Abstract
Purpose :
Herpes Simplex Virus 1 (HSV-1) is a prevalent pathogen that can lytically infect human eyes and maintain lifelong latency in sensory ganglia. A wide range of ocular diseases can be caused by viral reactivation, during which the virus transports from the ganglia to the eye using anterograde transport mechanisms. The Us7,8,9 gene cluster is required for HSV-1 anterograde transport, yet how their expression is initiated in reactivation is unknown. In latent HSV-1, we identified multiple chromatin loops and showed that disrupting one loop results in decreased anterograde transport gene expression and subsequent reactivation. Interestingly, this chromatin loop facilitates a cis-interaction between an enhancer (LTE) and the distal Us7,8,9 gene cluster. Therefore, we hypothesized that this loop positions LTE in spatial proximity of Us7,8,9 promoters to drive their transcription during reactivation. The aim of this study was to determine the role of LTE in anterograde transport gene expression and reactivation.
Methods :
We generated a mutant HSV-1 virus with LTE deleted (17ΔA). NZW rabbits were infected with wild-type (wt) or 17ΔA viruses and latency was established. Reactivation was induced by epinephrine and reactivation frequency was determined by detecting infectious viruses in the rabbit tear film. For gene expression experiments, either female BALB/c mice or human neuronal cells (LUHMES) were infected with wt or 17ΔA viruses and latency was established. Ex vivo reactivation was done in mice by explanting ganglia into cell culture media for 24 hrs. Gene expression was measured by qRT-PCR. Reactivation in LUHMES was induced by wortmannin.
Results :
We found no difference in latency establishment between the two viruses. However, only 25% of 17ΔA-infected rabbits reactivated compared to 100% in wt (p>0.05), confirming that LTE was required for reactivation. To test whether there was attenuated anterograde transport gene expression, we quantified Us8 expression in mice following reactivation and found that Us8 expression was 8-fold lower in mutant ganglia upon reactivation (p>0.01). Similar results were found in the LUHMES cell model.
Conclusions :
These results support the hypothesis that the distal HSV-1 enhancer controls the expression of at least one anterograde transport gene in reactivation, likely through a chromatin loop that brings to two sites in proximity.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.