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

Understanding the molecular mechanism of herpes simplex virus type 1 (HSV-1) latent infection and reactivation in neurons requires the use of model systems. However, establishing a quiescent infection in cultured neurons is problematic as infectious virus released from any productively infected neuron can superinfect the cultures. Here, we describe a new reporter virus, HSV-1 Stayput-GFP, that is defective for cell-to-cell spread and can be used to model latency and reactivation at the single neuron level. Importantly, quiescent infection of neurons with Stayput-GFP can be established without use of a viral DNA replication inhibitor. The establishment of a quiescent state requires a longer time frame than previous models of HSV latency using DNA replication inhibitors. This results in a decreased ability of the virus to reactivate, and use of multiple reactivation triggers is required. Using this system, we demonstrate that an initial Phase I wave of lytic gene expression occurs independently of histone demethylase activity and viral DNA replication but is dependent on the neuronal cell stress protein, DLK. 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 with 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.