Dual modulation of type I interferon response by bluetongue virus

Abstract

Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus that causes an economically important disease in ruminants. BTV infection is a strong inducer of type I interferon (IFN-I) in multiple cell types. It has been shown recently that BTV and, more specifically, the nonstructural protein NS3 of BTV are able to modulate the IFN-I synthesis pathway. However, nothing is known about the ability of BTV to counteract IFN-I signaling. Here, we investigated the effect of BTV on the IFN-I response pathway and, more particularly, the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription protein (STAT) signaling pathway. We found that BTV infection triggered the expression of IFN-stimulated genes (ISGs) in A549 cells. However, when BTV-infected cells were stimulated with external IFN-I, we showed that activation of the IFN-stimulated response element (ISRE) promoter and expression of ISGs were inhibited. We found that this inhibition involved two different mechanisms that were dependent on the time of infection. After overnight infection, BTV blocked specifically the phosphorylation and nuclear translocation of STAT1. This inhibition correlated with the redistribution of STAT1 in regions adjacent to the nucleus. At a later time point of infection, BTV was found to interfere with the activation of other key components of the JAK/STAT pathway and to induce the downregulation of JAK1 and TYK2 protein expression. Overall, our study indicates for the first time that BTV is able to interfere with the JAK/STAT pathway to modulate the IFN-I response.

Importance: Bluetongue virus (BTV) causes a severe disease in ruminants and has an important impact on the livestock economy in areas of endemicity such as Africa. The emergence of strains, such as serotype 8 in Europe in 2006, can lead to important economic losses due to commercial restrictions and prophylactic measures. It has been known for many years that BTV is a strong inducer of type I interferon (IFN-I) in vitro and in vivo in multiple cell types. However, the ability of BTV to interact with the IFN-I system remains unclear. Here, we report that BTV is able to modulate the IFN-I response by interfering with the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription protein (STAT) signaling pathway. These findings contribute to knowledge of how BTV infection interferes with the host's innate immune response and becomes pathogenic. This will also be important for the design of efficacious vaccine candidates.

FIG 1

BTV is sensitive to IFN-I and inhibits the IFN-I response. (A) Effect of IFN-I on the expression of BTV proteins. A549 cells were treated for 5 h with IFN-β (250 and 1,000 IU/ml) and infected with 0.05 TCID₅₀ of BTV-8/cell for 24 h. Cells were then lysed in RIPA buffer and analyzed by Western blotting. BTV VP7, VP5, NS1, and NS3 expression was detected to assess viral infection, and β-actin was detected as an internal control. (B and C) A549 (B) and Vero (C) cells were infected with 0.02 to 0.1 TCID₅₀ of BTV-8/cell for 18 h and left untreated (NT, not treated) or treated for 6 h with IFN-β (250 IU/ml). Cell lysates were extracted and used for detection of MDA5 and RIG-I. BTV NS3 expression was detected as a control for viral infection, and β-actin served as an internal control. Note that double bands were detected with the antibody raised against BTV NS3, as shown previously (39). These bands correspond to NS3 (higher band) and NS3A (lower band), a truncated form of NS3 that is synthesized from a second in-frame translation initiation codon within the BTV segment 10. (D and E) 293T cells were mock infected, infected with 0.001 or 0.01 TCID₅₀ of live BTV-8/cell, or infected with UV-inactivated BTV-8 (BT8-UV) using the same volume of inoculum as cells infected with 0.01 TCID₅₀ of live BTV-8/cell. Six hours later, cells were transfected with an ISRE reporter plasmid (pISRE-Luc) (D) or a CMV reporter plasmid (pCMV-Luc) (E) and a control vector (pRSV–β-Gal) to normalize for transfection efficiency. Eighteen h later, cells were left untreated (NT) or treated with IFN-β (1,000 IU/ml) for 7 h and lysed to determine β-galactosidase and luciferase activity. Mean ratios between luciferase and β-galactosidase activities of triplicate samples (± standard deviations) are presented. Results are representative of one experiment and were reproduced in two (CMV) or three (ISRE) independent experiments. *, P < 0.01; **, P < 0.001, compared to mock-infected cells for treated samples (unpaired t tests).

FIG 2

BTV infection interferes with STAT1 translocation. A549 cells were mock infected or infected with 0.05 TCID₅₀ of BTV-8/cell for 18 h and left untreated (NT) or treated with IFN-β (1,000 IU/ml) for 30 min. Cells were then washed, fixed, and stained with primary antibodies specific for VP7 and STAT1, followed by fluorescent dye-conjugated secondary antibodies. Intracellular localization of DAPI-stained nuclei (blue), VP7 (green), and STAT1 (red) was visualized by immunofluorescence microscopy (magnification, ×20). Scale bar, 50 μm.

FIG 3

BTV interferes with STAT1 phosphorylation. (A to C) A549 (A), HeLa (B), or Vero (C) cells were mock infected or infected with 0.01 to 0.1 TCID₅₀ of BTV-8/cell for 18 to 20 h and then left untreated (NT) or treated with IFN-α or IFN-β (1,000 IU/ml) for 30 min. (D) HeLa cells were infected with 0.05 TCID₅₀ of BTV-4/cell for 16 h and then left untreated (not treated, NT) or treated with IFN-β (1,000 IU/ml) for 30 min. For experiments shown in all panels, cells lysates were extracted and used for detection of total STAT1 and phospho-STAT1 (p-STAT1). Note the presence of double bands corresponding to the alpha form (around 91 kDa) and the beta form (around 84 kDa) of STAT1. BTV VP5 or NS3 expression was detected as a control for viral infection, and β-actin served as an internal control.

FIG 4

BTV replication is required for the inhibition of STAT1 phosphorylation. (A) A549 cells were mock infected, infected with 0.1 TCID₅₀/cell of BTV-8 cell, or infected with UV-inactivated BTV-8 (BT8-UV) using the same volume of inoculum as cells infected with 0.1 TCID₅₀ of live BTV-8/cell. Seventeen hour later, cells were left untreated (NT) or treated with IFN-β (1,000 IU/ml) for 30 min. (B) A549 cells were mock infected or infected with 0.1 TCID₅₀/cell of BTV-8 for 3 to 24 h and left untreated (NT) or treated with IFN-β (1,000 IU/ml) for 30 min. For experiments shown in both panels, cells lysates were extracted and used for detection of phospho-STAT1. BTV NS3 expression was detected as a control for viral infection, and β-actin served as an internal control. Note the presence of multiple bands detected with the NS3 antibody corresponding to NS3, NS3a, and their glycosylated forms.

FIG 5

BTV interferes specifically with the phosphorylation of STAT1. A549 cells and Vero cells were mock infected or infected with 0.1 TCID₅₀/cell of BTV-8 for 17 h and treated with IFN-β (1,000 IU/ml) for 15 min. Cells lysates were extracted and used for detection of total JAK1, phosphorylated TYK2 (p-TYK2), total TYK2, p-STAT1, total STAT1, phosphorylated STAT2 (p-STAT2), and total STAT2. BTV VP7 was detected as a control for viral infection, and β-actin served as an internal control.

FIG 6

BTV interferes with STAT1 distribution. A549 cells were mock infected or infected with 0.05 TCID₅₀/cell of BTV-8, BTV-4, or a vaccine strain of BTV-4 (BTV-4 VAC) for 18 h and left untreated (A) or treated with IFN-β (1,000 IU/ml) for 30 min (B). Cells were then washed, fixed, and stained with primary antibodies specific for VP7 and STAT1, followed by fluorescent dye-conjugated secondary antibodies. Intracellular localization of DAPI-stained nuclei (blue), STAT1 (red), and VP7 (green) was visualized by immunofluorescence microscopy (magnification, ×63). Examples of STAT1 accumulation in perinuclear regions are indicated by white arrows. Scale bar, 20 μm.

FIG 7

BTV downregulates TYK2 and JAK1 protein levels. (A) A549 cells and Vero cells were mock infected or infected for 42 h with 0.1 or 0.05 TCID₅₀/cell of BTV-8, respectively, and treated with IFN-β (1,000 IU/ml) for 15 min. Cells lysates were extracted and used for detection of total JAK1, p-TYK2, total TYK2, p-STAT1, total STAT1, p-STAT2, and total STAT2. BTV VP7 was detected as a control for viral infection, and β-actin served as an internal control. (B) A549 cells were mock infected (M) or infected with 0.05 TCID₅₀/cell of BTV-8 (BT) for 17 to 48 h. Cells lysates were extracted and used for detection of total TYK2, JAK1, STAT1, STAT2, and RIG-I. BTV VP7 was detected as a control for viral infection, and β-actin was used as an internal control.