Activation of the chicken type I interferon response by infectious bronchitis coronavirus

Abstract

Coronaviruses from both the Alphacoronavirus and Betacoronavirus genera interfere with the type I interferon (IFN) response in various ways, ensuring the limited activation of the IFN response in most cell types. Of the gammacoronaviruses that mainly infect birds, little is known about the activation of the host immune response. We show that the prototypical Gammacoronavirus, infectious bronchitis virus (IBV), induces a delayed activation of the IFN response in primary renal cells, tracheal epithelial cells, and a chicken cell line. In fact, Ifnβ expression is delayed with respect to the peak of viral replication and the accompanying accumulation of double-stranded RNA (dsRNA). In addition, we demonstrate that MDA5 is the primary sensor for Gammacoronavirus infections in chicken cells. Furthermore, we provide evidence that accessory proteins 3a and 3b of IBV modulate the response at the transcriptional and translational levels. Finally, we show that, despite the lack of activation of the IFN response during the early phase of IBV infection, the signaling of nonself dsRNA through both MDA5 and TLR3 remains intact in IBV-infected cells. Taken together, this study provides the first comprehensive analysis of host-virus interactions of a Gammacoronavirus with avian innate immune responses.

Importance: Our results demonstrate that IBV has evolved multiple strategies to avoid the activation of the type I interferon response. Taken together, the present study closes a gap in the understanding of host-IBV interaction and paves the way for further characterization of the mechanisms underlying immune evasion strategies as well as the pathogenesis of gammacoronaviruses.

FIG 1

IBV infection delays Ifnβ upregulation. Chicken embryo kidney (CEK) cells were infected with IBV M41 at an MOI of 0.1. (A) Replication of IBV was quantified by titration in cell culture supernatants of infected cells. In a parallel experiment, intracellular IBV RNA was quantified using RT-qPCR. (B) Ifnβ mRNA levels were determined using RT-qPCR, and IFN protein levels were determined using a chicken IFN-specific mx-luc cell-based bioassay. (C) Expression of genes involved in the antiviral response. All gene expressions were calculated as fold changes relative to uninfected control cells and normalized against an external reference gene (luciferase). For IBV total RNA, fold changes were calculated relative to a C_T of 30. Depicted are the results from a representative experiment out of three independent experiments.

FIG 2

Delayed induction of Ifnβ transcription is independent of cell type or IBV strain. (A) Epithelial cells from adult chicken trachea were infected with IBV M41 at an MOI of 0.1. (B) Fibroblast DF-1 cells were infected with IBV Beau-R at an MOI of 0.1. (C) CEK cells were infected with IBV strains M41, QX, and It02 at an MOI of 0.1. Intracellular IBV total RNA (open diamonds) and Ifnβ mRNA (bars) are depicted as fold changes as assessed by RT-qPCR. Gene expression of Ifnβ was calculated as fold changes relative to uninfected control cells and normalized against an external reference gene (luciferase). For IBV total RNA, fold changes were calculated relative to a C_T of 30. Error bars indicate standard deviations.

FIG 3

Chicken cells have the intrinsic ability to respond rapidly to dsRNA. (A) CEK cells were seeded in 24-well plates, and 48 h later they were stimulated with extracellular poly(I·C) for the indicated times. (B) DF-1 cells were infected with IPNV, a nonreplicating dsRNA virus, or stimulated with extracellular poly(I·C) (pI:C) or transfected poly(I·C). Four hours later, Ifnβ fold changes were determined by RT-qPCR. Bars represent the means (plus standard deviations) from triplicate wells from a representative experiment. Asterisks indicate significant differences (P < 0.01) with respect to the nonstimulated control as assessed by one-way ANOVA followed by a Bonferroni post hoc test. (C) CEK cells were infected with IBV M41 (MOI, 1), IBV Beau-R (MOI, 1), Sindbis-GFP (MOI, 1), IPNV (MOI, 50), and RVFV Cl13 (MOI, 5). Depicted are Ifnβ fold changes at 12 hpi relative to uninfected control cells as assessed by RT-qPCR.

FIG 4

MDA5, not TLR3, is the prime sensor of IBV. (A) CEK cells were infected with IBV M41 for 24 h in the presence or absence of RNase A. Ifnβ expression was analyzed by RT-qPCR. Stimulation with poly(I·C) in the presence or absence of RNase A was included as a positive control. DF-1 cells (B and D) and DF-1 Ifnβ-luc reporter cells (C) were transfected with siRNAs against Tlr3 and Mda5 or a control siRNA, and 48 h later they were infected with IBV M41 (MOI, 0.1). Ifnβ mRNA (B), Ifnβ-luciferase activity (C), and IBV titers and intracellular RNA (D) were analyzed 18 hpi. Bars represent the means (plus standard deviations) from triplicate wells from a representative experiment. Asterisks indicate significant differences (P < 0.01) with respect to the non-RNase A-treated control (A) or to the siRNA control (B and C), as assessed by one-way ANOVA followed by a Bonferroni post hoc test.

FIG 5

Early accumulation of dsRNA in IBV-infected cells does not result in the early induction of Ifnβ. CEK cells were infected with IBV M41 or IBV Beau-R at the indicated MOIs. At 12 hpi (A), dsRNA was visualized in M41-infected cells using an antibody against dsRNA. (B) Expression of Ifnβ mRNA was analyzed by RT-qPCR. (C) CEK cells were infected with IBV M41, and the accumulation of dsRNA was visualized at the indicated time postinfection. (D) RNA fluorescent in situ hybridization of Ifnβ mRNA in IBV M41-infected CEK cells. Open arrowheads indicate cells that contain dsRNA and no Ifnβ mRNA. Solid white arrowheads indicate cells that contain both dsRNA and Ifnβ mRNA.

FIG 6

Accessory proteins 3a and 3b are involved in regulation of IFN transcription and protein production. (A) CEK cells were infected with IBV Beau-R 3a/3b (scAUG3ab) or 5a/5b (scAUG5ab) null virus (MOI, 0.1). Ifnβ levels were determined using RT-qPCR. (B to D) CEK cells were infected with scAUG3a, scAUG3b, or scAUG3ab null IBV virus (MOI, 0.1). In the same cultures, Ifnβ mRNA (B), virus titers (C), and type I IFN protein (D) were quantified. Bars represent the means (plus standard deviations) from triplicate wells from a representative experiment. Significant differences (P < 0.01) relative to the Beau-R virus at the same time point (*) or between the indicated bars (#) as assessed by a two-way ANOVA followed by a Bonferroni post hoc test.

FIG 7

Signaling of nonself RNA remains intact in IBV-infected cells. (A) CEK cells were infected with IBV M41 for 3 h and stimulated with extracellular poly(I·C) (50 μg/ml) for an additional 3 h, after which Ifnβ transcription was analyzed by RT-qPCR. (B) DF-1 Ifnβ-luc reporter cells were transfected with siRNAs against Tlr3 and Mda5 or a control siRNA, and 48 h later they were infected with IPNV (MOI, 50); at 6 hpi luciferase activity was quantified. (C) Knockout (KO) and wild-type (wt) MEFs were infected with IPNV (MOI, 50) for 8 h. (D) CEK cells were infected with IBV M41 (MOI, 10) for 6 h and superinfected with IPNV or UV-inactivated IPNV (MOI, 50) for an additional 6 h. (E) CEK cells were coinfected with IBV M41 (MOI, 5) and RVFV clone 13 (MOI, 5) and sampled at 6 hpi. (F) DF-1 cells were infected with IBV Beau-R (MOI, 1) for 3 h and superinfected with IPNV (MOI, 50) or transfected with poly(I·C) (t[pI:C]; 500 ng/well) for an additional 4 h. (C to F) Ifnβ levels were quantified by RT-qPCR. Bars represent the means (plus standard deviations) from triplicate wells. Significant differences (P < 0.01) are indicated by an asterisk and were assessed by one-way ANOVA followed by a Bonferroni post hoc test.