Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Oct;85(20):10814-25.
doi: 10.1128/JVI.00104-11. Epub 2011 Aug 10.

Myxoma virus induces type I interferon production in murine plasmacytoid dendritic cells via a TLR9/MyD88-, IRF5/IRF7-, and IFNAR-dependent pathway

Peihong Dai  1 , Hua Cao, Taha Merghoub, Francesca Avogadri, Weiyi Wang, Tanvi Parikh, Chee-Mun Fang, Paula M Pitha, Katherine A Fitzgerald, Masmudur M Rahman, Grant McFadden, Xiaoyu Hu, Alan N Houghton, Stewart Shuman, Liang Deng
Affiliations

Myxoma virus induces type I interferon production in murine plasmacytoid dendritic cells via a TLR9/MyD88-, IRF5/IRF7-, and IFNAR-dependent pathway

Peihong Dai et al. J Virol. 2011 Oct.

Erratum in

  • J Virol. 2011 Dec;85(23):12835

Abstract

Poxviruses are large DNA viruses that replicate in the cytoplasm of infected cells. Myxoma virus is a rabbit poxvirus that belongs to the Leporipoxvirus genus. It causes a lethal disease called myxomatosis in European rabbits but cannot sustain any detectable infection in nonlagomorphs. Vaccinia virus is a prototypal orthopoxvirus that was used as a vaccine to eradicate smallpox. Myxoma virus is nonpathogenic in mice, whereas systemic infection with vaccinia virus can be lethal even in immunocompetent mice. Plasmacytoid dendritic cells (pDCs) are potent type I interferon (IFN)-producing cells that play important roles in antiviral innate immunity. How poxviruses are sensed by pDCs to induce type I IFN production is not well understood. Here we report that infection of primary murine pDCs with myxoma virus, but not with vaccinia virus, induces IFN-α, IFN-β, tumor necrosis factor (TNF), and interleukin-12p70 (IL-12p70) production. Using pDCs derived from genetic knockout mice, we show that the myxoma virus-induced innate immune response requires the endosomal DNA sensor TLR9 and its adaptor MyD88, transcription factors IRF5 and IRF7, and the type I IFN positive-feedback loop mediated by IFNAR1. It is independent of the cytoplasmic RNA sensing pathway mediated by the mitochondrial adaptor molecule MAVS, the TLR3 adaptor TRIF, or the transcription factor IRF3. Using pharmacological inhibitors, we demonstrate that myxoma virus-induced type I IFN and IL-12p70 production in murine pDCs is also dependent on phosphatidylinositol 3-kinase (PI3K) and Akt. Furthermore, our results reveal that the N-terminal Z-DNA/RNA binding domain of vaccinia virulence factor E3, which is missing in the orthologous M029 protein expressed by myxoma virus, plays an inhibitory role in poxvirus sensing and innate cytokine production by murine pDCs.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Innate immune responses of murine Flt3L-BMDCs to myxoma virus or vaccinia virus infections. Flt3L-BMDCs were generated from female C57B/6 mice by culturing bone marrow cells in the presence of Flt3L. (A) Cells (1 × 106) were infected with wild-type vaccinia virus (WT VAC) or myxoma virus at a multiplicity of infection (MOI) of 10 for 1 h. A mock-infected control (no virus) was included. Alternatively, cells were stimulated with TLR9 agonist CpG 2216 (10 μg/ml). Supernatants were collected 22 h later, and the concentrations of IFN-α, IFN-β, TNF, and IL-12p70 were determined by ELISA. Data are means ± standard errors of the means (SEM). The experiment was repeated at least twice, and the results of a representative are shown. (B) Cells (1 × 106) were infected with myxoma virus at an MOI of 10. Supernatants were collected at 1, 4, 8, 12, or 22 h postinfection. The concentrations of IFN-α, IFN-β, TNF, and IL-12p70 were determined by ELISA. Data are means ± SEM. A representative experiment is shown and repeated at least twice. (C) Cells (1 × 106) were infected with myxoma virus at increasing MOIs ranging from 0.25 to 10. Supernatants were collected at 22 h postinfection. The concentrations of IFN-α, IFN-β, TNF, and IL-12p70 were determined by ELISA. Data are means ± SEM. The experiment was repeated once, and the results of a representative are shown.
Fig. 2.
Fig. 2.
Myxoma virus infection of purified murine pDCs induces type I IFN induction. Flt3L-BMDCs were sorted using FACS to purify pDCs and cDCs to high purity (∼98%). pDCs (2 × 105) (CD11c+ B220+ PDCA-1+) and cDCs (1 × 106) (CD11c+ B220− PDCA-1−) were stimulated with CpG or infected with either WT VAC or myxoma virus at an MOI of 10. Supernatants were collected at 22 h postinfection. The concentrations of IFN-α (A) and IFN-β (B) were determined by ELISA. Data are means ± SEM. The experiment was repeated at least twice, and the results of a representative are shown.
Fig. 3.
Fig. 3.
Myxoma virus infection of purified murine pDCs induces the expression of IFNA4, IFNB, IL-12p35, and IL-12p40 genes. FACS-sorted pDCs (5 × 105) were infected with either WT VAC or myxoma virus at an MOI of 10. Cells were collected at 3 or 6 h postinfection. Real-time PCR analysis of IFNA4 (A), IFNB (B), IL-12p35 (C), and IL-12p40 (D) mRNAs was performed. Data are means ± SEM. The experiment was repeated twice, and the results of a representative are shown.
Fig. 4.
Fig. 4.
Myxoma virus induction of type I IFN, TNF, and IL-12p70 in murine pDCs requires TLR9/MyD88. Purified murine pDCs were obtained using FACS from Flt3L-BMDCs generated from MyD88−/− (A), TLR9−/− (B), and TLR7−/− (C) mice and their age-matched WT controls. pDCs (2 × 105) were stimulated with CpG or infected with myxoma virus at an MOI of 10. Supernatants were collected 22 h later. The concentrations of IFN-α/β, IL-12p70, and TNF were determined by ELISA. Data are means ± SEM. The combined results of three independently performed experiments are shown. **, P < 0.01; ***, P < 0.001; ns, nonsignificant (comparisons were made between WT cells and various knockout cells as indicated).
Fig. 5.
Fig. 5.
Myxoma virus-induced innate immune responses in murine pDCs are independent of MAVS, TRIF, or IRF3. Flt3L-BMDCs were generated from MAVS−/− (A), TRIF−/− (B), and IRF3−/− (C) mice and their age-matched WT controls. FLt3L-BMDCs (1 × 106) were infected with myxoma virus. Supernatants were collected 22 h later, and the concentrations of IFN-α/β were determined by ELISA. Data are means ± SEM. Results shown are representative of three experiments.
Fig. 6.
Fig. 6.
The interferon regulatory factors IRF5 and IRF7 and the type I IFN positive-feedback loop mediated by IFNAR1 are required for myxoma virus-induced innate immune response in murine pDCs. Purified murine pDCs were obtained using FACS from Flt3L-BMDCs generated from IRF5−/− (A), IRF7−/− (B), and IFNAR1−/− (C) mice and their age-matched controls. pDCs (2 × 105) were infected with myxoma virus at an MOI of 10. Supernatants were collected 22 h later. The concentrations of IFN-α/β, TNF, and IL-12p70 were determined by ELISA. Data are means ± SEM. The combined results of three independently performed experiments are shown. ***, P < 0.001 (comparisons were made between WT cells and various knockout cells as indicated).
Fig. 7.
Fig. 7.
Chloroquine, wortmannin, and 3-methyladenine block the induction of IFN-α/β and IL-12p70 in murine BMDCs by myxoma virus. Flt3L-BMDCs (1 × 106) were infected with myxoma virus at an MOI of 10 for 1 h. Cells were washed and incubated in fresh medium in the presence or absence of increasing concentrations of chloroquine (A), wortmannin (B), or 3-methyladenine (C). Supernatants were collected 22 h later. The concentrations of IFN-α/β and IL-12p70 were determined by ELISA. Data are means ± SEM. The combined results of three independently performed experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant (compared with results for no drug treatment).
Fig. 8.
Fig. 8.
Akt inhibitors block the induction of IFN-α/β and IL-12p70 in murine BMDCs by myxoma virus. Flt3L-BMDCs (1 × 106) were infected with myxoma virus at an MOI of 10 for 1 h. Cells were washed and incubated in fresh medium in the presence or absence of increasing concentrations of Akt inhibitor VIII or X. Supernatants were collected 22 h later. The concentrations of IFN-α (A), IFN-β (B), and IL-12p70 (C) were determined by ELISA. Data are means ± SEM. The combined results of three independently performed experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant (compared with results for no drug treatment).
Fig. 9.
Fig. 9.
The induction of IFN-α/β, TNF, and IL-12p70 in murine pDCs by CpG or myxoma virus is attenuated by vaccinia virus E3. (A) Purified murine pDCs (2 × 105) were stimulated with CpG alone or infected with WT vaccinia virus or ΔE3L, E3LΔ83N, E3LY48A, or E3LΔ26C mutant virus followed by the addition of CpG. (B) Purified murine pDCs (2 × 105) were infected with myxoma virus alone at an MOI of 10 or coinfected with myxoma virus and equal amounts of WT vaccinia virus or ΔE3L, E3LΔ83N, E3LY48A, or E3LΔ26C mutant virus. Supernatants were collected 22 h later. The concentrations of IFN-α, IFN-β, TNF, and IL-12p70 were determined by ELISA. Data are means ± SEM. The combined results of three independently performed experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant (compared with results for CpG treatment alone [A] or myxoma virus infection alone [B]).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alexopoulou L., Holt A. C., Medizhitov R., Flavell R. A. 2001. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 413:732–738 - PubMed
    1. Asselin-Paturel C., et al. 2001. Mouse type I IFN-producing cells are immature APCs with plasmacytoid morphology. Nat. Immunol. 2:1144–1150 - PubMed
    1. Au W. C., Moore P. A., Lowther W., Juang Y. T., Pitha P. M. 1995. Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes. Proc. Natl. Acad. Sci. U. S. A. 92:11657–11661 - PMC - PubMed
    1. Barnes B. J., Kellum M. J., Field A. E., Pitha P. M. 2002. Multiple regulatory domains of IRF-5 control activation, cellular localization, and induction of chemokines that mediate recruitment of T lymphocytes. Mol. Cell. Biol. 22:5721–5740 - PMC - PubMed
    1. Blommaart E. F., Krause U., Schellens J. P., Vreeling-Sindelarova H., Meijer A. J. 1997. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur. J. Biochem. 243:240–246 - PubMed

Publication types

MeSH terms

LinkOut - more resources