Inhibition of O-Linked N-Acetylglucosamine Transferase Reduces Replication of Herpes Simplex Virus and Human Cytomegalovirus

Abstract

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential cellular enzyme that posttranslationally modifies nuclear and cytoplasmic proteins via O-linked addition of a single N-acetylglucosamine (GlcNAc) moiety. Among the many targets of OGT is host cell factor 1 (HCF-1), a transcriptional regulator that is required for transactivation of the immediate-early genes of herpes simplex virus (HSV). HCF-1 is synthesized as a large precursor that is proteolytically cleaved by OGT, which may regulate its biological function. In this study, we tested whether inhibition of the enzymatic activity of OGT with a small molecule inhibitor, OSMI-1, affects initiation of HSV immediate-early gene expression and viral replication. We found that inhibiting OGT's enzymatic activity significantly decreased HSV replication. The major effect of the inhibitor occurred late in the viral replication cycle, when it reduced the levels of late proteins and inhibited capsid formation. However, depleting OGT levels with small interfering RNA (siRNA) reduced the expression of HSV immediate-early genes, in addition to reducing viral yields. In this study, we identified OGT as a novel cellular factor involved in HSV replication. Our results obtained using a small molecule inhibitor and siRNA depletion suggest that OGT's glycosylation and scaffolding functions play distinct roles in the replication cycle of HSV.

Importance: Antiviral agents can target viral or host gene products essential for viral replication. O-GlcNAc transferase (OGT) is an important cellular enzyme that catalyzes the posttranslational addition of GlcNAc sugar residues to hundreds of nuclear and cytoplasmic proteins, and this modification regulates their activity and function. Some of the known OGT targets are cellular proteins that are critical for the expression of herpes simplex virus (HSV) genes, suggesting a role for OGT in the replication cycle of HSV. In this study, we found that OGT is required for efficient expression of viral genes and for assembly of new virions. Thus, we identify OGT as a novel host factor involved in the replication of HSV and a potential target for antiviral therapy.

FIG 1

OSMI-1 inhibits OGT activity in HFFs. (A) Chemical structure of OSMI-1. (B) Effect of OSMI-1 on O-GlcNAcylation in HFF cells. HFFs were incubated with increasing concentrations of OSMI-1 for 24 h. Cell lysates were analyzed by immunoblotting using O-GlcNAc-specific RL2 antibody. (C) HFFs were treated with increasing concentrations of OSMI-1. After 24 h, cell viability was measured with CellTiter-Glo Luminescent Cell Viability assay (Promega) and expressed as a percentage of DMSO-treated control cells.

FIG 2

Effect of OSMI-1 on HSV-1 replication. (A) HFFs were infected with HSV-1 at an MOI of 0.1 and incubated with media containing increasing concentrations of OSMI-1 or vehicle (DMSO). At 48 hpi, viral yields were determined by plaque assay on Vero cells. (B) HFFs infected at MOIs of 0.1 and 5 and treated with 50 μM OSMI-1 or DMSO control for 48 and 24 h, respectively. Bars represent mean values of viral yields ± SEMs (n = 3). Statistical analysis was done using a multiple measurements one-way analysis of variance (ANOVA) followed by Bonferroni posttest. **, P < 0.001. (C to E) HeLa (C), HEp-2 (D), and HEK-293 (E) cells were infected with HSV-1 at an MOI of 5 and treated with increasing concentrations of OSMI-1 for 24 h. Viral titers were determined by plaque assays on Vero cells. (F) EC₅₀ for OSMI-1 was determined in HFFs infected with KOS at an MOI of 0.1 and treated for 24 h. Values are expressed as percent yield relative to cells treated with DMSO vehicle control. EC₅₀ was calculated using nonlinear regression curve fitting with a variable slope (GraphPad Prism 5 software). (G) HFFs were infected with HSV-1 at an MOI of 5 and treated with 50 μM PG34, OSMI-1, or DMSO for 24 h, and viral yields were measured by a plaque assay.

FIG 3

Effect of OSMI-1 on HSV-2 replication. (A) HFFs were infected with WT HSV-2 at MOIs of 0.1 and 5 and treated with 50 μM OSMI-1 or DMSO. Viral yields were determined at 24 hpi (MOI of 5) and 48 hpi (MOI of 0.1) by plaque assays on Vero cells. Means ± SEMs (n = 3) were analyzed by one-way ANOVA with repeated measures followed by Bonferroni posttest. *, P < 0.05. (B) HFFs were infected with WT HSV-2 or 186 TK⁻ virus at an MOI of 0.1, followed by addition of 50 μM OSMI-1, 100 μM acyclovir (ACV), or DMSO control. Virus yield was determined at 24 hpi by titration on Vero cells. Bars represent mean values ± SEMs from three independent experiments.

FIG 4

Effect of OSMI-1 on HCMV replication. HFFs were infected with HCMV AD169 at MOIs of 0.1 (A) and 1 (B) and treated with 50 μM OSMI-1 or DMSO. Viral yields were determined at 96 hpi by plaque assays on HFFs. Means ± SEMs (n = 2) of viral yields were analyzed by one-way ANOVA with repeated measures followed by Bonferroni posttest. *, P < 0.05.

FIG 5

Effect of OSMI-1 on HSV-1 gene expression. HFFs were infected with HSV-1 at MOI of 5 or mock infected and treated with a vehicle control or 50 μM OSMI-1. Total RNA was harvested at 3, 6, and 9 h postinfection, and the levels of transcripts from IE ICP27 (A), E ICP8 (B), and L gC (C) genes were measured by qRT-PCR. The mRNA levels were normalized to cellular 18S rRNA. The results are expressed as means ± SEMs from three independent experiments. (D) HFFs were infected with HSV-1 at an MOI of 5 and treated with 50 μM OSMI-1 or DMSO. Protein lysates were collected at 3, 6, and 9 hpi and analyzed for expression of ICP27, ICP8, and gC or GAPDH as a loading control by immunoblotting. (E) HFFs were treated as described for panel B and blotted for expression of gD, VP5, and GAPDH to serve as a loading control. (F) Time-of-addition assay. HFFs were infected with HSV-1 at an MOI of 5 and treated with DMSO or 50 μM OSMI-1, which was added at different times after infection, as indicated. HSV-1 replication was measured after 24 h by plaque assay. Results represent means ± SEMs from two independent experiments.

FIG 6

Effect of OGT inhibition on capsid assembly. (A) Fibroblasts were infected with K26GFP recombinant virus at an MOI of 10 and treated with 50 μM OSMI-1 or DMSO. Cells were fixed at 8 hpi, stained with an antibody against VP5, and imaged with a Nikon TE2000 w/C1 point scanning confocal microscope (60× objective). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). (B) Electron microscopy of viral capsids and virions in OMSI-1-treated cells. (a to d) HFF cells were infected with HSV-1 at an MOI of 5 and treated with 50 μM OSMI-1 (b and d) or a vehicle control (a and c) for 16 h. Infected cells were fixed and processed for EM. White arrows point to C capsids, black arrows point to A capsids, and arrowheads designate B capsids. N, nucleus; C, cytoplasm. (e and f) KOS-infected HFFs were treated with a vehicle (e) or OMSI-1 (f) for 24 h. Extracellular virus released in the supernatant was isolated by centrifugation and resuspended in PBS. Virions were stained by uranyl acetate and imaged by EM. (C) Capsids were quantified by counting the number and type of capsids in 15 random microscopic fields. Counts are generated from two independent experiments.

FIG 7

Effect of OGT depletion on HSV replication. (A) HFFs were transfected with a nontargeting or OGT-specific siRNA pools. At 72 hpi, the levels of OGT were measured by qRT-PCR and Western blotting. (B) siControl- or siOGT-transfected cells were infected with HSV-1 at an MOI of 0.1. At 24 hpi, viral yields were determined by plaque assay on Vero cells. The graph presents means (±SEMs) of viral yields from 4 independent experiments. *, P < 0.05. (C) Cell viability at 96 h after siRNA treatment was measured with CellTiter-Glo Luminescent Cell Viability assay (Promega) and expressed as percentage of siControl-treated cells. (D) siRNA-treated cells were infected with HSV-1 at an MOI of 5, and total protein was harvested at 6 hpi. Levels of viral IE (ICP27), E (ICP8), and L (gC) proteins were measured by Western blotting. Cellular actin was used as a loading control. (D) siRNA-treated cells were infected with HSV-1 at an MOI of 5, and total RNA was isolated at 4 hpi. Relative transcript levels of viral IE (ICP27), E (ICP8), and L (gC) genes were measured by qRT-PCR and normalized to cellular 18S rRNA. Values represent means from 3 independent experiments (±SEMs). (E) siControl- or siOGT-treated HFFs were infected with HSV-1 at an MOI of 5, and at 2 hpi, cells were fractionated into cytoplasmic and nuclear extracts. DNA isolated from nuclear fractions and total cellular DNA were analyzed for ICP27 levels by qRT-PCR. Viral gene copy numbers were normalized to cellular GAPDH pseudogene levels. Values represent means from three independent experiments (±SEMs).