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Innate immune response of human plasmacytoid dendritic cells to poxvirus infection is subverted by vaccinia E3 via its Z-DNA/RNA binding domain.

Cao H, Dai P, Wang W, Li H, Yuan J, Wang F, Fang CM, Pitha PM, Liu J, Condit RC, McFadden G, Merghoub T, Houghton AN, Young JW, Shuman S, Deng L - PLoS ONE (2012)

Bottom Line: Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1.These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway.Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists.

View Article: PubMed Central - PubMed

Affiliation: Dermatology Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

ABSTRACT
Plasmacytoid dendritic cells (pDCs) play important roles in antiviral innate immunity by producing type I interferon (IFN). In this study, we assess the immune responses of primary human pDCs to two poxviruses, vaccinia and myxoma virus. Vaccinia, an orthopoxvirus, was used for immunization against smallpox, a contagious human disease with high mortality. Myxoma virus, a Leporipoxvirus, causes lethal disease in rabbits, but is non-pathogenic in humans. We report that myxoma virus infection of human pDCs induces IFN-α and TNF production, whereas vaccinia infection does not. Co-infection of pDCs with myxoma virus plus vaccinia blocks myxoma induction effects. We find that heat-inactivated vaccinia (Heat-VAC; by incubating the virus at 55°C for 1 h) gains the ability to induce IFN-α and TNF in primary human pDCs. Induction of IFN-α in pDCs by myxoma virus or Heat-VAC is blocked by chloroquine, which inhibits endosomal acidification required for TLR7/9 signaling, and by inhibitors of cellular kinases PI3K and Akt. Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1. These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway. Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists. The myxoma virus ortholog of vaccinia E3 (M029) lacks the N-terminal Z-DNA/RNA binding domain, which might contribute to the immunostimulating properties of myxoma virus.

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N-terminal domain of vaccinia E3L mediates partial inhibition of IFN-α and TNF induction by myxoma virus and heat-inactivated vaccinia virus in human pDCs.(A) Human pDCs (2×105) were infected with myxoma virus alone, or co-infected with myxoma virus plus WT vaccinia, ΔE3L, E3LΔ83N, E3LY48A or E3LΔ26C. (B) pDCs were infected with Heat-VAC alone, or co-infected with Heat-VAC plus WT vaccinia, ΔE3L, E3LΔ83N, E3LY48A or E3LΔ26C; (C) pDCs were treated with CpG alone, or infected with WT vaccinia, ΔE3L, E3LΔ83N, E3LY48A or E3LΔ26C followed by addition of CpG. Supernatants were collected at 20 h post treatment. The IFN-α and TNF concentration values shown are averages of triplicate means (± SEM) of three independent experiments using human pDCs isolated from three different donors (*, p<0.05; **, p<0.01; ***, p<0.001).
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pone.0036823-g008: N-terminal domain of vaccinia E3L mediates partial inhibition of IFN-α and TNF induction by myxoma virus and heat-inactivated vaccinia virus in human pDCs.(A) Human pDCs (2×105) were infected with myxoma virus alone, or co-infected with myxoma virus plus WT vaccinia, ΔE3L, E3LΔ83N, E3LY48A or E3LΔ26C. (B) pDCs were infected with Heat-VAC alone, or co-infected with Heat-VAC plus WT vaccinia, ΔE3L, E3LΔ83N, E3LY48A or E3LΔ26C; (C) pDCs were treated with CpG alone, or infected with WT vaccinia, ΔE3L, E3LΔ83N, E3LY48A or E3LΔ26C followed by addition of CpG. Supernatants were collected at 20 h post treatment. The IFN-α and TNF concentration values shown are averages of triplicate means (± SEM) of three independent experiments using human pDCs isolated from three different donors (*, p<0.05; **, p<0.01; ***, p<0.001).

Mentions: To test whether the failure of untreated vaccinia to induce a response is due to the production of inhibitors, we performed a mixing experiment. When human pDCs were co-infected with live vaccinia plus an equivalent amount of Heat-VAC, the production of IFN-α was blocked and TNF secretion was reduced by 98% compared to the level induced by Heat-VAC alone (Fig. 8B). This result indicates that live vaccinia infection of pDCs introduces inhibitor(s) of poxvirus sensing pathway(s) in pDCs that are not generated during infection with Heat-VAC.


Innate immune response of human plasmacytoid dendritic cells to poxvirus infection is subverted by vaccinia E3 via its Z-DNA/RNA binding domain.

Cao H, Dai P, Wang W, Li H, Yuan J, Wang F, Fang CM, Pitha PM, Liu J, Condit RC, McFadden G, Merghoub T, Houghton AN, Young JW, Shuman S, Deng L - PLoS ONE (2012)

N-terminal domain of vaccinia E3L mediates partial inhibition of IFN-α and TNF induction by myxoma virus and heat-inactivated vaccinia virus in human pDCs.(A) Human pDCs (2×105) were infected with myxoma virus alone, or co-infected with myxoma virus plus WT vaccinia, ΔE3L, E3LΔ83N, E3LY48A or E3LΔ26C. (B) pDCs were infected with Heat-VAC alone, or co-infected with Heat-VAC plus WT vaccinia, ΔE3L, E3LΔ83N, E3LY48A or E3LΔ26C; (C) pDCs were treated with CpG alone, or infected with WT vaccinia, ΔE3L, E3LΔ83N, E3LY48A or E3LΔ26C followed by addition of CpG. Supernatants were collected at 20 h post treatment. The IFN-α and TNF concentration values shown are averages of triplicate means (± SEM) of three independent experiments using human pDCs isolated from three different donors (*, p<0.05; **, p<0.01; ***, p<0.001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351467&req=5

pone.0036823-g008: N-terminal domain of vaccinia E3L mediates partial inhibition of IFN-α and TNF induction by myxoma virus and heat-inactivated vaccinia virus in human pDCs.(A) Human pDCs (2×105) were infected with myxoma virus alone, or co-infected with myxoma virus plus WT vaccinia, ΔE3L, E3LΔ83N, E3LY48A or E3LΔ26C. (B) pDCs were infected with Heat-VAC alone, or co-infected with Heat-VAC plus WT vaccinia, ΔE3L, E3LΔ83N, E3LY48A or E3LΔ26C; (C) pDCs were treated with CpG alone, or infected with WT vaccinia, ΔE3L, E3LΔ83N, E3LY48A or E3LΔ26C followed by addition of CpG. Supernatants were collected at 20 h post treatment. The IFN-α and TNF concentration values shown are averages of triplicate means (± SEM) of three independent experiments using human pDCs isolated from three different donors (*, p<0.05; **, p<0.01; ***, p<0.001).
Mentions: To test whether the failure of untreated vaccinia to induce a response is due to the production of inhibitors, we performed a mixing experiment. When human pDCs were co-infected with live vaccinia plus an equivalent amount of Heat-VAC, the production of IFN-α was blocked and TNF secretion was reduced by 98% compared to the level induced by Heat-VAC alone (Fig. 8B). This result indicates that live vaccinia infection of pDCs introduces inhibitor(s) of poxvirus sensing pathway(s) in pDCs that are not generated during infection with Heat-VAC.

Bottom Line: Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1.These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway.Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists.

View Article: PubMed Central - PubMed

Affiliation: Dermatology Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

ABSTRACT
Plasmacytoid dendritic cells (pDCs) play important roles in antiviral innate immunity by producing type I interferon (IFN). In this study, we assess the immune responses of primary human pDCs to two poxviruses, vaccinia and myxoma virus. Vaccinia, an orthopoxvirus, was used for immunization against smallpox, a contagious human disease with high mortality. Myxoma virus, a Leporipoxvirus, causes lethal disease in rabbits, but is non-pathogenic in humans. We report that myxoma virus infection of human pDCs induces IFN-α and TNF production, whereas vaccinia infection does not. Co-infection of pDCs with myxoma virus plus vaccinia blocks myxoma induction effects. We find that heat-inactivated vaccinia (Heat-VAC; by incubating the virus at 55°C for 1 h) gains the ability to induce IFN-α and TNF in primary human pDCs. Induction of IFN-α in pDCs by myxoma virus or Heat-VAC is blocked by chloroquine, which inhibits endosomal acidification required for TLR7/9 signaling, and by inhibitors of cellular kinases PI3K and Akt. Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1. These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway. Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists. The myxoma virus ortholog of vaccinia E3 (M029) lacks the N-terminal Z-DNA/RNA binding domain, which might contribute to the immunostimulating properties of myxoma virus.

Show MeSH
Related in: MedlinePlus