PMC

Acyclovir-resistant VZV is susceptible to inhibition of the JNK pathway. (A) Representative image of plaque assays from neurons (1 × 10⁵) infected with two different input concentrations of VZVr at an MOI of 0.00073 (High) or 0.00036 (Low) and in the presence or absence of ACV or SP, as indicated. The dilution factors of infected neurons overlaid on MRC-5 cells are indicated on the left. (B) Quantification of plaque assays (*, P < 0.05; ***, P < 0.001; n.s., not significantly different; ANOVA with Bonferroni test) from neurons (1 × 10⁵) infected with VZV-pOka (VZV) or VZVr at an MOI of 0.0073. ACV, acyclovir.

JNK blockade inhibits VZV infection in sensory neurons. (A) Sensory neurons immunostained with antibodies to peripherin (red), which stains intermediate filaments, and Brn3a (green), which is a transcription factor in the nucleus of the cell. Scale bar, 50 μm. (B) Sensory neurons (2.5 × 10⁴/cm²) were infected with VZVORF66GFP at an MOI of 0.001 for 4 days in the absence (top) or presence (bottom) of JNK inhibitor. Scale bar, 50 μm. (C) Quantification of plaque assays from 1 × 10⁵ sensory neurons infected with VZVORF66GFP at an MOI of 0.001 in the absence (VZV) or presence (VZV+SP) of JNK inhibitor. **, P < 0.01; Student's t test. Plaque assays were performed as for and . The error bars indicate SEM.

JNK blockade inhibits VZV gE protein expression and replication in neurons. (A) Western blotting demonstrating a dose-dependent decrease in VZV gE expression in neurons treated with increasing doses of the JNK inhibitor SP600125 (lane 1, mock infection; lanes 2 to 6, VZV infected; lanes 3 to 6, SP600125 at 1, 3, 9, and 18 μM, respectively). (B) Western blotting demonstrating that VZV gE expression increases to a far lesser extent over time in neurons treated with 18 μM SP600125. (C) Quantification of gE expression in panel B, relative to GAPDH expression. (D) Neurons infected with VZVORF66GFP in the presence or absence of SP600125 were subjected to immunostaining for gE (red) at the indicated time points postinfection. Scale bar, 10 μm. (E) Neurons were infected with VZVORF66GFP for 48 h and immunostained for pJNK (red). Scale bar, 20 μm. Neuron density, 2.5 × 10⁴/cm²; VZVORF66GFP, MOI = 0.001.

Activation of the JNK pathway in neurons following VZV infection. (A) Human ES-derived neurons were infected with cell-free pOka VZV. (Top two rows) Immunostaining for IE62 and gE (red) revealing expression at 48 h p.i. and 72 h p.i., respectively (scale bar, 50 μm). (Bottom two rows) Immunostaining for pJNK (red) and pc-Jun (magenta) demonstrating upregulation beginning at 48 h p.i. Parallel images are depicted in each row (scale bar, 20 μm). The arrows indicate examples of individual cells or cell clusters with increased expression. Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole) (blue). (B) Analysis of the percentages of pOka VZV-infected cells expressing 1.5-fold-higher intensity than mock-infected cells, revealing increased expression of pJNK and pc-Jun at 48 h p.i. and 72 h p.i. (*, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to mock infection; analysis of variance [ANOVA] with Bonferroni test). Bars represent the mean ± SEM. (C) Immunostaining of neurons infected with VZVORF66GFP at 48 h p.i. demonstrating GFP expression in several cells expressing increased amounts of pJNK (red) and pc-Jun (purple). Scale bar, 10 μm. Neuron density, 2.5 × 10⁴/cm²; VZV pOkaORF66GFP, multiplicity of infection (MOI) = 0.001.

JNK blockade inhibits the production of infectious virus and shows inhibitory effects additive to those of acyclovir on VZV infection in neurons. (A and B) Neurons (2 × 10⁵ at a density of 5 × 10⁴/cm²) were infected with rVZV_LUCBAC at an MOI of 0.002 in the presence of inhibitors. Infected neurons at 72 h p.i. were harvested, diluted, and laid onto MRC-5 cells (1 × 10⁵/cm²) at the dilution factors indicated on the left (1:1 corresponds to 1 × 10⁵ infected neurons). The cells were fixed at 6 days p.i., and the plaques were visualized. (A) Vehicle (DMSO), JNK inhibitor SP600125 (SP) (18 μM), acyclovir (ACV) (60 μM), or a combination (SP + ACV) was used. (B) Vehicle (DMSO) and a combination of ACV (1 μM) and SP600125 (0, 4, and 8 μM) were used as inhibitors. Visualized plaques were counted. The experiment was performed twice, and each time triplicate wells were used. The error bars indicate SEM. *, P < 0.05; one-way ANOVA.

JNK blockade inhibits viral reactivation. (A) Schematic of an in vitro model of viral latency and reactivation. Neurons (purple) were cultured in microfluidic devices whose microchannels (M) allow fluidic isolation of axons in the axonal compartment (A) from their neuronal cell bodies in the somal compartment (S). (i and ii) Selective addition of virus (red) to the axonal side did not lead to productive infection (i), though some neurons harbored viral DNA in a circular configuration compatible with latency (indicated by red soma) (ii). (iii and iv) Anti-NGF (blue) was added to cultures for 7 days (iii), resulting in viral reactivation from some latently infected neurons (3rd neuron from the top) (iv). (B) Immunostaining for pJNK revealed intense expression in the positive control (anisomycin at 25 μg/ml for 30 min, which is known to activate JNK) and in neurons treated with anti-NGF (50 μg/ml). Scale bar, 50 μm. (C) Addition of JNK inhibitor markedly decreased the observed reactivation following anti-NGF treatment. The red numbers represent the numbers of individual microfluidic cultures employed for each condition. Neuron density, 5 × 10⁴/cm²; VZV pOka, 100 PFU/axonal compartment.