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Human TANK-binding kinase 1 is required for early autophagy induction upon herpes simplex virus 1 infection  Liyana Ahmad, PhD, Bayarchimeg Mashbat, PhD,

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Presentation on theme: "Human TANK-binding kinase 1 is required for early autophagy induction upon herpes simplex virus 1 infection  Liyana Ahmad, PhD, Bayarchimeg Mashbat, PhD,"— Presentation transcript:

1 Human TANK-binding kinase 1 is required for early autophagy induction upon herpes simplex virus 1 infection  Liyana Ahmad, PhD, Bayarchimeg Mashbat, PhD, Corwin Leung, MSc, Charlotte Brookes, MSc, Samar Hamad, MSc, Sina Krokowski, MSc, Avinash R. Shenoy, PhD, Lazaro Lorenzo, PhD, Michael Levin, MD, PhD, Peter O'Hare, PhD, Shen-Ying Zhang, MD, PhD, Jean- Laurent Casanova, MD, PhD, Serge Mostowy, PhD, Vanessa Sancho-Shimizu, PhD  Journal of Allergy and Clinical Immunology  Volume 143, Issue 2, Pages e7 (February 2019) DOI: /j.jaci Copyright © 2018 The Authors Terms and Conditions

2 Fig 1 TBK1+/− fibroblasts show impaired cytosolic dsDNA- and HSV1-induced autophagy. A, Immunofluorescence images of LC3B puncta (green) and 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI; blue) in fibroblasts. B-E, Quantification of LC3B puncta–positive fibroblasts stimulated with rapamycin (Fig 1, B), poly(I:C) (Fig 1, C), cdi-GMP or cdi-GMP control (Fig 1, D), and 60mer-dsDNA (Fig 1, E). F, Immunoblots and densitometric graphs of HSV1-infected fibroblasts. G, Immunoblot confirming TBK1 siRNA knockdown in control fibroblasts and immunoblots for LC3B, p62, and GAPDH in HSV1-infected TBK1 knockdown fibroblasts. Viral titer and immunofluorescence images of infected TBK1 knockdown fibroblasts. AU, Arbitrary units; GFP, green fluorescent protein; L, lipofectamine. All experiments were performed at least 3 times (means ± SEM). *P < .05, ***P < .001, and ****P < .0001. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2018 The Authors Terms and Conditions

3 Fig 2 TBK1+/− fibroblasts lack cytoplasmic LC3B puncta induced early in HSV1 infection. A-C, Immunofluorescence images of fibroblasts stained for HSV1 ICP4 (red), LC3B or LC3A (green), and 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI; blue). White arrows indicate cytoplasmic LC3B, and yellow arrows indicate perinuclear LC3A/B. Dashed lines mark the plaque boundary. D-G, IFN-β production (Fig 2, D), viral titer (Fig 2, E), cell viability (Fig 2, F), and cytoplasmic LC3B puncta–positive fibroblasts (Fig 2, G) were quantified. n.d., Not detectable. N = 3 (means ± SEMs). *P < .05, **P < .01, ***P < .001, and ****P < .0001. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2018 The Authors Terms and Conditions

4 Fig E1 TBK1 inhibition reduced cytoplasmic LC3B puncta formation but did not affect perinuclear LC3B formation after HSV1 infection. A, Control and TBK1+/− fibroblasts were pretreated with 1 μmol/L BX795 for 16 hours before infection with HSV1 (MOI 10) for the indicated length of time. Cells were fixed and stained for LC3B (green), ICP4 (red), or both. 4′-6-Diamidino-2-phenylindole dihydrochloride (DAPI; blue) was used for nuclear staining. The scale bar of each representative image is 20 μm. The inset represents the magnified view of the indicated area and has a scale bar of 10 μm. White arrows indicate cytoplasmic LC3B, whereas yellow arrows indicate perinuclear LC3B. B, The percentage of cells positive for cytoplasmic LC3B puncta in Fig E1, A, was counted on at least 100 cells. Images are representative of 3 independent experiments (n = 3). Data are represented as means ± SEMs and analyzed by using 2-way ANOVA. **P < .01. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2018 The Authors Terms and Conditions

5 Fig E2 TRIF−/− fibroblasts showed delayed cytoplasmic LC3B puncta formation. A, Fibroblasts grown on coverslips were infected with HSV1 (MOI 10) for indicated lengths of time before being fixed and stained for LC3B (green), HSV1 ICP4 (red), or both. 4′-6-Diamidino-2-phenylindole dihydrochloride (DAPI; blue) was used as the nuclear stain. The scale bar of each representative image is 20 μm. The inset represents the magnified view of the indicated area and has a scale bar of 10 μm. White arrows indicate cytoplasmic LC3B, whereas yellow arrows indicate perinuclear LC3B. B, The percentage of cells positive for cytoplasmic LC3B puncta was counted on at least 100 cells. Images are representative of 3 independent experiments (n = 3). Data are represented as means ± SEMs and were analyzed by using 2-way ANOVA. ***P < .001 and ****P < .0001. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2018 The Authors Terms and Conditions

6 Fig E3 Immunoblots of TBK1, IRF3, phosphorylated IRF3, and STING during HSV1 infection. Control, TRIF−/−, and TBK1+/− fibroblasts were infected with HSV1 (MOI 1) for the indicated lengths of time. Whole-cell lysates were electrophoresed and probed for endogenous TBK1, IRF3, and phosphorylated IRF3 (p-IRF3; A) and STING (B) proteins. GAPDH was used as a loading control. Relative level of STING to GAPDH was measured using densitometry (arbitrary units). Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2018 The Authors Terms and Conditions

7 Fig E4 TRIF−/− and TBK1+/− fibroblasts showed impaired IFN-β production after HSV1 infection. Control, TRIF−/−, and TBK1+/− fibroblasts were infected with HSV1 at indicated MOIs for 24 hours before collecting supernatants and measuring IFN-β levels by ELISA. Data are represented as means ± SEMs and were analyzed by using 2-way ANOVA (n = 3). *P < .05 and ****P < .0001. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2018 The Authors Terms and Conditions

8 Fig E5 Interferon-induced autophagy in fibroblasts. A, Control, TRIF−/−, and TBK1+/− fibroblasts were stimulated with 1 × 105 IU/mL IFN-α-2A for 24 hours before being fixed and stained for LC3B (green). 4′-6-Diamidino-2-phenylindole dihydrochloride (DAPI; blue) was used as the nuclear stain. The scale bar of each representative image is 20 μm. The inset represents the magnified view of the indicated area and has a scale bar of 10 μm. White arrows indicate LC3B puncta. Images are representative of 3 independent experiments (n = 3). Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2018 The Authors Terms and Conditions

9 Fig E6 Primary fibroblasts showed similar HSV1-induced autophagy phenotypes to SV40-transformed fibroblasts. Primary fibroblasts grown on coverslips were infected with HSV1 (MOI 10) for 3 or 8 hours before being fixed and stained for LC3B (green), HSV1 ICP4 (red), or both. 4′-6-Diamidino-2-phenylindole dihydrochloride (DAPI; blue) was used as the nuclear stain. The scale bar of each representative image is 20 μm. The inset represents the magnified view of the indicated area and has a scale bar of 10 μm. White arrows indicate cytoplasmic LC3B, whereas yellow arrows indicate perinuclear LC3B. Images are representative of 3 independent experiments (n = 3). Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2018 The Authors Terms and Conditions

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