Differential Sensitivity of HSV-1 and PRV to IFN-λ Reveals a Neuron-Specific Antiviral Role for RSAD2

Figures

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  Figure 1.. Delayed ISG activation characterizes the IFN-λ response in peripheral neurons. SCGS were treated with rat IFN-λ2 (200 ng/ml) or vehicle for (A) 3 h and (B) 24 h. Volcano plots represent differential gene expression normalized to GAPDH. Scattered points represent genes; the x-axis is the log 2-fold change, whereas the y-axis is the statistical significance in differential expression. Red dots highlight genes significantly over- or under-expressed after IFN-λ exposure. The abundance of genes that play a role in innate immune responses are determined by using gProfiler.

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  Figure 2.. Time-course qPCR analysis of ISG induction in IFN-λ-treated SCGs. Normalized gene expression (2−ddCt) in superior cervical ganglion (SCG) neurons following (A) rat IFN-λ2 treatment (200 ng/ml) or (B) rat IFN-β treatment (100 U/ml) at 3 h-, 8 h- 24 h-post treatment. GAPDH was used as a housekeeping gene. Untreated SCGs served as the control. Statistical significance analyzed via Student’s t test (n ≥ 3).

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  Figure 3.. Differential antiviral activity of IFN-λ across neuronal and non-neuronal cell types following HSV-1 infection. (A) SCGs or (C) Rat2 cells were pre-treated with rat IFN-λ2 (200 ng/mL) for 24 h prior to infection with OK-14 (MOI 1). (B) SK-N-SH cells or (D) HDFs were pre-treated with human IFN-λ2 (200 ng/mL) for 24 h prior to infection with OK-14 (MOI 1). Virus yield was determined as plaque forming units (pfu). Statistical significance analyzed via Student’s t test (n ≥ 3) is indicated as follows: ns, not significant; *, p < 0.05, ****, p < 0.00005.

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  Figure 4.. Loss of ICP34.5 renders HSV-1 more susceptible to IFN-λ-mediated restriction in neuronal cells. (A) Schematic comparison of the HSV-1 and PRV genomes, highlighting ICP34.5 that is missing in the PRV genome. HSV-1 mutant, Δ34.5 (originally named 17termA) illustration is adapted from Bolovan, et al.[51]. SCGs were pre-treated with rat IFN-λ2 (200 ng/mL) for 24 h prior to infection with (B) Δ34.5 or (C) 34.5R for 48 h. SK-N-SH cells were pre-treated with human IFN-λ2 (200 ng/mL) for 24 h prior to infection with (D) Δ34.5 or (E) 34.5R for 24 h. Statistical significance analyzed via Student’s t test (n ≥ 4) is indicated as follows: ns, not significant; *, p < 0.05, ****, p < 0.00005.

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  Figure 5.. Neuronal IFN-λ response does not alter HSV-1 transcription but interferes with viral protein synthesis when ICP34.5 is absent. qPCR quantification of viral immediate-early (ICP27) (A), early (ICP8) (B), and late (gC) (C) gene expression and protein accumulation (D) in SCGs pre-treated with rat IFN-λ2 (200 ng/ml) for 24 h and infected with Δ34.5 or 34.5R at MOI 1 for either 3 h-, 8 h-, or 24 hpi. Statistical significance was performed using Student’s t test: *, p < 0.05 (n=3 replicates). Immunoblotting was done using antibodies against ICP4, ICP8, poly-HSV-1, and SNAP25.

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  Figure 6.. RSAD2 expression increases in SCGs after IFN-λ treatment. (A) Immunofluorescence staining of RSAD2 in SCGs after 3 h- and 24 h-post-treatment with rat IFN-λ2 (scale bars, 10 μm). (B) Immunoblotting of RSAD2 in SCGs treated with rat IFN-λ2 for 3 or 24 hours. SNAP25 was used as a loading control. (C) Immunofluorescence staining of RSAD2 in SK-N-SH cells treated with human IFN-λ2 for 3 or 24 hours (scale bars, 20 μm). (D) Immunoblotting of RSAD2 in SK-N-SH cells treated with human IFN-λ2 for either 3 or 24 hours. Beta-actin was used as a loading control.

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  Figure 7.. ICP34.5 suppresses RSAD2 expression and disrupts ER localization in primary neurons. (A) SCGs were pretreated with rat IFN-λ2 prior to infection with either 34.5R or Δ34.5 at MOI 1. Cells were fixed at 24 hpi and stained with RSAD2 antibody (scale bars, 20 μm). (B) Normalized gene expression of RSAD2 in SCGs following infection with Δ34.5, or 34.5R at MOI 1 for 3 h or 24 hpi. Statistical significance was assessed using unpaired two-tailed t tests with Welch’s correction. (C) Immunoblotting of SCGs pretreated with rat IFN-λ2 (200 ng/ml) for 24 h and infected with either 34.5R or Δ34.5 at MOI 1 for 3 h, 8 h, or 24 h. Lysates were probed with antibodies against RSAD2 and SNAP25.

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  Figure 8.. ICP34.5 suppresses RSAD2 expression, disrupts ER localization and supports viral gene expression in SK-N-SH cells. (A) Immunofluorescence staining of SK-N-SH cells pretreated with human IFN-λ2 prior to infection with either 34.5R or Δ34.5 at MOI 1. Cells were fixed at 3 hpi and 24 hpi and stained for RSAD2 antibody (scale bars are 10 μm). (B) Normalized gene expression of RSAD2 in SK-N-SH cells following infections with either Δ34.5 or 34.5R at MOI 1 across three timepoints: 3, 8, and 24 hpi. Statistical significance was determined using an unpaired Student’s t test; *, p < 0.05. (C) Immunoblotting of SK-N-SH cells pretreated with human IFN-λ2 for 24 h and infected with either 34.5R or Δ34.5 at MOI 1 for 3, 8, and 24 h. Lysates were probed with antibodies against RSAD2, ICP4, ICP8, poly-HSV-1, and beta-actin.

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  Figure 9.. eIF2α phosphorylation sustains in IFN-λ-treated neurons during Δ34.5 infection. SCGs were pretreated with rat IFN-λ2 for 24 h and subsequently infected with either the Δ34.5 or 34.5R at MOI 1. (A) Cells were harvested 3-, 8-, and 24 h post-infection. Immunoblots were probed with antibodies against phosphorylated eIF2α (p-eIF2α) and Snap25. (B) SK-N-SH cells were pretreated with human IFN-λ2 for 24 h, and subsequently infected with Δ34.5 or 34.5R at MOI 1. Cells were harvested 3, 8, and 24 h post-infection for immunoblot analysis. Immunoblots were probed with antibodies against phosphorylated eIF2α (p-eIF2α) and beta-actin. (C) RSAD2 levels were normalized to beta-actin and each lane is analyzed for p-eIF2α levels comparing Δ34.5 or 34.5R infection time points (ImageJ, n=3). Statistical significance was determined using an unpaired Student’s t test: *, p < 0.05, **, p < 0.005, ***, p < 0.0005, ns, not significant.

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  Figure 10.. RSAD2 knockdown restores ICP34.5-deficient HSV-1 replication in IFN-λ-treated SCGs. (A) Representative immunoblot showing RSAD2 protein levels in SCGs following transfection with nontargeting (NT) or RSAD2-specific siRNA. Quantification of the bands shown below the blot using ImageJ. RSAD2 levels were normalized to SNAP25 and demonstrate efficient RSAD2 depletion (RSAD2, 0.061 ± 0.003 SEM relative to NT control; unpaired Student’s t test, **, p < 0.005). (B) SCGs were transfected with siNT or ]siRSAD2 for 48 h and pretreated with rat IFN-λ2 for 24 h prior to infection with the HSV-1 Δ34.5 at an MOI of 10. Viral titers were quantified at 24 h post-infection. Bars represent mean ± SEM. Individual points indicate biological replicates. Statistical significance was determined using Brown-Forsythe and Welch one-way ANOVA followed by Games-Howell’s multiple comparisons test. *, p < 0.05; ns, not significant.

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  Figure 11.. Model of ICP34.5-mediated regulation of RSAD2 during IFN-λ-induced antiviral responses in neurons. IFN-λ binding to its receptor initiates the intrinsic immune response (1), inducing the phosphorylation of JAK1/TYK2 and formation of STAT1 homodimers in peripheral neurons (2), which promote the expression of a subset of interferon stimulated genes (ISGs), including RSAD2 (3). RSAD2 localizes to the endoplasmic reticulum (ER) (4). RSAD2-mediated ER stress activates the PKR-like endoplasmic reticulum kinase (PERK) to eIF2α and translational arrest (5). This leads to ATF4 nuclear localization and induction of the unfolded protein response (UPR) (6A). ICP34.5 counteracts this antiviral response by recruiting PP1α, which dephosphorylates eIF2α, reversing translational shut off (6B).