VZV latency in peripheral neurons is difficult to study, and experimental reactivation in human neurons has not been achieved. In our human embryonic stem cell (hESC)-derived neuron model, cell associated or higher titer cell-free VZV infection leads to a productive and spreading infection. We show here that human embryonic stem cell-derived neurons can also host a non-productive, latent infection in which VZV can be reactivated to a productive infection.
Latent infections were established by exposing neurons to low titers of cell-free VZV in the presence of the viral DNA replication inhibitor acyclovir. Infection that included viral protein synthesis was detected by the expression of GFP tagged to ORF66 protein kinase. GFP- cultures one week after exposure to virus remained GFP- for up to 7 weeks after acyclovir removal, but maintained detectable VZV DNA in approximately 4% of neurons, and showed low levels of viral transcripts. Persistent infections could also be established without use of acyclovir by infecting axons in compartmented microfluidic chambers. Latently infected cultures treated with the HDAC inhibitor sodium butyrate, removal of growth factors from the medium, or by inhibition of PI3K activity all resulted in renewed expression of GFP, increased VZV genomes and higher viral transcription, although spread of virus between neurons was not observed. Strikingly, combination of PI3K-inhibition with an additional, non-pharmacological stimulus lead to more efficient VZV reactivation, including generation of infectious virus.
These results establish a model for VZV latency that can be experimentally reactivated that has great potential for development of improved vaccines and treatments for the widespread and painful disease, herpes zoster.
