De Novo Synthesis of VP16 Coordinates the Exit from HSV Latency In Vivo

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Abstract
The mechanism controlling the exit from herpes simplex virus latency (HSV) is of central importance to recurrent disease and transmission of infection, yet interactions between host and viral functions that govern this process remain unclear. The cascade of HSV gene transcription is initiated by the multifunctional virion protein VP16, which is expressed late in the viral replication cycle. Currently, it is widely accepted that VP16 transactivating function is not involved in the exit from latency. Utilizing the mouse ocular model of HSV pathogenesis together with genetically engineered viral mutants and assays to quantify latency and the exit from latency at the single neuron level, we show that in vivo (i) the VP16 promoter confers distinct regulation critical for viral replication in the trigeminal ganglion (TG) during the acute phase of infection and (ii) the transactivation function of VP16 (VP16TF) is uniquely required for the exit from latency. TG neurons latently infected with the VP16TF mutant in1814 do not express detectable viral proteins following stress, whereas viruses with mutations in the other major viral transcription regulators ICP0 and ICP4 do exit the latent state. Analysis of a VP16 promoter/reporter mutant in the background of in1814 demonstrates that the VP16 promoter is activated in latently infected neurons following stress in the absence of other viral proteins. These findings support the novel hypothesis that de novo expression of VP16 regulates entry into the lytic program in neurons at all phases of the viral life cycle. HSV reactivation from latency conforms to a model in which stochastic derepression of the VP16 promoter and expression of VP16 initiates entry into the lytic cycle. Herpes simplex virus (HSV) establishes life-long latent infections in sensory neurons of the human host. Periodically, HSV exits latency in an infected neuron and is transported to the body surface where it replicates, leading to recurrent disease and infection of new hosts. We do not currently understand how entry into the lytic cycle is blocked in neurons and latency is established. Nor do we know how, at some time in the future, the lytic program becomes activated in the one or two latently infected neurons which characterize a reactivation event. In tissue culture cells, and by analogy in cells at the body surface, the HSV replication program is initiated by the interaction of a virion protein, VP16 (brought in with the virus as a protein), with host cell factors. Here we show that the de novo synthesis of VP16 is required for efficient viral replication during the acute phase of infection in neurons. This indicates that latency is favored because VP16 may not be transported efficiently to the nerve cell nucleus. Once latency is established, the de novo expression of VP16 is an absolute and very early requirement for the exit from the latent state. Our data support a model of HSV reactivation in which the stochastic derepression of the VP16 promoter and resulting expression of VP16 starts the viral lytic program.

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