Volume 68, Issue 2, Pages 308-322.e4 (October 2017) Tetherin Suppresses Type I Interferon Signaling by Targeting MAVS for NDP52- Mediated Selective Autophagic Degradation in Human Cells  Shouheng Jin, Shuo Tian, Man Luo, Weihong Xie, Tao Liu, Tianhao Duan, Yaoxing Wu, Jun Cui  Molecular Cell  Volume 68, Issue 2, Pages 308-322.e4 (October 2017) DOI: 10.1016/j.molcel.2017.09.005 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 68, 308-322.e4DOI: (10.1016/j.molcel.2017.09.005) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Tetherin Negatively Regulates RLR-Induced Type I IFN Signaling Pathway (A) Flag-Tetherin inducible A549 cells were treated with doxycycline (Doxy) (200 ng/mL) for 12 hr. The transcription levels of IFNB and replication of virus after SeV or VSV infection were analyzed by qRT-PCR. (B) Luciferase activity in 293T cells transfected with ISRE (up) or IFN-β (down) promoter-driven luciferase reporters and together with plasmid encoding RIG-I (N) and increasing amount of plasmid expressing Tetherin. (C) Immunoblot analysis of the knockdown efficiency of Tetherin by Tetherin-specific siRNAs. (D) 293T cells were transfected with scrambled siRNA or Tetherin siRNA; luciferase activity transfected with ISRE (up) or IFN-β (down) promoter-driven luciferase reporters after induced with intracellular (IC) poly (I:C) (10 μg/mL) or 5′-ppp-RNA (2 μg/mL) for indicated time points. (E) A549 cells were treated with scrambled siRNA or Tetherin siRNA. The cells were then treated with IC poly (I:C) (10 μg/mL) or 5′-ppp-RNA (2 μg/mL) and the transcription levels of IFNB and ISGs were analyzed by qRT-PCR. (F) THP-1 cells were transfected with control or Tetherin-specific siRNA, followed by treatment with SeV (MOI = 1) at different time points; the lysates were analyzed with indicated antibodies. (G) PBMCs were transfected with control or Tetherin-specific siRNA, followed by treatment with influenza A/Puerto Rico/8/34 (H1N1) (PR8) (MOI = 5) at different time points; the lysates were analyzed with indicated antibodies. (H) PBMCs transfected with control or Tetherin-specific siRNA were infected with H1N1 virus (MOI = 5) at different time points. Relative expression levels of IFNB, ISG54, and ISG56 mRNA and H1N1 nucleoprotein (NP) RNA were measured by real-time PCR. Data in (A), (B), (D), (E), and (H) are expressed as means ± SEM of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; NS, not significant (two-tailed Student’s t test). See also Figure S1. Molecular Cell 2017 68, 308-322.e4DOI: (10.1016/j.molcel.2017.09.005) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Tetherin Interacts with the Signaling Adaptor MAVS (A and B) 293T cells were transfected with the ISRE (A) or IFN-β (B) luciferase reporter, together with the indicated plasmids along with empty vector (no wedge) or increasing amounts (wedge) of expression vector for HA-Tetherin. (C) 293T cells were transfected with plasmids encoding HA-Tetherin and Flag-tagged key proteins in RLR-mediated type I IFN signaling (Flag-RIG-I, Flag-MDA5, Flag-MAVS, Flag-TBK1, Flag-IKKε, and Flag-IRF3), followed by IP with anti-Flag beads and immunoblot analysis with anti-HA. Throughout was the immunoblot analysis of whole-cell lysates (WCLs) without immunoprecipitation. (D) Extracts of PBMCs infected with H1N1 for various times (above lanes) were subjected to IP with anti-MAVS and immunoblot analysis with indicated antibodies (shown on the left). (E) Confocal microscopy of HeLa cells transfected IC poly (I:C) (10 μg/mL) for 6 hr, followed by labeling of Tetherin and MAVS with specific primary antibody and a CF350 donkey anti-rabbit-IgG secondary antibody (blue) and Alexa Fluor 488 conjugated anti-mouse-IgG secondary antibody (green). The mitochondria were stained by mito-Tracker (red). Scale bars, 200 μm. (F) Quantitative analysis of similar samples as in (E) (no less than ten cells per sample). (G) CoIP and immunoblot analysis of 293T cells transfected with Tetherin and its indicated mutants along with vector encoding HA-MAVS. (H) CoIP and immunoblot analysis of 293T cells transfected with MAVS and its indicated mutants along with vector encoding HA-Tetherin. Arrow denotes nonspecific bands. Data in (A) and (B) are expressed as means ± SEM of at least three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; NS, not significant (two-tailed Student’s t test). See also Figure S2. Molecular Cell 2017 68, 308-322.e4DOI: (10.1016/j.molcel.2017.09.005) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Tetherin Promotes the Autophagic Degradation of MAVS (A) Immunoblot analysis of extracts of 293T cells transfected with expression vector for Flag-MAVS and increasing doses of expression vector for HA-Tetherin (wedge). (B) Flag-Tetherin inducible A549 cells were treated with indicated concentration of Doxy for 12 hr, and the protein was harvested for immunoblot analysis. (C) MAVS mRNA levels of the same sample (B) were detected by real-time PCR. Data represented mean ± SEM of triplicate samples. (D) THP-1 cells were transfected with Tetherin-specific or scramble siRNA and treated with SeV (MOI = 1) for indicated time points. The protein was harvested for immunoblot analysis. (E) 293T cells were transfected with plasmids encoding Flag-MAVS together with HA-Tetherin plasmid treated with MG132 (10 μM), 3-methyladenine (3MA) (10 mM), chloroquine (CQ) (50 μM), bafilomycin A1 (Baf A1) (0.2 μM),or NH4Cl (20 mM) for 6 hr. The cell lysates were analyzed by immunoblot. (F) 293T cells were transfected with plasmid encoding HA-Tetherin, then were cultured in EBSS for indicated time points, and the protein expression levels of MAVS were detected by immunoblot. (G and H) WT and BECN1 KO (G) or ATG5 KO (H) 293T cells were treated with CHX (100 μg/mL) for indicated time points, and the cell lysates were analyzed by immunoblot. (I) WT, BECN1, and ATG5 KO 293T cells were transfected with plasmid encoding HA-Tetherin; the lysates were analyzed with each antibody. See also Figure S3. Molecular Cell 2017 68, 308-322.e4DOI: (10.1016/j.molcel.2017.09.005) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Tetherin Enhances the Recognition of MAVS by Cargo Receptor NDP52 (A) 293T cells were transfected with vectors encoding HA-Tetherin and indicated Flag-tagged cargo receptors, followed by IP with anti-Flag beads and immunoblot analysis with anti-HA. Arrow denotes nonspecific bands. (B) Lysates of THP-1 cells transfected with scramble or Tetherin-specific siRNA and treated with SeV (MOI = 1) for indicated time points with Baf A1 (0.2 μM). The lysates were subjected to IP with anti-MAVS and immunoblot analysis with indicated antibodies (shown on the left). (C) WT and NDP52 KO 293T cells were treated with CHX (100 μg/mL) for indicated time points. The protein expression levels of MAVS were detected by immunoblot. (D) WT and NDP52 KO 293T cells were transfected with plasmid for HA-Tetherin. Cell lysates were subjected to immunoblot analysis. (E and F) WT and NDP52 KO 293T cells transfected with an ISRE (E) or IFN-β (F) promoter-driven luciferase reporter with or without the plasmid encoding Tetherin; luciferase activity was detected after infection with SeV for indicated time points. (G) Relative expression levels of IFNB mRNA and IFN-stimulated genes, including ISG54 and ISG56 in WT, NDP52, and SQSTM1 KO cells with or without Tetherin. Data in (E)–(G) are expressed as means ± SEM of at least three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; NS, not significant (two-tailed Student’s t test). See also Figure S4. Molecular Cell 2017 68, 308-322.e4DOI: (10.1016/j.molcel.2017.09.005) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Tetherin Increases the K27-Linked Ubiquitination of MAVS (A) Domain organization of NDP52 protein. (B) CoIP and immunoblot analysis of 293T cells transfected with deletion mutants of NDP52 plasmids along with vector encoding HA-MAVS. (C) THP-1 cells were transfected with scrambled or Tetherin siRNA, then infected with SeV (MOI = 1) for 18 hr and protein extracts were harvested after Baf A1 (0.2 μM) treatment for 6 hr. Protein extracts were immunoprecipitated using anti-MAVS antibody or with IgG as a negative control and analyzed by immunoblot using anti-ubiquitin and anti-MAVS antibodies. (D) Lysates of 293T cells transfected with plasmids expressing Flag-MAVS and HA-tagged ubiquitin (Ub) and its indicated mutants, together with the empty vector or expression vector of Myc-Tetherin, and treated with Baf A1 (0.2 μM) were immunoprecipitated with anti-Flag and immunoblotted with anti-HA. (E) 293T cells were transfected with Tetherin siRNA and plasmids encoding Flag-MAVS, plus ubiquitin K27 mutant, and infected with SeV (MOI = 1) for 18 hr. Protein was harvested after Baf A1 (0.2 μM) treatment for 6 hr and immunoprecipitated with anti-Flag and immunoblotted with anti-HA. See also Figure S5. Molecular Cell 2017 68, 308-322.e4DOI: (10.1016/j.molcel.2017.09.005) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Tetherin Recruits MARCH8 to Ubiquitinate MAVS (A) Alignment of MARCH8 sequences of different species with human herpesvirus 8 V5. (B) THP-1 cells were transfected with scrambled siRNA or Tetherin siRNA, then infected with SeV (MOI = 1) for 18 hr and protein extracts were harvested after Baf A1 (0.2 μM) treatment for 6 hr. Protein extracts were immunoprecipitated using anti-MAVS antibody or with IgG as a negative control and analyzed by immunoblot using indicated antibodies. (C) Lysates of 293T cells transfected with plasmids expressing Flag-MAVS and HA-tagged ubiquitin and its indicated mutants, together with the empty vector or expression vector of Myc-MARCH8, and treated with Baf A1 (0.2 μM) were immunoprecipitated with anti-Flag and immunoblotted with anti-HA. (D) WT and NDP52 KO 293T cells were transfected with Flag-MAVS and HA-K27-linked-Ub, together with empty vector or expression vector for Myc-Tetherin, and were immunoprecipitated with anti-Flag and immunoblotted with anti-HA. (E) Protein lysates of WT and MARCH8 KO 293T cells infected with SeV for indicated time points were immunoblotted with indicated antibodies. (F and G) Luciferase activity in WT and MARCH8 KO 293T cells transfected with an ISRE (F) or IFN-β (G) promoter-driven luciferase reporter and together with the plasmid encoding MAVS with or without the plasmid encoding HA-Tetherin. Data are expressed as means ± SEM of at least three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; NS, not significant (two-tailed Student’s t test). (H) Protein lysates of WT and MARCH8 KO 293T cells transfected with plasmid encoding HA-Tetherin were immunoblotted with indicated antibodies. (I) 293T cells were transfected with scramble or Tetherin siRNA and plasmid encoding HA-MARCH8 and infected with SeV (MOI = 1) for 18 hr. Protein was harvested for immunoblot analysis. See also Figure S6. Molecular Cell 2017 68, 308-322.e4DOI: (10.1016/j.molcel.2017.09.005) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 K7 Is Critical for the Selective Autophagic Degradation of MAVS (A) Domain organization of MAVS. (B) IP and immunoblot analysis of 293T cells transfected with vectors expressing Flag-MAVS and HA-K27-linked ubiquitin. (C) IP and immunoblot analysis of 293T cells transfected with vectors expressing Flag-MAVS and HA-K27-linked ubiquitin in the presence of MARCH8. (D) 293T cells were transfected with plasmids encoding HA-NDP52 and Flag-MAVS (WT, K7R, K10R, or K136R), followed by IP with anti-Flag beads and immunoblot analysis with anti-HA. (E) 293T cell were transfected with Flag-MAVS (WT or K7R) and treated with CHX (100 μg/mL) for indicated time points. The expression levels of Flag-MAVS were analyzed by immunoblot. (F) Quantification of the protein levels of WT and K7R Flag-MAVS. (G) Immunoblot analysis of protein extracts of 293T cells transfected with empty vector or vector for HA-Tetherin, together with plasmid expressing WT or K7R, K10R, or K136R mutants of Flag-MAVS. (H) MAVS KO 293T cells were transfected with vectors encoding WT and K7R Flag-MAVS, and then infected with SeV for indicated time points. The cell lysates were analyzed by immunoblot. (I) MAVS KO 293T cells restored with WT and K7R Flag-MAVS were transfected with plasmids encoding Tetherin or Tetherin ΔGPI, and then infected with SeV for indicated time points. Relative expression levels of IFNB and IFN-stimulated genes, including ISG54 and ISG56, were analyzed. (J) A proposed working model to illustrate how the Tetherin-MARCH8-NDP52 axis negatively regulates type I IFN signaling. Data in (I) are expressed as means ± SEM of at least three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; NS, not significant (two-tailed Student’s t test). See also Figure S7. Molecular Cell 2017 68, 308-322.e4DOI: (10.1016/j.molcel.2017.09.005) Copyright © 2017 Elsevier Inc. Terms and Conditions