Inhibition of type III interferon activity by orthopoxvirus immunomodulatory proteins

Abstract

The type III interferon (IFN) family elicits an antiviral response that is nearly identical to that evoked by IFN-alpha/beta. However, these cytokines (known as IFN-lambda1, 2, and 3) signal through a distinct receptor, and thus may be resistant to the evasion strategies used by some viruses to avoid the IFN-alpha/beta response. Orthopoxviruses are highly resistant to IFN-alpha/beta because they encode well-characterized immunomodulatory proteins that inhibit IFN activity. These include a secreted receptor (B18R) that neutralizes IFN-alpha/beta, and a cytoplasmic protein (E3L) that blocks IFN-alpha/beta effector functions in infected cells. We therefore determined the ability of these immunomodulators to abrogate the IFN-lambda-induced antiviral response. We found that (i) vaccinia virus (VACV) replication is resistant to IFN-lambda antiviral activity; (ii) neither VACV B18R nor the variola virus homolog B20R neutralizes IFN-lambda; (iii) VACV E3L inhibits the IFN-lambda-mediated antiviral response through a PKR-dependent pathway; (iv) VACV infection inhibits IFN-lambdaR-mediated signal transduction and gene expression. These results demonstrate differential sensitivity of IFN-lambda to multiple distinct evasion mechanisms employed by a single virus.

FIG. 1.

Interferon-λ (IFN-Δ) weakly inhibits vaccinia virus (VACV) replication. (A) A549 cells were treated with the indicated concentrations of IFN-λ1 for 18 h prior to infection with VACV at MOI = 1 or 0.1. Extracellular virus titers were measured in the cell culture medium 48 h post-infection. (B) A549 cells were treated with the indicated concentrations of IFN-λ1 for 18 h prior to infection with VACV at MOI = 0.1. Intracellular virus replication was measured 48 h post-infection. For both A and B, data are displayed as the mean of 2–3 independent experiments, and error bars represent standard error of the mean (SEM). (C) A549 cells were treated with increasing concentrations (0.025–250 ng/mL in 10× steps) of IFN-λ1 and IFN-α or IFN-γ alone or in combination for 18 h prior to infection with VACV at MOI = 1. Six untreated control wells were left uninfected. Cells were stained with 5% crystal violet 48 h post-infection to visualize virus-induced cytopathic effects.

FIG. 2.

Vaccinia virus (VACV) B18R and VARV B20R both neutralize interferon (IFN)-α/β but not IFN-λ. DMEM or conditioned medium from HEK293T cells transfected with B18R-FLAG, B20R-FLAG, or empty expression vectors was incubated with the indicated concentrations of either (A) IFN-α2a or (B) IFN-λ1 for 30 min on ice before adding to Huh7 cells. (C) Conditioned medium diluted 1:10 was incubated with 5 ng of the indicated type I IFN. (D) Conditioned medium was diluted by the indicated ratio prior to Western blot analysis using a FLAG-specific antibody. Diluted conditioned media was incubated with 10 ng of IFN-α2a prior to addition to cells. (E) DMEM or conditioned medium from VACV-infected or -uninfected (uninf) HeLa S3 cells was incubated with either 0.5 ng/mL or 5 ng/mL IFN-α or IFN-λ1 for 30 min on ice prior to addition to Huh7 cells. For (A–E), induction of the representative IFN-stimulated gene MxA was measured 24 h after addition of IFN plus conditioned medium by quantitative RT-PCR. Expression is displayed as fold induction relative to untreated controls, and is normalized to GAPDH. All data are displayed as the mean of 3 independent experiments, and error bars represent SEM.

FIG. 3.

Vaccinia virus (VACV) infection rescues VSV from the antiviral activity of IFN-λ. (A) Immortalized mouse hepatocytes were treated with 2.5 ng IFN-α(A/D)/mL, 10 ng IFN-λ2/mL, or were left untreated for 24 h prior to infection with VACV (MOI = 1). Two hours later cells were infected with VSV-EGFP at MOI = 1 for 18 h. (A) Microscopic visualization of GFP+ cells. Numbers indicate mean ± SEM of GFP+ cells per field in 4 independent experiments. (B) Titer of VSV in infected culture medium displayed as plaque-forming units (PFU) per milliliter. Data are displayed as the mean of 4 independent experiments, and error bars represent SEM.

FIG. 4.

Inhibition of interferon-λ (IFN-Δ) antiviral activity requires vaccinia E3L. (A) Immortalized mouse hepatocytes were treated with 2.5 ng IFN-α(A/D)/mL, 10 ng IFN-λ2/mL, or were left untreated for 18 h prior to infection with wild-type vaccinia virus (VACV) or VACVΔE3L (MOI = 1). Two hours later cells were treated with 5 µg actinomycin D/mL to prevent late VACV gene expression, and were infected with VSV-EGFP at MOI = 1 for 18 h. GFP+ cells were quantified by flow cytometry. Data are displayed as the mean of 3 independent experiments, and error bars represent SEM. (B) Western blot analysis of E3L protein expression in wild-type VACV and VACVΔE3L-infected cells. Lysate from HEK293 cells transfected with a FLAG-tagged E3L expression vector is shown as a positive control. (C) VACVΔE3L is sensitive to the antiviral activity of IFN-λ. Huh7 cells were treated with 2.5 ng IFN-α/mL or 250 ng IFN-λ1/mL for 18 h prior to infection with VACV or VACVΔE3L (MOI = 1). Graph bars represent the mean extracellular viral titers 24 and 72 h post-infection from 3 independent experiments, and error bars indicate SEM.

FIG. 5.

Inhibition of VACVΔE3L by interferon-λ (IFN-Δ) requires PKR. Huh7 cells were transfected with a nontargeting negative control or PKR-specific siRNA 24 h prior to treatment with 2.5 ng IFN-α/mL or 250 ng IFN-λ1/mL. Eighteen hours post-treatment with IFN, cells were infected with VACVΔE3L at an MOI = 1. (A) PKR siRNA reduces the amount of PKR (arrow) as compared to cells transfected with a negative control siRNA as shown by Western blot. (B) Titers of VACVΔE3L in culture media collected at 1 and 3 days post-infection, determined by plaque assay. Graph bars represent the mean of 3 independent experiments and error bars indicate the standard error of the mean. (C) PKR siRNA restores virus-induced CPE in VACVΔE3L-infected cells treated with IFN-α or IFN-λ1 as determined by crystal violet staining 72 h post-infection.

FIG. 6.

Vaccinia infection blocks interferon-λ (IFN-Δ)–mediated STAT phosphorylation and gene expression. A549 cells were infected with VACV for 1 h (MOI = 5) prior to treatment with 2.5 ng IFN-α/mL or 5 ng IFN-λ1/mL. Cells were harvested for (A) Western blot analysis of STAT-1 phosphorylation 1 and 4 h post-treatment or (B) quantitative RT-PCR analysis of IFN-stimulated gene expression 4 h post-treatment. Graph bars represent the mean of 3 independent experiments, and error bars indicate SEM.

FIG. 7.

Summary of interferon-λ (IFN-Δ) neutralization by vaccinia virus (VACV). Although IFN-α and IFN-λ have low amino acid sequence homology and bind to distinct receptors, these receptors signal through pathways that are nearly identical. Therefore, the intracellular, but not extracellular, mechanisms used by VACV to block the IFN-α/β response also inhibit IFN-λ.