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SARS-Cov-2 Membrane Protein Inhibits Type I Interferon Production Through Ubiquitin-Mediated Degradation of TBK1

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SARS-Cov-2 Membrane Protein Inhibits Type I Interferon Production Through Ubiquitin-Mediated Degradation of TBK1 ORIGINAL RESEARCH published: 18 May 2021 doi: 10.3389/fimmu.2021.662989 SARS-CoV-2 Membrane Protein Inhibits Type I Interferon Production Through Ubiquitin-Mediated Degradation of TBK1 Liyan Sui 1, Yinghua Zhao 1, Wenfang Wang 2, Ping Wu 3, Zedong Wang 1, Yang Yu 4, Zhijun Hou 3, Guangyun Tan 5* and Quan Liu 1,6* 1 Laboratory of Emerging Infectious Disease, Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, China, 2 College of Basic Medical Science, Jilin University, Changchun, China, 3 College of Wildlife and Protected Area, Northeast Forestry University, Harbin, China, 4 Hospital of Stomatology, Jilin University, Changchun, China, 5 Department of Immunology, Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, China, 6 School of Life Sciences and Engineering, Foshan University, Foshan, China The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative Edited by: pathogen of current COVID-19 pandemic, and insufficient production of type I interferon Kai Deng, Sun Yat-Sen University, China (IFN-I) is associated with the severe forms of the disease. Membrane (M) protein of SARS- Reviewed by: CoV-2 has been reported to suppress host IFN-I production, but the underlying Shuai Chen, mechanism is not completely understood. In this study, SARS-CoV-2 M protein was Sun Yat-Sen University Cancer Center confirmed to suppress the expression of IFNb and interferon-stimulated genes induced by (SYSUCC), China Chi Ping Chan, RIG-I, MDA5, IKKϵ, and TBK1, and to inhibit IRF3 phosphorylation and dimerization The University of Hong Kong, caused by TBK1. SARS-CoV-2 M could interact with MDA5, TRAF3, IKKϵ, and TBK1, and Hong Kong induce TBK1 degradation via K48-linked ubiquitination. The reduced TBK1 further *Correspondence: – – e Guangyun Tan impaired the formation of TRAF3 TANK TBK1-IKK complex that leads to inhibition of [email protected] IFN-I production. Our study revealed a novel mechanism of SARS-CoV-2 M for negative Quan Liu regulation of IFN-I production, which would provide deeper insight into the innate [email protected] immunosuppression and pathogenicity of SARS-CoV-2. Specialty section: Keywords: SARS-CoV-2, membrane protein, type I interferon, TBK1, ubiquitination This article was submitted to Viral Immunology, a section of the journal Frontiers in Immunology INTRODUCTION Received: 02 February 2021 Accepted: 31 March 2021 The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the Published: 18 May 2021 coronavirus disease 2019 (COVID-19) pandemic is a novel viral member in the genus Citation: Betacoronavirus of the family Coronaviridae (1, 2), which is the third coronavirus associated Sui L, Zhao Y, Wang W, Wu P, with severe respiratory diseases, following SARS-CoV and Middle East respiratory syndrome Wang Z, Yu Y, Hou Z, Tan G and Liu Q coronavirus (MERS-CoV) (3, 4). As of January 25, 2021, there are more than 100 million confirmed (2021) SARS-CoV-2 Membrane cases of COVID-19, with 2 million deaths all over the world (https://coronavirus.jhu.edu/). SARS- Protein Inhibits Type I Interferon CoV-2 has a single stranded, positive-sense RNA genome, which contains approximately 29.7 kb Production Through Ubiquitin- Mediated Degradation of TBK1. nucleotides, with at least 12 open reading frames (ORFs) encoding 16 nonstructural proteins Front. Immunol. 12:662989. (NSPs), seven accessory proteins and four structural proteins (envelope, spike, membrane, and doi: 10.3389/fimmu.2021.662989 nucleocapsid) (1, 5). Frontiers in Immunology | www.frontiersin.org 1 May 2021 | Volume 12 | Article 662989 Sui et al. SARS-CoV-2 M Degrades TBK1 via Ubiquitination Innate immune response is considered as the first host CoraLite488-conjugated Goat Anti-Rabbit IgG antibodies were defense against viral infections, which initiates antiviral purchased from Proteintech, Wuhan, China; anti-IRF3 responses through the pattern recognition receptors (PRRs) of antibody was obtained from the Cell Signaling, Danvers, hosts. The double-strand RNA, resulting from coronavirus USA. MG132 was purchased from Sigma, St Louis, USA. genome replication and transcription, is first recognized by Z-VAD-FMK was obtained from Promega, Madison, USA. host PRRs, including the retinoic acid-inducible gene-I (RIG-I) Chloroquine was purchased from MCE, Monmouth, USA. like receptors (RLRs), such as RIG-I and melanoma differentiation associated gene 5 (MDA5) (6, 7). Activated ELISA RLRs trigger TANK-binding kinase 1 (TBK1) activation Flag tagged empty vector or Flag-M and MDA5, TBK1 or IKKϵ through the key adaptor mitochondrial antiviral signaling expression plasmids were co-transfected into HEK293T cells. (MAVS) (8), further activating the transcription factor After 24 h, culture supernatant was harvested and secreted IFNb interferon regulation factor 3 (IRF3) to induce production of was detected by ELISA according to the manufacturers’ protocol type I interferon (IFN-I) and downstream interferon-stimulated from Proteintech, Wuhan, China. genes (ISGs), the critical host antiviral factors (9, 10). Viruses have evolved elaborate mechanisms to evade host Luciferase Reporter Assay antiviral immunity, with a common strategy of virus-encoded HEK293T cells (2 × 105, 24-well plate) were transfected with IFN antagonists (11). SARS-CoV-2 encoded proteins, such as reporter plasmid of 200 ng IFNb-Luc or ISRE-Luc and 40 ng ORF6, NSP13, membrane (M), and nucleocapsid (N) proteins Renilla-Luc, together with expressing plasmids of RIG-I, MDA5, have been shown to possess the IFN-antagonizing properties TBK1, IKKϵ,or1mg/ml poly(I:C), and plasmids expressing viral (12–14). The SARS-CoV-2 M protein can interact with MAVS proteins. Cells were harvested after 24 h, and cell lysates were and impede the formation of MAVS–TRAF3–TBK1 complex to used to determine individually for IFNb or ISRE luciferase using antagonize IFN-I production (15, 16). However, whether SARS- a Dual Luciferase Reporter Assay System (Promega, Madison, CoV-2 M interacts with RIG-I, MDA5, or TBK1 is in dispute (15, USA). Relative luciferase activity was calculated by normalize 16), and its association with TRAF3 and IKKϵ remains to be firefly luciferase activity to Renilla luciferase activity recovered investigated, which would contribute to understanding of the from cell lysate. immune evasion mediated by the SARS-CoV-2 M protein. In this study, we reported that the SARS-CoV-2 M protein RNA Isolation and Quantitative PCR suppressed IFN-I production by interacting with TBK1 and (qPCR) promoting its degradation via K48-linked ubiquitination, and The cDNA was synthesized after total RNA extraction. M protein could also interact with MDA5, TRAF3 and IKKϵ. Quantitative cDNA amplification was performed using ABI The reduced TBK1 impaired the formation of TRAF3–TANK– Plus one (Applied Biosystems, Foster City, USA); primers used TBK1-IKKe complex, resulting to the inhibition of IRF3 were listed in Supplementary Table 1. Each PCR reaction of 20 activation and further IFN-I production. This study reveals a µl contains 8 µl of ddH2O, 10 µl of SYBR green premix, 1 µl of novel mechanism for SARS-CoV-2 M protein to inhibit IFN-I cDNA, and 0.5 µl of each forward and reverse primer (10 µM). production, which provides in-depth insight into the innate Amplification conditions were as follows: 5 min denaturation at immunosuppression and pathogenicity of SARS-CoV-2. 95°C, 40 cycles of PCR for the quantitative analysis (95°C for 10 s and 60°C for 30 s). The relative expression of each gene was −DD analyzed by 2 CT method. MATERIALS AND METHODS Immunofluorescence Plasmids To assess the colocation of SARS-CoV-2 M with TRAF3, TBK1 and The SARS-CoV M protein (NC_004718), SARS-CoV-2 M protein IKKϵ,animmunofluorescence assay was carried out as described of IPBCAMS-WH-01/2019 strain (no. EPI_ISL_402123), TBK1 elsewhere (19). After fixed with 4% paraformaldehyde for 30 min, the genes and their truncations were cloned into vector VR1012 with cells were permeabilized with 1% Triton X-100/PBS for 15 min and the Flag-tag or GST-tag (Sangon Biotech, Shanghai, China). The blocked with blocking buffer (PBS +1% bovine serum albumin) for expression vectors of Flag-Ubi, Flag-K48-Ubi, Flag-K63-Ubi, 1 h, cells were then incubated with primary and secondary antibodies pIFNb-Luc, ISRE-luc and Renilla luc were constructed in the and stained for nuclear. Fluorescence was captured on OLYMPUS previous study (17, 18). The expression plasmids for IRF3, FV3000 confocal microscope (Olympus, Shinjuku, Japan). TANK, IKKϵ, RIG-I and TRAF3 were purchased from PPL Biotech, Jiangsu, China. The expression plasmid for TBK1 and Co-Immunoprecipitation and Immunoblot MDA5 was purchased from Miaoling Biotech, Wuhan, China. Analysis Co-immunoprecipitation and immunoblot analysis were Antibodies and Drugs performed as described elsewhere (18). Briefly, cells were lysed Anti-Flag, anti-HA, anti-Myc, anti-GST tag antibodies, anti- with cell lysis buffer and boiled for 10 min. The cell lysates added Phospho-IRF3 (S396) antibody, anti-GAPDH and anti-actin anti-Flag or anti-HA agarose were incubated on a roller at 4°C antibodies, CoraLite594-conjugated goat anti‐rabbit IgG, and overnight. The immunoprecipitants or cell lysates were subjected Frontiers in Immunology | www.frontiersin.org 2 May 2021 | Volume 12 | Article 662989 Sui et al. SARS-CoV-2 M Degrades TBK1 via Ubiquitination to electrophoresis on a 12% SDS-PAGE gel and transferred onto SARS-CoV-2 M Protein Antagonizes PVDF membranes for immunoblot analysis. The membranes IRF3 Activation were blocked and incubated with primary and HRP-conjugated The phosphorylation and nuclear translocation of IRF3 is the key secondary antibodies. Chemi-luminescence was tested using ECL step for IRF3 activation and IFN production (21). Thus, we (Thermo, Waltham, USA) and protein bands were visualized by explored the effect of M protein on IRF3 activation. Biorad CHemiDoc XRS (Biorad, California, USA).
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