Increased neurovirulence and reactivation of the herpes simplex virus type 1 latency-associated transcript (LAT)-negative mutant dLAT2903 with a disrupted LAT miR-H2

Abstract

At least six microRNAs (miRNAs) appear to be encoded by the latency associated transcript (LAT) of herpes simplex virus type 1 (HSV-1). The gene for ICP0, an important immediate early (IE) viral protein, is antisense to, and overlaps with, the region of LAT from which miRNA H2 (miR-H2) is derived. We recently reported that a mutant (McK-ΔH2) disrupted for miR-H2 on the wild type HSV-1 strain McKrae genomic background has increased ICP0 expression, increased neurovirulence, and slightly more rapid reactivation. We report here that HSV-1 mutants deleted for the LAT promoter nonetheless make significant amounts of miR-H2 during lytic tissue culture infection, presumably via readthrough transcription from an upstream promoter. To determine if miR-H2 might also play a role in the HSV-1 latency-reactivation cycle of a LAT negative mutant, we constructed dLAT-ΔH2, in which miR-H2 is disrupted in dLAT2903 without altering the predicted amino acid sequence of the overlapping ICP0 open reading frame. Similar to McK-ΔH2, dLAT-ΔH2 expressed more ICP0, was more neurovirulent, and had increased reactivation in the mouse TG explant induced reactivation model of HSV-1 compared to its parental virus. Interestingly, although the increased reactivation of McK-ΔH2 compared to its parental wt virus was subtle and only detected at very early times after explant TG induced reactivation, the increased reactivation of dLAT-ΔH2 compared to its dLAT2903 parental virus appeared more robust and was significantly increased even at late times after induction. These results confirm that miR-H2 plays a role in modulating the HSV-1 reactivation phenotype.

Mutations Inactivating Herpes Simplex Virus 1 MicroRNA miR-H2 Do Not Detectably Increase ICP0 Gene Expression in Infected Cultured Cells or Mouse Trigeminal Ganglia

“…These authors stated that they did not observe any miR-H2 expression following transfection of their ICP0-expressing plasmid; however, the type and sensitivity of the assay(s) used were not specified. None of the previous studies experimentally addressed the concern of mutant miR-H2 expression (37)(38)(39).…”

“…Regardless, the different results could be due to different viral and mouse strains. Interestingly, a mutant with the Mck-ΔH2 miR-H2 mutations introduced into an LAT deletion background (dLAT-ΔH2), which severely decreases miR-H2 expression during latency (25) but has little or no effect on miR-H2 levels during acute infection or in cell culture (25,39), exhibited similar phenotypic changes in virulence and an even more apparent effect on reactivation compared to the mutant in the WT McKrae background (39). Regardless, phenotypes exhibited by miR-H2 mutant viruses would be unlikely to be due to derepression of ICP0, as our studies have found no evidence that miR-H2 represses ICP0 expression during infection.…”

“…ICP0 is an E3 ubiquitin ligase that counteracts cellular repressive functions, including intrinsic and innate immunity (27)(28)(29)(30)(31), and is important for viral replication and gene expression in many cell types and for reactivation from latency (32)(33)(34)(35)(36). The complementarity of miR-H2 and ICP0 mRNA, prior results showing that one or more products of the LAT locus repress lytic gene expression in mouse neurons (6)(7)(8), and transienttransfection experiments showing that miR-H2 can repress ICP0 expression in cell culture (3,37,38) led to the attractive hypothesis that miR-H2 could promote latency by repressing ICP0 expression, thereby globally repressing lytic gene expression (3,(37)(38)(39). HSV-2, which is closely related to HSV-1, also encodes a miR-H2 (alternatively known as miR-III) that is completely complementary to coding sequences in the HSV-2 ICP0 mRNA and can similarly repress ICP0 expression in transfection assays (40).…”

“…Some mutational studies investigating miR-H2 have been published (37)(38)(39). Two miR-H2 mutant viruses derived from strain McKrae were reported to exhibit increased ICP0 protein expression 4 and 6 h after infection of rabbit skin (RS) cells and increased viral virulence and reactivation rates in a mouse model of ocular inoculation (37,39).…”

“…Two miR-H2 mutant viruses derived from strain McKrae were reported to exhibit increased ICP0 protein expression 4 and 6 h after infection of rabbit skin (RS) cells and increased viral virulence and reactivation rates in a mouse model of ocular inoculation (37,39). A mutant virus in a strain 17 background was reported to show increased ICP0 expression at both the mRNA and protein level 8 h after infection of 293T cells but reduced viral DNA replication in Neuro-2A cells following infection at a low but not a high multiplicity of infection (MOI) (38).…”