Herpes Simplex Virus 1 MicroRNA miR-H8 Is Dispensable for Latency and Reactivation In Vivo

The regulatory functions of 10 individual viral microRNAs (miRNAs) that are abundantly expressed from the herpes simplex virus 1 (HSV-1) latency-associated transcript (LAT) region remain largely unknown. Here, we focus on HSV-1 miRNA miR-H8, which is within the LAT 3p exon, antisense to the first intron of ICP0, and has previously been shown to target a host glycosylphosphatidylinositol (GPI)-anchoring pathway. However, the functions of this miRNA have not been assessed in the context of the viral genome during infection. Therefore, we constructed a recombinant virus lacking miR-H8 (17dmiR-H8) and compared it to the parental wild-type and rescue viruses to characterize phenotypic differences. In rabbit skin cells, 17dmiR-H8 exhibited only subtle reductions in viral yields. In contrast, we found significant decreases in both viral yields (8-fold) and DNA replication (9.9-fold) in murine neuroblastoma cells, while 17dmiR-H8 exhibited a 3.6-fold increase in DNA replication in differentiated human neuronal cells (Lund human mesencephalic [LUHMES] cells). These cell culture phenotypes suggested potential host- and/or neuron-specific roles for miR-H8 in acute viral replication. To assess whether miR-H8 plays a role in HSV latency or reactivation, we used a human in vitro reactivation model as well as mouse and rabbit reactivation models. In the LUHMES cell-induced reactivation model, there was no difference in viral yields at 48 h postreactivation. In the murine dorsal root ganglion explant and rabbit ocular adrenergic reactivation models, the deletion of miR-H8 had no detectable effect on genome loads during latency or reactivation. These results indicate that miR-H8 is dispensable for the establishment of HSV-1 latency and reactivation.IMPORTANCE Herpesviruses have a remarkable ability to sustain lifelong infections by evading host immune responses, establishing a latent reservoir, and maintaining the ability to reactivate the lytic cascade to transmit the virus to the next host. The HSV-1 latency-associated transcript region is known to regulate many aspects of HSV-1 latency and reactivation, although the mechanisms for these functions remain unknown. To this end, we characterize an HSV-1 recombinant containing a deletion of a LAT-encoded miRNA, miR-H8, and demonstrate that it plays no detectable role in the establishment of latency or reactivation in differentiated human neurons (LUHMES cells) and mouse and rabbit models. Therefore, this study allows us to exclude miR-H8 from phenotypes previously attributed to the LAT region. Elucidating the genetic elements of HSV-1 responsible for establishment, maintenance, and reactivation from latency may lead to novel strategies for combating persistent herpesvirus infections.