DLK-dependent biphasic reactivation of herpes simplex virus latency established in the absence of antivirals

Abstract

An understanding of the molecular events that regulate entry of herpes simplex virus type 1 (HSV-1) into a latent infection of neurons and subsequent reactivation requires model systems. However, establishing a latent-like infection in cultured neurons is problematic as the release of any infectious virus can potentially superinfect the cultures. Here, we describe a new reporter virus, HSV-1 Stayput-GFP, that is defective for cell-to-cell spread. Use of this virus permits the establishment of a quiescent infection of neurons without the need for a viral DNA replication inhibitor. As such, Stayput-GFP provides a new model to study latency establishment and reactivation at the single neuron level. We demonstrate that reactivation involves an initial wave of viral gene expression consistent with the Phase I previously observed in primary neuron models where a viral DNA replication inhibitor is used during latency establishment. Phase I gene expression was dependent on the cell stress protein DLK but independent of histone demethylase activity and viral DNA replication. Progression into the later, Phase II wave of reactivation, characterized by detectable late viral protein synthesis and a requirement for histone demethylase activity, is also observed in the Stayput-GFP model system. These data demonstrate that the two waves of viral gene expression following HSV-1 reactivation are independent of secondary infection and occur when latent infections are established in the absence of a viral DNA replication inhibitor.

Co-option of mitochondrial nucleic acid sensing pathways by HSV-1 UL12.5 for reactivation from latent Infection

“…However, during the productive replication cycle, the VP16 gene is expressed with late kinetics. During Phase I, VP16, like other late genes, is expressed in the absence of viral DNA replication 33,36,37 . Further, work from the Sawtell and Thompson labs has shown that elements within the VP16 promoter can function prior to immediate-early gene expression, specifically in neurons, and are involved in the initial depression of viral gene expression during acute infection of the ganglia 55,65 .…”

“…So far, the host proteins known to function during Phase I to induce lytic gene expression are DLK and JNK 32,34,36,37 . Experiments performed here with the DLK inhibitors suggest that DLK may be downstream of STING activation.…”

“…A further minor weakness is that we used acyclovir to help promote latency establishment. However, we previously published that all the criteria for Phase I gene expression occur when latency is established in the absence of acyclovir 36 . We did not use that system here because we wished to control for equivalent latent viral loads following infection with the UL12.5 mutant viruses.…”

“…HSV-1 reactivation occurs in a biphasic manner. Phase I consists of a burst of lytic gene expression from all three gene classes (IE, E, L) independent of DNA replication, while the subsequent Phase II recapitulates the ordered viral gene cascade observed in lytic replication and results in the production of infectious virus [32][33][34][35][36][37] . Phase I gene expression is thought to be independent of the synthesis of viral proteins, and so far, no viral protein is known to function in Phase I to promote lytic gene expression 33 .…”

“…The absence of viral proteins during latency necessitates that HSV exploits existing neuronal signaling pathways to initiate and progress reactivation. Several stimuli have been identified that trigger reactivation, including nerve growth factor (NGF) deprivation 48,49 , DNA damage 50 , and neuronal hyperexcitability 32 with a notable convergence on the neuronal stress pathway mediated by dual leucine zipper kinase (DLK) and JNK 32,34,36,37,50,51 . Our recent work has identified interleukin-1β (IL-1β), a major inflammatory cytokine, in triggering HSV reactivation via hyperexcitability 32 .…”