AAV-Mediated Ribozyme Targeting of HSV-1 Latency-Associated Transcript RNAs Reveals Distinct Functions of the LAT Intron and Exon In Vivo

Abstract

Herpes Simplex Virus 1 (HSV-1) establishes lifelong latency in sensory neurons of the trigeminal ganglia (TG), from which periodic reactivation causes recurrent disease. The latency-associated transcript (LAT) has been implicated in latency establishment, neuronal survival, and reactivation; however, most functional studies have relied on viral deletion mutants that alter genomic architecture and disrupt overlapping noncoding RNAs, making transcript-specific functions difficult to define. To investigate LAT RNA function independent of viral genome modification, we used recombinant adeno-associated virus (rAAV) vectors to deliver hammerhead ribozymes targeting either the LAT exon or the stable LAT intron in a rabbit model of HSV-1 infection. We found that knocking down the LAT intron prior to HSV-1 infection significantly increased acute disease morbidity and reduced latent viral genome loads in the TG, whereas depletion of the LAT 5' exon had no detectable effect on latency establishment. LAT intron targeting was also associated with reduced abundance of both the intron and exon LAT transcripts during latency. In contrast, post-establishment depletion of either the LAT exon or intron significantly reduced reactivation-associated virus shedding following induced reactivation. These findings provide direct evidence that LAT-derived RNAs contribute to multiple stages of the HSV-1 life cycle. Furthermore, the differential effects of LAT exon and intron depletion suggest that functions required for efficient latency establishment localize more closely to the LAT intron, whereas both LAT exon and intron transcripts contribute to efficient reactivation. More broadly, this study demonstrates the utility of transcript-targeting approaches for dissecting the functions of complex herpesvirus noncoding RNA loci.