Neuronal miR-138 Represses HSV-2 Lytic Infection by Regulating Viral and Host Genes with Mechanistic Differences from HSV-1

Herpes simplex virus 2 (HSV-2) establishes latent Infection in dorsal root ganglion (DRG) neurons after productive (lytic) Infection in peripheral tissues. A neuron-specific MicroRNA, miR-138, favors HSV-1 latency by repressing viral ICP0 and host Oct-1 and Foxc1 genes, yet the role of miR-138 in HSV-2 Infection was unknown. The ICP0 mRNAs of HSV-1, HSV-2, and chimpanzee herpesvirus each have one to two canonical miR-138 binding sites. The sites are 100% conserved in 308 HSV-1 and 300 HSV-2 published sequences of clinical isolates. In cotransfection assays, miR-138 repressed HSV-2 ICP0 expression through the seed region and surrounding interactions that are different from HSV-1. An HSV-2 mutant with disrupted miR-138 binding sites on ICP0 showed increased ICP0 expression in Neuro-2a cells. Photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation confirmed miR-138 binding to HSV-2 ICP0 and identified UL19 and UL20 as additional targets whose expression was repressed by miR-138 during cotransfection. In Neuro-2a cells, transfected miR-138 and its antagomir decreased and increased HSV-2 replication, respectively, and a knockout experiment showed that miR-138's host targets OCT-1 and FOXC1 were important for HSV-2 replication. In primary mouse DRG neurons, both ICP0 and FOXC1 positively regulated HSV-2 replication, but both overexpressed and endogenous miR-138 suppressed HSV-2 replication primarily by repressing ICP0 expression. Thus, miR-138 can suppress HSV-2 neuronal replication through multiple viral and host pathways. These results reveal functional similarities and mechanistic differences in how miR-138 regulates HSV-1 and HSV-2 Infection and indicate an evolutionary advantage of using miR-138 to repress lytic Infection in neurons. IMPORTANCE HSV-1 and HSV-2 are closely related viruses with major differences. Both viruses establish latency in neurons from which they reactivate to cause disease. A key aspect of HSV latency is repression of productive Infection in neurons. Based on previous work with HSV-1, we investigated the role of a neuron-specific MicroRNA, miR-138, in HSV-2 Infection and established it as a repressor of HSV-2 productive Infection in neuronal cells. This repression is mediated mainly by targeting viral ICP0 and host Foxc1 mRNAs, but other pathways also contribute. Despite functional conservation of the role of miR-138 between HSV-1 and HSV-2, many molecular mechanisms differ, including how miR-138 represses ICP0 expression and miR-138 targeting of HSV-2 but not HSV-1 UL19 and UL20. To our knowledge, this study provides the first example of host MicroRNA regulation of HSV-2 Infection.