factors trigger expression of key viral transcriptional regulatory genes. To explore this hypothesis, RNA-sequencing studies compared viral gene expression in PT during early stages of dexamethasone-induced reactivation from latency. Strikingly, RNA encoding infected cell protein 4 (bICP4), which is translated into an essential viral transcriptional regulatory protein, was detected 30 min after dexamethasone treatment. Ninety minutes after dexamethasone treatment bICP4 and, to a lesser extent, bICP0 RNA were detected in PT. All lytic cycle viral transcripts were detected within 3 h after dexamethasone treatment. Surprisingly, the latency related (LR) gene, the only viral gene abundantly expressed in latently infected TG neurons, was not detected in PT during latency. In TG neurons, bICP0 and the viral tegument protein VP16 are expressed before bICP4 during reactivation, suggesting distinct viral regulatory genes mediate reactivation from latency in PT versus TG neurons. Finally, these studies confirm PT is a biologically relevant site for BoHV-1 latency, reactivation from latency, and virus transmission.IMPORTANCE BoHV-1, a neurotropic herpesvirus, establishes, maintains, and reactivates from latency in neurons. BoHV-1 DNA is also detected in pharyngeal tonsil (PT) from latently infected calves. RNA-sequencing studies revealed the viral infected cell protein 4 (bICP4) RNA was expressed in PT of latently infected calves within 30 min after dexamethasone was used to initiate reactivation. As expected, bICP4 RNA was not detected during latency. All lytic cycle viral genes were expressed within 3 h after dexamethasone treatment. Conversely, bICP0 and the viral tegument protein VP16 are expressed prior to bICP4 in trigeminal ganglionic neurons during reactivation. The viral latency related gene, which is abundantly expressed in latently infected neurons, was not abundantly expressed in PT during latency. These studies provide new evidence PT is a biologically relevant site for BoHV-1 latency and reactivation. Finally, we predict other alphaherpesvirinae subfamily members utilize PT as a site for latency and reactivation.