Abstract
Purpose :
HSV-1 latency associated transcript (LAT) plays a major role in establishing latency and reactivation; however, the mechanism by which LAT controls these processes is largely unknown. The LAT locus contains two sncRNA sequences (sncRNA1 and sncRNA2) that are not miRNAs. We recently reported that sncRNA1 is more important for in vitro activation of the herpes virus entry mediator (HVEM) than sncRNA2, but its in vivo function is not known. In this study, we sought to establish the role of sncRNA1 during HSV-1 ocular infection by constructing a recombinant HSV-1 lacking sncRNA1.
Methods :
The 62 bp sncRNA1 sequence in HSV-1 strain McKrae was deleted by homologous recombination using HSV-1 strain McKrae and a plasmid containing the region of LAT from -161 to +1667 relative to the LAT transcription start site and lacking sncRNA1 region, generating recombinant virus ΔsncRNA1. Deletion of the sncRNA1 in ΔsncRNA1 virus was confirmed by complete sequencing of ΔsncRNA1 virus and its parental virus (i.e., McKrae). Mice were ocularly infected with 2x105 PFU HSV-1 McKrae, dLAT2903 (LAT-minus) or ΔsncRNA1 virus. Virus titers in tear films were determined by standard plaque assay. Mouse survival, level of latency and reactivation and extent of eye disease were monitored, and expression of viral and host transcripts were measured by qRT-PCR and Nanostring assays.
Results :
Replication of ΔsncRNA1 in tissue culture or in the eyes of infected mice was similar to McKrae and dLAT2903 viruses. Absence of sncRNA1 reduced LAT expression in trigeminal ganglia (TG), but not in corneas of infected mice by day 5 post infection (PI). Levels of eye disease in mice infected with ΔsncRNA1 and McKrae viruses were similar, and absence of sncRNA1 did not affect latency or ex-vivo reactivation. However, mice infected with ΔsncRNA1 virus were more susceptible to ocular infection than their WT counterparts. Expression of host immune response genes in corneas and TG of infected mice during primary infection showed reduced IFNβ and IFNγ expression and altered activation of key innate immune pathways, such as the JAK-STAT pathway in ΔsncRNA1 virus compared with parental WT virus.
Conclusions :
Our results reveal novel functions for sncRNA1 in up-regulating host immune response and suggest sncRNA1 has a protective role during primary ocular HSV-1 infection.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.