Chikungunya virus induces IPS-1-dependent innate immune activation and protein kinase R-independent translational shutoff

Abstract

Chikungunya virus (CHIKV) is an arthritogenic mosquito-transmitted alphavirus that is undergoing reemergence in areas around the Indian Ocean. Despite the current and potential danger posed by this virus, we know surprisingly little about the induction and evasion of CHIKV-associated antiviral immune responses. With this in mind we investigated innate immune reactions to CHIKV in human fibroblasts, a demonstrable in vivo target of virus replication and spread. We show that CHIKV infection leads to activation of the transcription factor interferon regulatory factor 3 (IRF3) and subsequent transcription of IRF3-dependent antiviral genes, including beta interferon (IFN-β). IRF3 activation occurs by way of a virus-induced innate immune signaling pathway that includes the adaptor molecule interferon promoter stimulator 1 (IPS-1). Despite strong transcriptional upregulation of these genes, however, translation of the corresponding proteins is not observed. We further demonstrate that translation of cellular (but not viral) genes is blocked during infection and that although CHIKV is found to trigger inactivation of the translational molecule eukaryotic initiation factor subunit 2α by way of the double-stranded RNA sensor protein kinase R, this response is not required for the block to protein synthesis. Furthermore, overall diminution of cellular RNA synthesis is also observed in the presence of CHIKV and transcription of IRF3-dependent antiviral genes appears specifically blocked late in infection. We hypothesize that the observed absence of IFN-β and antiviral proteins during infection results from an evasion mechanism exhibited by CHIKV that is dependent on widespread shutoff of cellular protein synthesis and a targeted block to late synthesis of antiviral mRNA transcripts.

FIG. 1.

CHIKV infection triggers MOI-dependent transcription of IFN-β and ISGs. Primary HFs were infected with CHIKV at the indicated MOIs for 24 h. Values presented are fold changes in the expression of IFN-β, Viperin, and ISG56 mRNA relative to untreated cells as determined by qPCR. The data are representative of results from duplicate experiments.

FIG. 2.

CHIKV infection triggers Ser398 phosphorylation and nuclear accumulation of IRF3. (A) IRF3 Ser398 phosphorylation in primary HFs at 16 h postinfection with CHIKV at MOIs of 0.1, 1, and 10. (B) IRF3 Ser398 phosphorylation in primary HFs infected with CHIKV (MOI = 10) between 2 and 24 h postinfection. (C) Fold change relative to untreated cells as determined by qPCR of IFN-β mRNA in HFs infected with CHIKV (MOI = 1) for 4, 8, 16, and 24 h. (D) Nuclear accumulation of IRF3 in primary HFs infected with CHIKV (MOI = 10) at 16 h postinfection.

FIG. 3.

IRF3 is essential for CHIKV-induced IFN-β/ISG transcription. (A) Immunoblot showing GAPDH and total IRF3 protein from HFs in the presence or absence of stably expressed NPro or transiently transfected nonspecific (NS) or IRF3-directed siRNA as described in text. (B) Accumulation of IFN-β, ISG56, or Viperin mRNA in the indicated cell type following treatment with IFN-β (1,000 U/ml), infection with SeV (160 HA units/ml), or infection with CHIKV (MOI = 10) for 16 h. CHIKV-induced accumulation of IFN-β, ISG56, or Viperin mRNA is also shown in HFs after transfection of NS or IRF3-directed siRNA. The values presented are the mRNA fold changes relative to untreated cells as determined by qPCR and are representative of experiments performed at least in duplicate. (C) Accumulation of Mx1 mRNA in HF following treatment with SeV (160 HA units/ml), IFN-β (1,000 U/ml), or CHIKV (MOI = 10) for 16 h. The values presented are mRNA fold changes relative to untreated cells as determined by qPCR and are representative of experiments performed in duplicate.

FIG. 4.

CHIKV infection of HF leads to accumulation of dsRNA and IRF3-dependent transcription via IPS1. (A) Indirect immunofluorescence showing dsRNA in CHIKV-infected HFs (MOI = 10) at 0, 2, 4, 6, and 8 h postinfection. (B) Immunoblot showing levels of IPS1, Ser398-phosphorylated IRF3, total IRF3, and GAPDH after treatment of HFs with NS or IPS1-directed siRNA in the presence or absence of CHIKV infection (MOI = 10) at 24 h postinfection. (C) Transcriptional induction of IFN-β, Viperin, and ISG56 as measured by qPCR after treatment of HFs with NS or IPS1-directed siRNA. The cells were either left uninfected or were infected with SeV (░⃞) or CHIKV (▪; MOI = 10), and total RNA was harvested at 24 h postinfection. Values presented are normalized to transcriptional induction in the presence of NS siRNA (set to 1) and are representative of experiments performed in duplicate.

FIG. 5.

CHIKV infection of HFs does not induce secretion of IFN-α/β or synthesis of ISG proteins. (A) Expression of IFN-α/β-dependent luciferase from reporter cells after exposure to media collected from HF that were left untreated, treated with media containing 1,000 U of IFN-β/ml, infected with SeV (160 HA units/ml), or infected with SINV or CHIKV at indicated MOIs for 24 h. Treatments were performed in quadruplicate, and media were transferred to confluent THF-ISRE in six wells of a 96-well plate per treatment. Values presented are average luminescence readings from four separate treatments ± the standard error of the mean (SEM). (B) Immunoblot for Viperin, ISG56, GAPDH, and CHIKV capsid proteins after a 24-h treatment with IFN-β, SeV, or CHIKV at the indicated MOIs.

FIG. 6.

CHIKV infection of HFs induces cellular translational shutoff. An immunoblot shows the puromycin incorporated into newly synthesized protein in untreated HF or after exposure to 200 μg of cycloheximide (CHX) ml⁻¹ for 6 h or CHIKV (MOI = 10) (top), CHIKV capsid protein (middle), and GAPDH (bottom) for the indicated durations.

FIG. 7.

CHIKV infection and infection-associated RNA induce phosphorylation of PKR. (A) Immunoblot showing PKR phosphorylated on Thr446, total PKR, CHIKV capsid, and GAPDH after infection of HFs with CHIKV for the indicated duration (left) and densitometry ratio of phosphorylated PKR to total PKR with ratio in untreated cells set to 1 (right). (B) Immunoblot showing phosphorylated PKR, total PKR, and GAPDH from HFs treated only with transfection reagent (TF) or transfected for 6 h with total RNA harvested from HFs infected with CHIKV (MOI = 10) for the indicated duration.

FIG. 8.

CHIKV induces PKR-dependent phosphorylation of eIF2α. Immunoblots show PKR, Ser51-phosphorylated eIF2α, total eIF2α, CHIKV capsid, and GAPDH in HFs and HFs stably expressing shRNA directed against PKR following no infection or 24 h postinfection with CHIKV at the indicated MOIs (top), the corresponding densitometry ratio of phosphorylated eIF2α to total eIF2α in parental HFs (░⃞), and HFs expressing shRNA directed against PKR (▪) in response to CHIKV at an MOI of 0 (ratio set to 1), an MOI of 1, an MOI of 10, and an MOI of 100 (right).

FIG. 9.

CHIKV-associated cellular translational shutoff occurs in the absence of PKR. (A) Immunoblot showing PKR, CHIKV capsid, GAPDH, and puromycin incorporated into newly synthesized protein in HFs either left untreated or infected with CHIKV for 4, 8, 12, or 16 h at an MOI of 10 following the transfection of nonspecific (NS) or PKR-directed siRNA. (B) Immunoblot showing PKR, ISG56, Viperin, CHIKV capsid, and GAPDH in HF or HF-shPKR following no treatment, transfection with poly(I:C), or infection with CHIKV for 24 h. (C) Expression of IFN-α/β-dependent luciferase from reporter cells after exposure to media collected from HFs or HF-shPKRs that were left untreated or infected with SeV (160 HA units/ml) or CHIKV (MOI = 10) for 24 h. Treatments were performed in quadruplicate, and media were transferred to confluent THF-ISRE in six wells of a 96-well plate per treatment. Values presented are average luminescence readings from four separate treatments ± the SEM.

FIG. 10.

Infection with CHIKV or SINV reduces levels of cellular transcription. (A) Ratio of biotinylated (newly synthesized) to total RNA in HF following (i) no treatment, (ii) exposure to 5 μg of actinomycin D (ActD) ml⁻¹, or (iii) infection with CHIKV or SINV (MOI = 10) for the indicated durations. The ratio obtained from untreated cells was set to 1. (B) Agarose gel showing amplification products of RT-PCR generated using biotinylated or unbiotinylated RNA fractions as templates that were harvested from HFs either (i) left untreated, (ii) exposed to 5 μg of ActD ml⁻¹, or (iii) infected with CHIKV or SINV (MOI = 10) for 6 or 24 h.