Fig 1.
MDV suppresses the induction of IFN-β and downstream antiviral genes during the late phase of viral infection.
(A-C) CEFs were infected with the virulent MDV GA strain at a multiplicity of infection (MOI) of 0.1. The mRNA levels of IFN-β (A) and IFN-stimulated genes (ISGs) chicken ZAP (chZAP) (B) and chicken IFN-inducible transmembrane protein 3 (chIFITM3) (C) were measured by real-time qPCR from 4 to 72 hpi. The expression of MDV protein Meq and gI during viral infection was monitored by western blotting. (D-F) One-day-old specific pathogen-free chickens were inoculated subcutaneously with 2000 PFUs of MDV GA virus, and the mRNA levels of IFN-β (D) and chicken ISGs ZAP (E) and IFITM3 (F) in the spleen samples were measured by real-time qPCR. The relative amounts of IFN-β, ZAP, and IFITM3 mRNA were normalized to the actin mRNA level in each sample, and the fold differences were compared with those in the mock samples. *: p < 0.05, **: p < 0.01, ***: p < 0.001; ns: no significant difference.
Fig 2.
Screening of MDV open reading frames (ORFs) that modulate the cGAS-STING pathway.
(A) DF-1 cells were cotransfected with IFN-β promoter luciferase reporter and various plasmids (pCAGGS or pCAGGS-cGAS-HA and pCAGGS-STING-HA combined). The luciferase activity was measured at 36 h posttransfection. (B) Schematic of the screening assay. DF-1 cells were transfected with the same amount of cGAS and STING expression plasmid, plus each of MDV ORF expression plasmid or the empty vector. (C) Heat map of the effects of MDV ORFs on the cGAS-STING pathway. Higher IFN-β promoter luciferase activation levels are indicated by red, whereas lower levels are indicated by blue, which corresponds to a higher degree of inhibition. (D) The top five MDV ORF inhibitors and the MDV gI ORF were cotransfected with cGAS and STING expression plasmids into DF-1 cells. At 36 h posttransfection, IFN-β mRNA levels were measured by real-time qPCR. The relative amount of IFN-β mRNA was normalized to the actin mRNA level in each sample, and the fold changes were compared with those in the mock controls. (E) The top five MDV ORF inhibitors and the gI ORF were cotransfected with cGAS and STING expression plasmids into DF-1 cells, and IFN-β protein levels were measured by enzyme-linked immunosorbent assay 36 h posttransfection. (F) Varying doses of the top five MDV ORF inhibitors and the gI ORF were cotransfected with cGAS and STING expression plasmids, and IFN-β promoter luciferase activity was measured at 36 h posttransfection. (G) The top five MDV ORF inhibitors and the gI ORF were transfected into DF-1 cells, and IFN-β promoter luciferase activity was measured at 36 h posttransfection. *: p < 0.05, **: p < 0.01, ***: p < 0.001; ns: no significant difference.
Fig 3.
MDV Meq suppresses IFN-β induction in response to viral DNA and promotes viral replication.
(A, B) DF-1 cells were transfected with empty vector or Meq expression plasmid. After 24 h, they were transfected with IFN stimulatory DNA (ISD) fragments or poly(dA:dT). IFN-β mRNA was measured by real-time qPCR 8 h post ISD or poly(dA:dT) transfection (A), and IFN-β protein levels were measured by enzyme-linked immunosorbent assay (ELISA) 24 h post ISD or poly(dA:dT) transfection (B). (C) The expression of Meq in DF-1 cells transduced with empty vector or Meq-expressing lentivirus was monitored by western blotting. (D, E) DF-1 cells transduced with empty vector or Meq-expressing lentivirus were left uninfected or infected with HVT (multiplicity of infection (MOI) = 0.1). IFN-β mRNA in these cells was measured by real-time qPCR 12 hpi (D), and IFN-β protein was measured by ELISA 24 hpi (E). (F) Transduced DF-1 cells were infected with varying doses of HVT (MOI = 1, 0.1, or 0.01). At 48 hpi, the HVT viral titer was tested with real-time qPCR. The relative level of IFN-β mRNA was normalized to actin in each sample, and the fold differences between the treated samples and the mock samples were calculated. *: p < 0.05, **: p < 0.01, ***: p < 0.001; ns: no significant difference.
Fig 4.
Meq deficiency enhances MDV-triggered induction of IFN-β and downstream antiviral genes.
(A) Real-time qPCR and western blot analysis of the CEFs lentivirally transduced with Meq-specific small hairpin RNAs (shMeq) or a control shRNA (shNC) after 48 h of MDV infection. (B, C) CEFs transduced with shMeq or shNC were infected with MDV (multiplicity of infection (MOI) = 0.1) for the indicated times. IFN-β mRNA levels were measured by real-time qPCR (B), and IFN-β protein levels were measured by enzyme-linked immunosorbent assay (C). (D, E) CEFs transduced with shNC or shMeq were infected with MDV (MOI = 0.1) for the indicated times. The mRNA levels of chicken ZAP (chZAP) (D) and chicken IFN-inducible transmembrane protein 3 (chIFITM3) (E) were measured by real-time qPCR. (F) Schematic diagram of the recombinant fosmids for constructing the wild-type MDV (MDV-WT) and the Meq-deficient MDV (MDV-dMeq) viruses. (G) PCR analyses of the Meq-deficient MDV. (H) Western blot analysis of the CEFs infected with MDV-WT or MDV-dMeq using the indicated antibodies. (I-K) Effects of Meq deficiency on transcription of IFN-β and downstream antiviral genes in vitro. CEFs were infected with MDV-WT or MDV-dMeq (multiplicity of infection (MOI) = 0.1) for 12 h prior to analysis of IFN-β (I), chZAP (J), and chIFITM3 (K) mRNA levels. The amounts of IFN-β, chZAP, or chIFITM3 mRNA were normalized to the actin mRNA level in each sample, and the fold difference relative to the mock controls at each time point was determined. *: p < 0.05, **: p < 0.01, ***: p < 0.001; ns: no significant difference.
Fig 5.
Meq interacts with STING and IRF7.
(A) The IFN-β-luc, IRF7-luc, or NF-κB-luc reporter was cotransfected with cGAS and STING constructs as well as Meq-Flag plasmid or empty vector into DF-1 cells. After 36 h, cells were harvested and analyzed using the dual-luciferase reporter assay. (B) DF-1 cells were transfected with cGAMP or plasmid expressing TBK1 or IRF7, together with IFN-β-luc reporter and Meq-Flag plasmid or an empty vector. The dual-luciferase reporter assay was performed 36 h posttransfection, and the fold relative to the mock controls was determined. (C) HEK293T cells were transfected with the indicated plasmids for 36 h before coimmunoprecipitation and immunoblot analysis with the indicated antibodies. (D) Coimmunoprecipitation and immunoblot analyses were performed with the endogenous proteins from the CEFs left uninfected or infected with MDV. (E) Purified GST, GST-STING, or GST-IRF7 were used to pull down transiently expressed Meq-Flag as indicated. (F) Full length Meq (Meq-FL), and N- (Meq-N) or C-terminally truncated forms of Meq (Meq-C) were transfected together with STING-HA or IRF7-HA plasmids into HEK293T cells for 36 h before coimmunoprecipitation and immunoblot analysis with the indicated antibodies. (G) DF-1 cells were transfected with IFN-β-luc reporter, and expression plasmids for cGAS, STING, IRF7, Meq, and its truncation mutants for 36 h before luciferase assays. ***: p < 0.001; ns: no significant difference.
Fig 6.
Meq impairs the recruitment of TBK1 and IRF7 to STING adaptor.
(A) DF-1 cells were transfected with the indicated plasmids for 36 h before coimmunoprecipitation and immunoblot analysis with the indicated antibodies. (B) The STING-HA plasmid was cotransfected with STING-Flag with or without Meq-Myc into DF-1 cells. After 36 h of transfection, coimmunoprecipitation and immunoblot were performed with the indicated antibodies. (C) The CEFs were first mock infected or infected with wild-type MDV (MDV-WT) or Meq-deficient MDV (MDV-dMeq) and then transfected with ISD for another 12 h. The cells were lysed and subjected to immunoprecipitation assays with the indicated antibodies.
Fig 7.
Meq inhibits the phosphorylation and nuclear translocation of IRF7.
(A) DF-1 cells were left untreated or transfected with the empty vector or Meq-Flag plasmid and treated with IFN stimulatory DNA (ISD) for 12 h. Cell lysates were left untreated or treated with calf intestine alkaline phosphatase (CIP) for 1 h, and western blotting was performed with the indicated antibodies. The protein levels of phosphorylated TBK1 (p-TBK1) and phosphorylated IRF7 (p-IRF7) were normalized to those of actin; the p-IRF7 protein is indicated by an asterisk (*). (B) CEFs were infected with MDV-WT or MDV-dMeq (MOI = 0.1) for 12 h before immunoblot analysis with the indicated antibodies. (C) DF-1 cells were transfected with the indicated plasmids for 36 h before coimmunoprecipitation and immunoblot analysis with the indicated antibodies. (D) DF-1 cells were transfected with an empty vector or Meq-Flag plasmid, and 24 h later, cells were either left untreated or transfected with ISD for 12 h before confocal microscopy. (E) DF-1 cells were transfected and treated with ISD as indicated and the cell lysates were separated into cytoplasmic and nuclear extracts. The IRF7 protein levels in the cytoplasm and nucleus were analyzed by western blotting. The data represent results from one of the triplicate experiments. **: p < 0.01, ***: p < 0.001.
Fig 8.
Meq deficiency facilitates IFN-β induction and host CD8+ T cell responses.
(A-C) Chickens were infected with 2000 PFUs of wild-type MDV (MDV-WT) or Meq-deficient MDV (MDV-dMeq), and the mRNA levels of IFN-β (A), chicken ZAP (chZAP) (B), and chicken IFN-inducible transmembrane protein 3 (chIFITM3) (C) in the spleen samples were measured by real-time qPCR at the indicated times postinfection. (D) The CEFs transduced with Meq-specific small hairpin RNAs (shMeq) or a control shRNA (shNC) were infected with MDV (MOI = 0.01) for 48 h before the detection of MDV viral titers with real-time qPCR. (E) The indicated cells were infected with wild-type MDV or MDV-dMeq, respectively, and the MDV viral titers were tested using a plaque assay at the indicated time points after infection. (F) One-day-old specific pathogen-free chickens were left untreated or inoculated with MDV-WT or MDV-dMeq, and virus genome copy numbers in the spleen were monitored by real-time qPCR at the indicated time points. (G) Chicken peripheral blood lymphocytes were obtained to analyze the percentage of CD8+ T cells at the indicated time points after infection. (H) The survival rate of chickens after infection. *: p < 0.05, **: p < 0.01, ***: p < 0.001; ns: no significant difference.
Fig 9.
Schematic model of Meq-mediated inhibition of the cGAS-STING pathway during MDV infection.
MDV DNA or tumor-derived DNA is recognized by cytosolic DNA sensor cGAS, which produces cGAMP for STING activation and IFN-β production. However, the MDV oncoprotein Meq interacts with STING and IRF7, which disrupts assembly of the STING-TBK1-IRF7 complex, thereby leading to the inhibition of IRF7 activation and IFN-β induction during MDV infection. Additionally, STING is involved in the melanoma differentiation-associated gene 5 (MDA5) signaling pathway against RNA viruses as well as the innate immunity against bacteria. As an inhibitor of the cGAS-STING signaling, Meq could exert its immunomodulatory functions in the innate immune responses against not only DNA viruses but also RNA-related pathogens and various bacteria.