The EBV-encoded dUTPase activates NF-kappa B through the TLR2 and MyD88-dependent signaling pathway

Abstract

The innate immune response plays a key role as the primary host defense against invading pathogens including viruses. We have previously shown that treatment of human monocyte-derived macrophages with EBV-encoded dUTPase induces the expression of proinflammatory cytokines through the activation of NF-kappaB. However, the receptor responsible for EBV-encoded dUTPase-mediated biological effects is not known. In this study, we demonstrate that the purified EBV-encoded dUTPase activates NF-kappaB in a dose-dependent manner through TLR2 and requires the recruitment of the adaptor molecule MyD88 but not CD14. Furthermore, activation of NF-kappaB was abrogated by anti-TLR2, anti-EBV-encoded dUTPase blocking Abs and the overexpression of a dominant negative construct of MyD88 in human embryonic kidney 293 cells expressing TLR2. In addition, treatment of human monocyte-derived macrophages with the anti-EBV-encoded dUTPase Ab 7D6 or the anti-TLR2 Ab blocked the production of IL-6 by the EBV-encoded dUTPase. To our knowledge, this is the first report demonstrating that a nonstructural protein encoded by EBV is a pathogen-associated molecular pattern and that it has immunomodulatory functions. Although additional studies are necessary to define the signaling pathways activated by the EBV-encoded dUTPase and to determine its role in modulating immune responses to EBV infection, our results suggest that the dUTPase could be a potential target for the development of novel therapeutic agents against infections caused by EBV.

FIGURE 1.

EBV-encoded dUTPase-mediated activation of NF-κB does not occur through TLR4, TLR4/MD2, or TLR3. TLR4-, TLR4/MD2-, and TLR3-expressing HEK293 cells were transiently transfected with NF-κB luciferase reporter plasmid. After 24–36 h, TLR4-, TLR4/CD14 (A)- and TLR3 (B)-expressing HEK293 cells were treated with EBV-encoded dUTPase (10 μg/ml), LPS (100 ng/ml), or poly(IC) (10 μg/ml) or left untreated for 8 h, and luciferase reporter gene activity was measured. Luciferase activity values are expressed as the ratio between the firefly and the Renilla luciferase. Data are the mean fold induction ± SD relative to control levels. Values represent the average of three independent experiments.

FIGURE 2.

EBV-encoded dUTPase stimulates NF-κB activity in a dose-dependent manner through TLR2. Dose-response activation of NF-κB by EBV-encoded dUTPase in TLR2- and TLR2/CD14-expressing HEK293 cells. HEK cells were transiently transfected with NF-κB luciferase reporter plasmid. After 24–36 h, TLR2 (A) and TLR4/CD14 (B) HEK293-expressing cells were treated with various concentration of EBV-encoded dUTPase (0–10 μg/ml) or zymosan (10 μg/ml) or left untreated for 8 h, and luciferase reporter gene activity was measured. C, EBV-encoded dUTPase does not activate NF-κB in HEK293 wild-type cells. HEK293 wild-type cells were transfected with NF-κB reporter gene plasmid and either empty vector or pCMV-NF-κBp65 (p65). After 24–36 h, cells were treated with EBV-encoded dUTPase (10 μg/ml) or zymosan or left untreated for 8 h, and luciferase reporter gene activity was measured. Data are expressed as the mean fold induction ± SD relative to control levels. Values represent the average of three independent experiments.

FIGURE 3.

EBV-encoded dUTPase signals through TLR2 and MyD88. A, TLR2-expressing HEK293 cells were transiently transfected with NF-κB luciferase reporter plasmid and cotransfected with either empty vector or MyD88DN (0.5 μg). After 24–36 h, TLR2-expressing HEK293 cells were treated with EBV-encoded dUTPase (10 μg/ml) or zymosan (10 μg/ml) or left untreated for 8 h, and luciferase reporter gene activity was measured. B, Anti-TLR2 blocking experiments inhibit NF-κB activation by the EBV-encoded dUTPase. HEK293 cells expressing TLR2 were transfected with NF-κB reporter gene plasmid. After 24–36 h, cells were preincubated with isotype control or anti-TLR2 mAbs (20 μg/ml) for 1 h followed by treatment with either EBV-encoded dUTPase (10 μg/ml) or zymosan for 8 h or left untreated. C, Anti-EBV-encoded dUTPase blocking experiments inhibit NF-κB activation through TLR2 signaling. TLR2-HEK293 cells were preincubated with isotype control or 7D6 Abs (20 μg/ml) for 1 h followed by treatment with EBV-encoded dUTPase (10 μg/ml) for 8 h or left untreated. Data are expressed as the mean fold induction ± SD relative to control levels. Values represent the average of three independent experiments.

FIGURE 4.

EBV-encoded dUTPase-mediated cytokine secretion in hMDM is TLR2 dependent. hMDMs from a single individual were incubated with anti-TLR2, EBV-encoded dUTPase-specific 7D6 or isotype control Abs (10–20 μg/ml) as described in Materials and Methods and treated with full-length EBV-encoded dUTPase protein (10 μg/ml) or left untreated. After 24 h, cell culture supernatants from control and treated samples were collected and analyzed for IL-6 production using the BD Human Inflammation Cytometric Bead Array Kit. Data are means ± SD (picograms per milliliter) from two independent experiments.

FIGURE 5.

TLR6 is a negative regulator of EBV-encoded dUTPase-mediated induction of NF-κB activity through TLR2 whereas TLR1 plays no role. A, TLR2-expressing HEK293 cells were transiently transfected with NF-κB luciferase reporter plasmid and cotransfected with 0.5 μg of empty vector, TLR1, or TLR6. After 24–36 h, cells were treated with EBV-encoded dUTPase (10 μg/ml) or left untreated for 8 h, and NF-κB luciferase levels were measured. Zymosan (10 μg/ml), FSL-1, and Pam3CsK4 (0.1 μg/ml respectively) were used as positive controls for TLR2, TLR6, and TLR1, respectively. B, TLR2-HEK293 cells were transiently transfected with increasing concentrations (0–2 μg) of pCMV-TLR6. After 24–36 h, cells were treated with EBV-encoded dUTPase (10 μg/ml) or FSL-1 or left untreated for 8 h, and luciferase reporter gene activity was measured. Data are expressed as the mean fold induction ± SD relative to control levels. Values represent the average of three independent experiments. C, EBV-encoded dUTPase-mediated induction of IL-6 in hMDM is regulated by TLR6. hMDM from a single individual were incubated with IgG1 isotype control or anti-TLR6 Abs (10 μg/ml) followed by treatment with full-length EBV-encoded dUTPase protein (10 μg/ml) or left untreated. After 24 h, cell culture supernatants from control and treated samples were collected and analyzed for IL-6 production using the BD Human Inflammation Cytometric Bead Assay Kit. Data are means ± SD (picograms per milliliter) from two independent experiments. WT, Wild type.