A Varicella Zoster Virus latency model in human embryonic stem cell-derived neurons using conditional translation of a viral protein.

Varicella Zoster Virus (VZV) infection causes chickenpox followed by latency in peripheral neurons. Reactivation results in Herpes Zoster, a painful disease often followed by chronic pain (post-herpetic neuralgia). Our in-vitro model for VZV latency and reactivation uses the antiviral drug acyclovir to block lytic infection, but acyclovir can damage latent viral DNA, complicating interpretation. We present here a novel model for VZV latency and reactivation using a conditional replication virus.

A VZV pOkaBAC expressing eGFP was engineered to contain a degron from dihydrofolate reductase (DHFR) of E. coli fused to ORF4. The cell-free virus made in presence of trimethoprim (TMP), an inhibitor of the degron, was used to infect in human embryonic stem cell (hESC) - derived neurons in the presence or absence of TMP.

6 days post-infection, neurons maintained in the presence of TMP showed a productive, spreading infection, visualized by GFP fluorescence. By contrast, no GFP was observed in neurons infected without TMP in the medium. PCR of the GFP-negative cultures showed that these neurons contained viral genomes. Strikingly, treating GFP-negative infected neurons with sodium butyrate that “reactivates” VZV in our acyclovir-based model, in the presence of TMP, renewed GFP expression and virus spread, simulating reactivation. TMP alone did not result in renewed viral replication.

Thus, infection with VZV containing a DHFR degron for an essential viral protein eliminates the requirement for antiviral drugs to block lytic neuronal infection in-vitro. This conditional infection system is potentially an important advance in studying VZV latency and reactivation.