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Published byΑνδρομέδη Ξάνθη Λόντος Modified over 6 years ago
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Volume 21, Issue 2, Pages 231-243 (February 2017)
Human Cytomegalovirus Tegument Protein UL82 Inhibits STING-Mediated Signaling to Evade Antiviral Immunity Yu-Zhi Fu, Shan Su, Yi-Qun Gao, Pei-Pei Wang, Zhe-Fu Huang, Ming-Ming Hu, Wei-Wei Luo, Shu Li, Min-Hua Luo, Yan-Yi Wang, Hong-Bing Shu Cell Host & Microbe Volume 21, Issue 2, Pages (February 2017) DOI: /j.chom Copyright © 2017 Elsevier Inc. Terms and Conditions
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Cell Host & Microbe 2017 21, 231-243DOI: (10.1016/j.chom.2017.01.001)
Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 1 Identification of HCMV UL82 as an Inhibitor of DNA-Triggered Signaling (A) UL82 inhibits cGAS-STING-induced ISRE activation in a dose-dependent manner. HEK293 cells were transfected with ISRE-luc reporter, expression plasmids for cGAS, STING, and increased amounts of the indicated HCMV proteins for 20 hr before luciferase assays. The lower blots show the expression levels of the transfected UL proteins. (B) UL82 inhibits HCMV- and HSV-1-induced transcription of antiviral genes in HFFs. UL82 stable HFFs were uninfected or infected with HCMV (MOI = 1) and HSV-1 (MOI = 1) for the indicated times before qPCR analysis. (C) M82 inhibits HCMV- and MCMV-induced transcription of antiviral genes in MLFs. M82-stable MLFs were un-infected or infected with HCMV or MCMV for the indicated times before qPCR analysis. (D) UL82 inhibits DNA-induced transcription of antiviral genes in HFFs. UL82 stable HFFs were transfected with HSV120 (2 μg/mL) or B-DNA (2 μg/mL) for the indicated times before qPCR analysis. (E) UL82 impairs HCMV- and HSV-1-induced phosphorylation of downstream components. UL82 stable HFFs were untreated or infected with HCMV (MOI = 1) or HSV-1 (MOI = 1) for the indicated times before immunoblot analysis. The lower panels are results of RT-PCR analysis for viral or cellular RNAs. (F) UL82 impairs HSV120 (2 μg/mL)- and B-DNA (2 μg/mL)-induced phosphorylation of downstream components. UL82 stable HFFs were transfected with HSV120 or B-DNA for the indicated times before immunoblot analysis. Graphs show mean ± SD, n = 3. ∗p < 0.05, ∗∗p < See also Figures S1 and S2. Cell Host & Microbe , DOI: ( /j.chom ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 2 Knockdown of UL82 Increases HCMV-Induced Expression of Downstream Antiviral Genes (A) Effects of UL82-RNAi plasmids on HCMV- and HSV-1-induced transcription of antiviral genes. UL82-RNAi stable HFFs were infected with HCMV (MOI = 1) or HSV-1 (MOI = 1) for the indicated times before qPCR analysis. (B) Effects of UL82-RNAi plasmids on HCMV- and HSV-1-induced phosphorylation of downstream components. UL82-RNAi stable HFFs were infected with HCMV (MOI = 1) or HSV-1 (MOI = 1) for the indicated times before immunoblot analysis. Graphs show mean ± SD, n = 3. ∗p < 0.05, ∗∗p < 0.01. See also Figure S3. Cell Host & Microbe , DOI: ( /j.chom ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 3 UL82 Acts at the Level of STING
(A) Effects of UL82 on ISRE activation mediated by various components. HEK293 cells were transfected with ISRE reporter, the indicated expression plasmids, and increased amounts of UL82 expression plasmid for 20 hr before luciferase assays. (B) UL82 inhibits cGAMP-induced transcription of antiviral genes in HFFs. Control or UL82-transduced HFFs were transfected with cGAMP (0.2 μg/mL) for 6 hr before qPCR analysis of IFN-β, ISG56, and TNF mRNA abundance. (C) Association of UL82 or M82 with human or murine STING. HEK293T cells were transfected with the indicated plasmids for 20 hr before coimmunoprecipitation and immunoblot analysis with the indicated antibodies. (D) Association of endogenous UL82 with STING in HFFs. The HFF cells were left untreated or infected with HCMV for 12 hr before coimmunoprecipitation and immunoblot analysis with the indicated antibodies. (E) Domain mapping of the UL82-STING interaction. The experiments were performed similar to those in (B). Schematic representations are also shown. (F) The effects of UL82 mutants on cGAS-STING-mediated activation of ISRE. HEK293T cells were transfected with ISRE reporter, and mammalian expression plasmids for cGAS, STING, UL82, and its truncation mutants for 20 hr before luciferase assays. Graphs show mean ± SD, n = 3. ∗p < See also Figure S4. Cell Host & Microbe , DOI: ( /j.chom ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 4 UL82 Impairs the Trafficking of STING
(A and B) UL82 is localized to the ER (A), but not to mitochondria (B). HEK293 were transfected with UL82-Flag, ER-GFP, or Mito-RFP as indicated for 18 hr before confocal microscopy. (C) Colocalization of UL82 with STING. HEK293 cells were transfected with UL82-Flag and STING-HA as indicated for 18 hr before confocal microscopy. (D) UL82 is localized to the ER in Sting+/+ or Sting−/− MLFs. The experiments were performed similar to those in (C). (E–G) UL82 impairs HCMV-, HSV-1-, or HSV120-triggered trafficking of STING. UL82 stably transduced Sting−/−-MLF-Sting-Flag cells were infected with HCMV (MOI = 1) (E) or HSV-1 (MOI = 1) (F) or transfected with HSV120 (G) for 4 hr before confocal microscopy. (H) UL82 disrupts the STING-iRhom2-TRAPβ complex. HEK293T cells were transfected with the indicated plasmids for 20 hr before coimmunoprecipitation and immunoblot analysis with the indicated antibodies. Graphs show mean ± SD, n = 3. ∗p < 0.05, ∗∗p < 0.01. Cell Host & Microbe , DOI: ( /j.chom ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 5 UL82 Impairs the Recruitment of TBK1 and IRF3 to STING
(A) UL82 disrupts the associations of STING-TBK1 and STING-IRF3. HEK293T cells were transfected with the indicated plasmids for 20 hr before coimmunoprecipitation and immunoblot analysis with the indicated antibodies. (B) UL82 inhibits the endogenous association of STING-TBK1-IRF3. UL82 stable HFFs were untreated or infected with HCMV for the indicated times before coimmunoprecipitation and immunoblot analysis with the indicated antibodies. (C) UL82 impairs the interaction of STING with TBK1 or IRF3 in vitro. Purified GST- GST-STING (151–379) was used to pull down transiently expressed Flag-TBK1, Flag-IRF3, and/or UL82-HA as indicated. See also Figure S5. Cell Host & Microbe , DOI: ( /j.chom ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 6 UL82 Deficiency Facilitates Innate Antiviral Responses
(A and B) Effects of UL82 deficiency on transcription of downstream antiviral genes. HFFs (A) or THP1 cells (B) were infected with wild-type HCMV (MOI = 1) or HCMV-ΔUL82 (MOI = 1) for the indicated times before qPCR analysis. (C) HCMV-ΔUL82 viruses elicit stronger immune responses in primary human monocyte-derived macrophages and dendritic cells (DCs) than those elicited by wild-type HCMV. Cells were infected with wild-type HCMV (MOI = 1) or HCMV-ΔUL82 (MOI = 1) for the indicated times before qPCR analysis. (D) Inhibition of UL82 on IFNB1 transcription induced by UV-inactivated HCMV. Control or UL82 stable cells were infected with wild-type or UV-inactivated HCMV before qPCR analysis. (E) Effects of UL82 deficiency on phosphorylation of downstream components. The indicated cells were infected with wild-type HCMV (MOI = 1) or HCMV-ΔUL82 (MOI = 1) for the indicated times before immunoblot analysis. (F) Effects of UL82 deficiency on the association of STING-TBK1-IRF3. HFFs were infected with wild-type HCMV (MOI = 1) or HCMV-ΔUL82 (MOI = 1) for the indicated times before coimmunoprecipitation and immunoblot analysis. Graphs show mean ± SD, n = 3. ∗p < 0.05, ∗∗p < 0.01. Cell Host & Microbe , DOI: ( /j.chom ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 7 Roles of UL82 on HCMV Replication
(A) UL82 enhances HCMV replication. The indicated cells were infected with HCMV (MOI = 1), and the supernatants were harvested at 48, 60, 72, and 96 hr post infection, respectively, for measurements of viral titers with standard TCID50 assays. (B) Effects of UL82 deficiency on the replication of HCMV. The indicated cells were infected with wild-type HCMV or HCMV-ΔUL82, respectively, for 48, 60, 72, and 96 hr. The supernatants were then harvested for measurements of the viral titers with standard TCID50 assays. Cell Host & Microbe , DOI: ( /j.chom ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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