Abstract
Purpose :
Herpes simplex virus-1 (HSV-1) infection is a classic example of chronic and latent viral infection in both human and experimental animal models. Latency associated transcript (LAT) plays a major role in establishment of latency and recurrences and has antiapoptotic functions. LAT is known to encode two small non-coding RNAs (sncRNAs) and whether the absence of these two sncRNAs will alter latency-reactivation and apoptosis in latently infected mice is unknown.
Methods :
We constructed a HSV-1 recombinant virus lacking sncRNA1&2 with McKrae background (i.e., ΔsncRNA1&2). Replication of the ΔsncRNA1&2 virus was measured by standard plaque assay and apoptosis, LAT and viral transcripts expression levels of infected RS and Neuro2A cells were measured by qRT-PCR. WT C57BL/6 mice were infected ocularly with 2 X 105 PFU/eye of WT (LAT-plus) HSV-1 strain McKrae, dLAT2903 (LAT-minus), or ΔsncRNA1&2 (LAT-plus; sncRNA1&2-minus). Virus replication in the eyes (days 1-7) and eye disease (day 28) were determined. Levels of latency, expression levels of viral and host transcripts and reactivation from latency were determined on day 28 post infection.
Results :
Deletion of the 62 bp and 36 bp sncRNA1 and sncRNA2 sequences, respectively was verified by whole genome sequencing of the mutant virus. ΔsncRNA1&2 replicated similar to dLAT2903 and WT McKrae in vitro and in vivo. There were no differences in the eye disease or survival between ΔsncRNA1&2 infected mice or control viruses. Levels of apoptosis in ΔsncRNA1&2 infected Neuro2A and RS cells were similar to dLAT2903 infected cells, and both were significantly higher than McKrae infected cells. Similarly, levels of gB DNA in TG of latently infected ΔsncRNA1&2 virus were the same as dLAT2903 and both were significantly lower than McKrae infected mice. Finally, the absence of sncRNA1&2 in ΔsncRNA1&2 infected mice did not affect reactivation time compared with McKrae and both were significantly different from dLAT2903.
Conclusions :
Here we demonstrated a novel function for sncRNA1&2 in reducing antiapoptotic functions and levels of LAT in wild-type HSV-1. We have also shown that two sncRNAs encoded within the 1.5 kb of LAT do not play any role in duration of time to reactivation.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.