Baculovirus induces type I interferon production through toll-like receptor-dependent and -independent pathways in a cell-type-specific manner

Abstract

Autographa californica nuclear polyhedrosis virus (AcNPV) is a double-stranded-DNA virus that is pathogenic to insects. AcNPV was shown to induce an innate immune response in mammalian immune cells and to confer protection of mice from lethal viral infection. In this study, we have shown that production of type I interferon (IFN) by AcNPV in murine plasmacytoid dendritic cells (pDCs) and non-pDCs, such as peritoneal macrophages and splenic CD11c+ DCs, was mediated by Toll-like receptor (TLR)-dependent and -independent pathways, respectively. IFN regulatory factor 7 (IRF7) was shown to play a crucial role in the production of type I IFN by AcNPV not only in immune cells in vitro but also in vivo. In mouse embryonic fibroblasts (MEFs), AcNPV produced IFN-beta and IFN-inducible chemokines through TLR-independent and IRF3-dependent pathways, in contrast to the TLR-dependent and IRF3/IRF7-independent production of proinflammatory cytokines. Although production of IFN-beta and IFN-inducible chemokines was severely impaired in IFN promoter-stimulator 1 (IPS-1)-deficient MEFs upon infection with vesicular stomatitis virus, AcNPV produced substantial amounts of the cytokines in IPS-1-deficient MEFs. These results suggest that a novel signaling pathway(s) other than TLR- and IPS-1-dependent pathways participates in the production of type I IFN in response to AcNPV infection.

FIG. 1.

Involvement of the TLR pathway in the production of type I IFN by AcNPV in immune cells and in mice. (A) PECs (2 × 10⁵ cells/well) prepared from wild-type, MyD88-deficient, or TLR9-deficient mice were stimulated with AcNPV (10 μg/ml) at the indicated concentrations of chloroquine. These cells were also treated with LPS (10 μg/ml) in the presence (+) or absence (−) of chloroquine (Chl) (12.5 μg/ml) (right). After 24 h of incubation, the production of IFN-α, IFN-β, and IL-12 in culture supernatants was determined by ELISA. (B) PECs (2 × 10⁵ cells/well) prepared from wild-type, TLR3-deficient, or TLR7-deficient mice were stimulated with AcNPV (10 μg/ml), LPS (10 μg/ml), VSV (NCP mutant, MOI of 0.1), or poly(I:C) (pIC) (50 μg/ml). After 24 h of incubation, production of IFN-α in culture supernatants was determined by ELISA. (C) PECs prepared as described for panel A were transfected with AcNPV DNA (Ac) (25 μg/ml), E. coli DNA (Ec) (25 μg/ml), or mCpG (CpG) (1 μg/ml). After 24 h of incubation, production of IFN-α, IFN-β, and IL-12 in the culture supernatants was determined by ELISA. (D) Splenic pDCs (2 × 10⁵ cells/well) prepared from wild-type, MyD88-deficient, or TLR9-deficient mice were stimulated with AcNPV (10 μg/ml). After 24 h of incubation, production of IFN-α in the culture supernatants was determined by ELISA. (E) AcNPV (100 μg/mouse) was intraperitoneally inoculated into wild-type, MyD88-deficient, or TLR9-deficient mice, and levels of IFN-α and IL-12 production in sera were determined by ELISA at the indicated time points. Data are shown as the means ± standard deviations.

FIG. 2.

IRF7 plays a crucial role in the production of type I IFN by AcNPV in immune cells and in mice. (A) PECs and splenic CD11c⁺ DCs (2 × 10⁵ cells/well) prepared from wild-type, IRF3-deficient, or IRF7-deficient mice were stimulated with the indicated amounts of AcNPV or VSV (NCP mutant, MOI of 0.1). After 24 h of incubation, the production of IFN-α, IFN-β, and IL-12 in culture supernatants was determined by ELISA. (B) Splenic pDCs (2 × 10⁵ cells/well) prepared from wild-type or IRF7-deficient mice were stimulated with the indicated amounts of AcNPV. After 24 h of incubation, production of IFN-α in culture supernatants was determined by ELISA. (C) AcNPV (100 μg/mouse) was intraperitoneally inoculated into wild-type and IRF7-deficient mice, and levels of IL-12, IL-6, and IFN-α production in sera were determined by ELISA at the indicated times. Data are shown as the means ± standard deviations.

FIG. 3.

Involvement of the IFNR signaling pathway in the production of type I IFN by AcNPV. (A and B) PECs (A) and splenic CD11c⁺ DCs (B) (2 × 10⁵ cells/well) prepared from wild-type and IFNR-deficient mice were stimulated with the indicated amounts of AcNPV or poly(I:C) (pIC) (50 μg/ml). After 24 h of incubation, the production of IFN-α, IFN-β, or IL-12 in culture supernatants was determined by ELISA. (C) AcNPV (100 μg/mouse) was intraperitoneally inoculated into wild-type and IFNR-deficient mice, and levels of IFN-α and IL-12 production in sera were determined by ELISA at the indicated time points. Data are shown as the means ± standard deviations.

FIG. 4.

Immune activation by AcNPV is not mediated by gp64. (A) His-gp64ΔTM expressed in Sf-9 cells was purified and subjected to sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis under reducing conditions. Molecular size markers (lane M), purified AcNPV particles (lanes 1), and His-gp64ΔTM (lanes 2) were visualized by Coomassie blue (CBB) staining (left) and immunoblotting using antihexahistidine monoclonal antibody (middle) and anti-gp64 antibody (AcV5) (right). (B) PECs and splenic CD11c⁺ DCs (2 × 10⁵ cells/well) prepared from wild-type mice were stimulated with AcNPV (10 μg/ml) or His-gp64ΔTM (gp64) (20 μg/ml). After 24 h of incubation, production of IL-12 and IFN-α in culture supernatants was determined by ELISA. (C) MEFs (3 × 10⁵ cells/well) prepared from wild-type mice were stimulated with AcNPV (10 μg/ml) or His-gp64ΔTM (20 μg/ml). At 4 h or 8 h poststimulation, total RNA was extracted and expression of mRNA of IFN-β, IL-6, MCP-1, RANTES, and IP-10 was determined by real-time PCR. Data are shown as the means ± standard deviations.

FIG. 5.

AcNPV produces IFN-β and IFN-inducible chemokines through a TLR-independent and IRF3-dependent pathways in MEFs. (A) MEFs (2 × 10⁴ cells/well) prepared from wild-type or MyD88/TRIF double knockout mice were stimulated with AcNPV (10 μg/ml), VSV (NCP mutant, MOI of 0.1), LPS (10 μg/ml), or poly(I:C) (pIC) (50 μg/ml). After 24 h of incubation, production of IL-6 and IFN-β in culture supernatants was determined by ELISA. (B) MEFs (3 × 10⁵ cells/well) prepared from wild-type or MyD88/TRIF double knockout mice were stimulated with AcNPV (10 μg/ml). Total RNA was extracted at the indicated time points, and the expression of mRNA of IFN-β, MCP-1, RANTES, and IP-10 was determined by real-time PCR. (C) MEFs (2 × 10⁴ cells/well) prepared from wild-type, IRF3-deficient, or IRF7-deficient mice were stimulated with AcNPV (10 μg/ml), LPS (10 μg/ml), or VSV (NCP mutant, MOI of 0.1). After 24 h of incubation, the production of IL-6 and IFN-β in culture supernatants was determined by ELISA. (D) MEFs (3 × 10⁵ cells/well) prepared from wild-type, IRF3-deficient, or IRF7-deficient mice were stimulated with AcNPV (10 μg/ml). Total RNA was extracted at the indicated time points, and the expression of mRNA of IFN-β, MCP-1, RANTES, and IP-10 was determined by real-time PCR. Data are shown as the means ± standard deviations.

FIG. 6.

AcNPV induces antiviral status in MEFs through an IRF3-dependent pathway. (A) MEFs (3 × 10⁵ cells/well) prepared from wild-type and IRF3-deficient mice were transfected with AcNPV DNA (25 μg/ml). Total RNA was extracted at the indicated time points, and the expression of mRNA of IFN-β, IFN-α1, MCP-1, RANTES, IL-6, and IP-10 was determined by real-time PCR. (B) MEFs (2 × 10⁴ cells/well) prepared from wild-type mice were stimulated with AcNPV (10 μg/ml) or LPS (10 μg/ml) in the presence of the indicated concentrations of chloroquine. After 24 h of incubation, production of IL-6 and IFN-β in culture supernatants was determined by ELISA. (C) MEFs (2 × 10⁴ cells/well) prepared from wild-type and IRF3-deficient mice were incubated with AcNPV (0.016 μg/ml to 2 μg/ml) or poly(I:C) (0.2 μg/ml to 25 μg/ml). After 24 h of incubation, cells were washed extensively with warm medium and infected with VSV (GLPLF mutant, MOI of 0.1). Cell viability was determined at 24 h postinfection by crystal violet staining and quantitated by spectroscopy. (D) MEFs (2 × 10⁴ cells/well) prepared from wild-type, IRF3-deficient, or IRF7-deficient mice were incubated with serial dilutions of murine IFN-α (10¹ to 10⁴ U/ml). After 24 h of incubation, cells were washed extensively with warm medium and infected with VSV (GLPLF strain, MOI of 0.1). Cell viability was determined at 24 h postinfection by crystal violet staining and quantitated by spectroscopy. Values are plotted as means from the triplicate wells. Data are shown as means ± standard deviations. (E) Microscopic observation of MEFs from wild-type mice, showing the antiviral status against VSV infection by the treatment with AcNPV or poly(I:C) in a dose-dependent manner. PC, infected cells; Mock, mock-infected cells. Samples are shown at a magnification of ×40.

FIG. 7.

Role of IPS-1 in immune activation by AcNPV. (A) PECs and splenic CD11c⁺ DCs (2 × 10⁵ cells/well) prepared from wild-type and IPS-1-deficient mice were stimulated with AcNPV (10 μg/ml), VSV (NCP mutant, MOI of 0.1), or poly(I:C) (pIC) (50 μg/ml). After 24 h of incubation, production of IFN-α and IFN-β in culture supernatants was determined by ELISA. (B) MEFs (3 × 10⁵ cells/well) prepared from wild-type and IPS-1-deficient mice were stimulated with AcNPV (10 μg/ml) or VSV (NCP mutant, MOI of 0.1). Total RNA was extracted at the indicated time points, and the expression of mRNA of IFN-β, MCP-1, RANTES, IL-6, and IP-10 was determined by real-time PCR. Data are shown as the means ± standard deviations.