Abstract
Purpose :
Herpes Simplex Virus-1 (HSV-1) is a leading cause of infectious blindness due to repeated reactivation of latent virus from the neuron to the eye. However, the mechanisms that govern the establishment of latency and reactivation are not well characterized. We have shown that a viral long noncoding RNA called the latency associated transcript (LAT) and its intron are required for efficient reactivation. Degradation of these LAT elements results in an attenuated reactivation phenotype. However, the roles that the expression and accumulation of LAT have on the establishment of latency are poorly understood, and previous studies aimed at understanding LAT have only leveraged mutant viruses with large deletions. In this work, we use the topical corneal delivery of Adeno-associated virus (rAAV) vectors containing a LAT intron targeting ribozyme to determine how intron expression dictates the establishment of latency.
Methods :
New Zealand White (NZW) rabbits were ocularly administered a rAAV vector containing a ribozyme specific for either the LAT intron (LATRz992) or a Rhodopsin control (N=8). Rabbits were subsequently infected with HSV-1 and maintained for 28 days post-infection, at which time latency is considered to be established. Trigeminal ganglia (TG) were harvested and subjected to either qRT-PCR for gene expression or qPCR to determine viral genome loads. All expression was normalized to the endogenous control GAPDH. Two-tailed Student’s t-test was used for statistical analysis of PCR and Cox regression was used for survival analysis.
Results :
Establishment of latency, shown by genomic load in TG, was significantly reduced in rabbits pretreated with LATRz992 (5.44±0.17 Fold change) compared to control (p=0.01) . Overall intron expression was reduced (10.2±0.08 Fold change) when compared to control. LATRz992 treated rabbits had significantly higher mortality rates (p=0.031) following infection with HSV-1 when compared to control, suggesting that lytic replication is occurring in the neuron.
Conclusions :
We found that the LAT intron is essential for the establishment of latency in neurons. These data further suggest that future strategies aimed at preventing lifelong HSV-1 latency target the LAT intron during the initial infection.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.