key: cord-0875579-yhmchydw
authors: Han, Lulu; Zhuang, Meng‐Wei; Deng, Jian; Zheng, Yi; Zhang, Jing; Nan, Mei‐Ling; Zhang, Xue‐Jing; Gao, Chengjiang; Wang, Pei‐Hui
title: SARS‐CoV‐2 ORF9b antagonizes type I and III interferons by targeting multiple components of the RIG‐I/MDA‐5–MAVS, TLR3–TRIF, and cGAS–STING signaling pathways
date: 2021-05-09
journal: J Med Virol
DOI: 10.1002/jmv.27050
sha: da3684d0ceef38213ae796e9fa538a74295bcf99
doc_id: 875579
cord_uid: yhmchydw

The suppression of types I and III interferon (IFN) responses by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) contributes to the pathogenesis of coronavirus disease 2019 (COVID‐19). The strategy used by SARS‐CoV‐2 to evade antiviral immunity needs further investigation. Here, we reported that SARS‐CoV‐2 ORF9b inhibited types I and III IFN production by targeting multiple molecules of innate antiviral signaling pathways. SARS‐CoV‐2 ORF9b impaired the induction of types I and III IFNs by Sendai virus and poly (I:C). SARS‐CoV‐2 ORF9b inhibited the activation of types I and III IFNs induced by the components of cytosolic dsRNA‐sensing pathways of RIG‐I/MDA5‐MAVS signaling, including RIG‐I, MDA‐5, MAVS, TBK1, and IKKε, rather than IRF3‐5D, which is the active form of IRF3. SARS‐CoV‐2 ORF9b also suppressed the induction of types I and III IFNs by TRIF and STING, which are the adaptor protein of the endosome RNA‐sensing pathway of TLR3‐TRIF signaling and the adaptor protein of the cytosolic DNA‐sensing pathway of cGAS–STING signaling, respectively. A mechanistic analysis revealed that the SARS‐CoV‐2 ORF9b protein interacted with RIG‐I, MDA‐5, MAVS, TRIF, STING, and TBK1 and impeded the phosphorylation and nuclear translocation of IRF3. In addition, SARS‐CoV‐2 ORF9b facilitated the replication of the vesicular stomatitis virus. Therefore, the results showed that SARS‐CoV‐2 ORF9b negatively regulates antiviral immunity and thus facilitates viral replication. This study contributes to our understanding of the molecular mechanism through which SARS‐CoV‐2 impairs antiviral immunity and provides an essential clue to the pathogenesis of COVID‐19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID- 19) , is a novel emerging coronavirus that is spreading globally, might be lethal to humans and other animals, and thus poses significant threats to public health worldwide. 1 ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c, and ORF10. 4 Although the accessory proteins of coronaviruses are not essential for viral replication and virion assembly, they contribute to virulence by affecting the release, stability, and pathogenesis of the virus. 5 To date, the function of SARS-CoV-2 accessory proteins in immune evasion still needs to be addressed.

Although the SARS-CoV-2 infection-mediated dysregulation of the immune system, which involves the suppression of antiviral immunity and the elevation of inflammatory responses, contributes to the pathogenesis of COVID-19, 6-11 the mechanism through which the recently emerged SARS-CoV-2 is recognized by innate immunity has not been clarified. Double-stranded RNA (dsRNA), which is produced by many viruses during replication, is a common viral pathogen-associated molecular pattern that is sensed by pattern recognition receptors. 12 Cytosolic retinoic acid-inducible gene (RIG)-I-like receptors, including RIG-I and MDA-5, and endosomal Toll-like receptor 3 (TLR3) recognize dsRNAs from intermediates generated during viral replication, and this recognition results in the serial activation of innate antiviral signaling cascades via induction of the production of types I and III IFNs. 12, 13 The coronaviruses have homologous genomes, similar replication intermediates, and the same lifecycles; thus, it appears that SARS-CoV-2 can be recognized by RNA sensors similarly to other coronaviruses to elicit innate antiviral immunity. 14 RIG-I participates in the immune sensing of murine coronavirus mouse hepatitis virus (MHV) in oligodendrocyte cells. 15 MDA5 can recognize MHV in brain macrophages, microglial cells, and oligodendrocyte cells. 15, 16 The sensing of cytosolic dsRNA by RIG-I/MDA-5 recruits the adaptor protein MAVS (also known as VISA, Cardiff, or IPS-1), which activates TANK-binding kinase 1 (TBK1)/inhibitor of κB kinase epsilon (IKKε) and then induces the phosphorylation and subsequent nuclear translocation of the transcription factor IFN regulatory factor 3 (IRF3), and nuclear IRF3 together with nuclear factor-κB (NF-κB), which is also activated by RIG-I/MDA-5 signaling, initiates the transcription of types I and III IFNs and other proinflammatory cytokines, which lead to antiviral immune responses. 12 TLR3 is involved in the defense against SARS-CoV-1 infection. 17 dsRNA-activated TLR3 activates IRF3 and NF-κB signaling via TIR-domain-containing adapter-inducing interferon-β (TRIF)-TBK1/IKKε signaling cascades, which results in the production of types I and III IFNs and other pro-inflammatory cytokines. 12 Although the involvement of the cytosolic DNA-sensing pathway of cGAS-stimulator of IFN genes (STING) signaling in the recognition of coronaviruses has not been elucidated, the papain-like protease domain from SARS-CoV-1 can act as an antagonist of IFNs by targeting STING, 18, 19 which suggests that the cGAS-STING pathway should play a vital role in the defense against certain coronaviruses. STING is activated by the second messenger 2ʹ−3ʹcGAMP produced by DNA-activated cGAS. 20 Subsequently, STING recruits TBK1, which phosphorylates IRF3, and this phosphorylation leads to the translocation of IRF3 into the nucleus to induce the expression of types I and III IFNs and other proinflammatory cytokines. 20 The RIG-I/MDA-5-MAVS, TLR3-TRIF, and cGAS-STING signaling pathways converge at TBK1/IKKε, which catalyzes IRF3 phosphorylation and the subsequent transcription of types I and III IFNs. 21 Secreted type I and III IFNs bind to their receptors and then activate Janus kinase/signal transducers and activators of transcription signaling to drive the expression of IFN-stimulated genes (ISGs), which can initiate antiviral states by suppressing viral replication and spreading, activating immune cells, and causing the death of infected cells. 13, 22 The types I and III IFN response is the essential action of host antiviral immunity in the clearance of virus infection. 13, 22 To establish a successful infection of host cells, viruses, including coronaviruses, have developed various strategies to antagonize the IFN response. 14 Previous studies have proposed that the accessory proteins of SARS-CoV-1, such as open reading frame 3b (ORF3b), ORF6, and ORF9b, inhibit the production of type I IFNs. 14 In COVID-19 patients, the induction of types I and III IFNs is suppressed. 7, 8, 23 The replenishment of types I or III IFNs can significantly contribute to the clearance of SARS-CoV-2 and to COVID-19 symptom relief. [24] [25] [26] Compared with type I IFNs, type III IFNs exhibit some advantages in COVID-19 treatment regarding the induction of a longer-lasting antiviral state and a less proinflammatory response. 27 Although SARS-CoV-2 infection impairs the antiviral immunity elicited by types I and III IFNs in COVID-19 patients and cell models, 7, 8, 23 the mechanism through which the recently emerged SARS-CoV-2 blocks the induction of types I and III IFNs remains elusive. Therefore, dissecting the molecular mechanism through which SARS-CoV-2 evades types I and III IFN responses will improve the understanding of the pathogenesis of COVID19 and provide therapeutic strategies for counteracting SARS-CoV-2 infections. SARS-CoV-1 ORF9b was reported to suppress IFN production; however, whether SARS-CoV-2 ORF9b could evade host antiviral innate immunity is still unknown; thus, we explored the effect of SARS-CoV-2 ORF9b on IFN production and the potential mechanism.

We found that the SARS-CoV-2 accessory protein ORF9b, which is encoded by an alternative ORF within the N gene, can remarkably suppress RIG-I/MDA-5-MAVS, TLR3-TRIF, and cGAS-STING signaling-activated types I and III IFN production by targeting multiple molecules of these innate antiviral pathways.

Protein A/G beads were purchased from Santa Cruz Biotechnology, and the anti-Flag magnetic beads were purchased from Bimake. Poly Table S1 by polymerase chain reaction (PCR) and cloned into the pCAG mammalian expression vector with a C-terminal Flag-tag.

HEK-293T, HeLa, and Vero E6 cells were obtained from the American Type Culture Collection (ATCC) and maintained according to the culture methods provided by the ATCC. All these cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS) at 37°C in a humidified incubator with 5% CO 2 .

The plasmids were transiently transfected into the cells using Lipofectamine 3000 (Invitrogen) or Polyethylenimine "Max" (Polysciences, Inc.) following the manufacturer's instruction. Poly (I:C) and 2′−3′ cGAMP were delivered into cells using Lipofectamine 2000 (Invitrogen) as described previously. 28 2.5 | RNA extraction and real-time quantitative PCR Total RNA was extracted with TRIzol reagent (Invitrogen) and then was reverse-transcribed into first-strand cDNA with the HiScript III 1st Strand cDNA Synthesis Kit with gDNA wiper (Vazyme) following the manufacturer's protocol. The SYBR Greenbased RT-qPCR kit UltraSYBR Mixture (CWBIO) was used to perform real-time quantitative PCR (RT-qPCR) assays using primers of each gene (Table S1 ) by a Roche LightCycler96 system according to the manufacturer's instructions. The relative expression of the indicated genes was normalized to the mRNA level of glyceraldehyde 3-phosphate dehydrogenase, one of the internal housekeeping genes in human cells. A comparative C t method (ΔΔC t method) was used to calculate the fold changes by normalizing to that of genes expressed in the control group as described previously. 31 

The concentration of secreted IFN-β in culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) according to the manufacturer′s instructions. control. Thirty-six hours later, the cells were harvested to assess the luciferase activities using the Dual-Luciferase Reporter Assay Kit (Vazyme) as described in our previous studies. [31] [32] [33] The luciferase activity was measured in a Centro XS3 LB 960 microplate luminometer (Berthold Technologies). The activity of firefly luciferase was normalized to that of Renilla luciferase to calculate the relative luciferase activity. 

HeLa cells were seeded on 12-well slides 24 h before transfection.

Each well was transfected with the indicated plasmids (1 µg each).

Transfected or infected HeLa cells were subject to fix, permeabilization, and blocking as described in the previous paper. The fixation, permeabilization, and blocking buffer were all purchased from Beyotime Biotechnology. The cells were then incubated with indicated primary antibodies at 4°C overnight, rinsed, and incubated with corresponding secondary antibodies (Invitrogen).

Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (Abcam). Images were captured with a Zeiss LSM880 confocal microscope.

flow cytometer with at least 10,000 cells per sample as described in our previous publication. 34 

Vero-E6 cells were used to perform plaque assays to determine the titer of VSV-eGFP. Vero cells at approximately 100% confluency cultured in 24-well plates were infected with serial dilutions of VSV-eGFP. 0.5 h later, the culture medium was discarded, and then DMEM containing 0.5% agar and 2% FBS overlaid. After 20 h culture, the cells were fixed with a 1:1 methanol-ethanol mixture and then visualized with 0.05% crystal violet. The plaques on the monolayer were used to determine the titer of VSV-eGFP as described in our previous publication. 34 

The statistical analyses were performed using two-tailed unpaired 3 | RESULTS

To explore the function of SARS-CoV-2 ORF9b in viral infection, HEK-293T cells expressing SARS-CoV-2 ORF9b were infected with SeV. RT-qPCR analysis revealed that the induction of IFN-β, IFN-λ1, and two ISGs called ISG56 and CXCL10 after SeV infection was suppressed in SARS-CoV-2 ORF9b-expressing cells compared with the control HEK-293T cells that did not express any viral protein ( Figure 1A ). The ELISA assays showed that less IFN-β is released into the culture supernatant from SARS-CoV-2 ORF9b-expressing cells than that from the control cells ( Figure 2 ). Similar results were observed with HEK-293T cells that were transfected with the dsRNA mimic poly (I:C) to stimulate antiviral immunity ( Figure 1B) . However, SARS-CoV-2 ORF9c, another alternative ORF within the N gene, exerted no effect on either SeV infection-or poly (I:C) transfection-induced types I and III IFN production ( Figures S1 and S2) .

We subsequently attempted to map the layer where SARS-CoV-2 ORF9b exerts its inhibitory effect on IFN production. We co- Figure 5F ), which is consistent with the results from the colocalization studies ( Figure 4 ). SARS-CoV-2 ORF9b prevents the association between TRIF and TBK1 but has no effect on the interactions between TBK1 and IRF3 ( Figure 5H and I). These data showed that SARS-CoV-2 ORF9b might target multiple molecules of the RIG-I/MDA-5-MAVS, TLR3-TRIF, and cGAS-STING signaling pathways to suppress IFN production.

The F I G U R E 2 SARS-CoV-2 ORF9b inhibits SeV-induced IFN-β secretion. HEK-293T cells were transfected with plasmids of the pcDNA6B empty vector (500 ng) or SCV2-ORF9b (500 ng). Twentyfour hours later, the cells were infected with SeV as indicated, and 9 and 12 h after stimulation, the culture supernatant was collected for ELISA assays. The results from one representative experiment are shown to represent three independent biological replicates. The error bars indicate the SD. ELISA, enzyme-linked immunosorbent assay; IFN, interferon; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2

The transcription of IFNs is initiated by phosphorylated IRF3 after its translocation into the nucleus. The retention of IRF3 in the cytosol and thus the inhibition of its nuclear translocation arrests its action on IFN induction. As SARS-CoV-2 ORF9b inhibits IRF3 phosphorylation, we subsequently examined the effect of SARS-CoV-2 ORF9b on SeV-induced IRF3 nuclear translocation. In resting cells, IRF3 was primarily distributed in the cytosol regardless of the expression of SARS-CoV-2 ORF9b ( Figure 7B ). After SeV infection, IRF3 was translocated into the nucleus of the control cells; however, IRF3 was restricted in the cytosol of cells expressing SARS-CoV-2 ORF9b ( Figure 7B ). These data support the hypothesis that SARS-CoV-2 ORF9b inhibits the nuclear translocation of IRF3 upon SeV infection ( Figure 7C ). 

The COVID-19 pandemic is affecting the economy, transport, and relationships of countries as well as people′s lives and health worldwide, and researchers worldwide are attempting to find various strategies for the treatment of COVID-19. The immune system is essential for defense against virus infection; unfortunately, SARS-CoV-2 infection subverts this system by suppressing types I and III responses and elevating the proinflammatory response, which will accelerate viral replication and damage the host tissues and organs. 8 Types I and III IFN responses play a critical role in human antiviral F I G U R E 4 (A-C) Subcellular localization of SARS-CoV-2 ORF9b. HeLa cells seeded on 12-well coverslips were transfected with the indicated plasmids. Twenty hours after transfection, the cells were subjected to immunofluorescence staining with mouse anti-Myc antibody and rabbit antibodies against the corresponding organelle marker. Scale bar = 10 μm. (D-I) Relative localization of SARS-CoV-2 ORF9b protein with signaling molecules, including RIG-I, MDA5, MAVS, TBK1, TRIF, and STING. The seeding and transfection of HeLa cells were performed as described in (A). After transfection, ORF9b was stained with a rabbit anti-Myc antibody, and the signaling molecules were reacted with mouse antibodies against the indicated tags. Scale bar = 10 μm. TOM20, Mitochondria marker; Calnerxin, ER marker; GM130, Golgi marker. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TBK1, TANK-binding kinase 1; TRIF, TIR-domain-containing adapter-inducing interferon-β immunity against SARS-CoV-2, and clinical trials have shown that the restoration of types I and III IFNs in COVID-19 patients is an effective therapeutic option; thus, further investigation of the mechanism through which SARS-CoV-2 evades antiviral immunity is warranted. 13, 25, 26, 35 Here, we found that SARS-CoV-2 ORF9b antagonizes types I and III IFNs and impairs host antiviral immunity. amino acid identity. Surprisingly, a recent screening study showed that SARS-CoV-2 ORF9b does not affect IFN activation induced by RIG-I signaling. 37 Thus, further investigation of whether SARS-CoV-2

ORF9b is also involved in the suppression of IFNs would be F I G U R E 5 SARS-CoV-2 ORF9b interacts with RIG-I, MDA-5, MAVS, TBK1, STING, and TRIF but not IRF3. HEK-293T cells were transfected with the indicated plasmids for 24 h before coimmunoprecipitation by the indicated antibody-conjugated beads. The input and immunoprecipitates were reacted with the indicated antibodies. IRF3, IFN regulatory factor 3; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TBK1, TANK-binding kinase 1; TRIF, TIR-domain-containing adapter-inducing interferon-β interesting. During the preparation of this manuscript, another study showed that SARS-CoV-2 ORF9b suppresses type I IFN production by targeting TOM70. 38 Although the molecular mechanism through which SARS-CoV-2 ORF9b inhibits the type I IFN response by interacting with TOM70 has not been investigated, it has been proposed that SARS-CoV-2 ORF9b might compete with HSP90 for binding to TOM70 or might induce the production of lactic acid, which has been proven to inhibit the IFN response. 38 Due to our lack of a biosafety level-3 laboratory, we had to use another RNA virus, SeV, in the viral infection studies, and we found that the overexpression of SARS-CoV-2 ORF9b significantly reduced the production of IFN-β, IFN-λ1, ISG56, and CXCL10 stimulated by SeV infection (Figure 1 ). We also found that SARS-CoV-2 ORF9b inhibited the SeV-induced phosphorylation and nuclear translocation of IRF3 (Figure 7) . Moreover, the overexpression of SARS-CoV-2

ORF9b in HEK-293T cells facilitated the replication of VSV-eGFP, which is sensitive to the activation of IFN signaling; thus, SARS-CoV-2 ORF9b might enhance VSV-eGFP replication by suppressing IFN production ( Figure 8 ). Although SARS-CoV-1 ORF9b reportedly inhibits IFN production, its role in viral infection is currently unknown; thus, this report provides the first demonstration that coronavirus ORF9b suppresses SeV-induced type I and III IFN production and promotes VSV-eGFP replication.

Luciferase reporter assays showed that SARS-CoV-2 ORF9b where it degrades the MAVS signalosome, is essential for its IFN inhibitory function. We found that in addition to its mitochondrial localization, SARS-CoV-2 also localizes to the ER and Golgi (Figure 4 ).

The ER is an important platform for TRIF and STING, whereas the Golgi is an important platform for TBK1. [40] [41] [42] Thus, these findings explain the colocalization and association of SARS-COV-2 ORF9b with TRIF, STING, and TBK1. The localization of SARS-CoV-2 ORF9b in the ER and Golgi might provide a platform for its inhibitory effects on TRIF-, STING-, and TBK1-induced IFN production. Thus, this study extends our understanding of the molecular mechanisms through which coronavirus ORF9b mediates the antagonizing of IFN.

SARS-CoV-2 is more sensitive to IFN treatment than other coronaviruses. 26 Multiple viral proteins that suppress IFN production at different steps become more critical to ensure that the production and function of IFNs are minimized during SARS-CoV-2 infection. SARS-COV-2 ORF9b targets multiple proteins of distinct antiviral signaling pathways and thus suppresses IFN signaling at different steps. Similarly, SARS-CoV-2 ORF6 and MERS-CoV ORF4b are capable of perturbing multiple antiviral signaling pathways by targeting various components of these pathways. 37, 43, 44 Although cGAS-STING is a cytosolic dsDNA sensing pathway, coronaviruses, a family of RNA viruses, also encode viral proteins such as papain-like protease to impair STING function; thus, this pathway is essential for F I G U R E 8 SARS-CoV-2 ORF9b overexpression impairs TBK1-dependent antiviral immunity. Plasmids were transfected into HEK-293 cells as indicated. Twenty-four hours after transfection, the cells were infected with vesicular stomatitis virus (VSV)-enhanced green fluorescent protein (eGFP) (MOI = 0.001). Ten hours after infection, (A) the GFP-positive cells were observed and analyzed with fluorescence microscopy and flow cytometry, and (B) the culture supernatant (20 h postinfection) was harvested for plaque assays to measure the titer of extracellular VSV-eGFP. The fluorescence imaging results are representative of two independent experiments. Scale bar = 50 μm. In Panel b, the results from one representative experiment are shown, three independent biological replicates were analyzed, and the error bars indicate the SD. The statistical significance is shown as indicated. E.V., empty vector; IRF3, IFN regulatory factor 3; MOI, multiplicity of infection; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TBK1, TANK-binding kinase 1 defense against coronavirus infection. 18, 19 The inhibition of the cGAS-STING pathway by SARS-CoV-2 ORF9b may suggest that this pathway may play a role in SARS-CoV-2 clearance; thus, drugs or chemicals, such as 2′−3′cGAMP that activate this pathway may be considered to be used in COVID-19 treatment.

Distinct methods have shown that SARS-CoV-2 ORF9b inhibits type I and III IFN production by targeting multiple proteins of antiviral signaling pathways. We should be conscious that the transfection system might differ from real viral infection. Therefore, further studies should be conducted in the context of real SARS-CoV-2 infection experiments. The accessory proteins of coronaviruses have been proposed to be not essential for viral replication. These proteins are not directly involved in viral assembly 5 ; thus, theoretically, ORF9b-null SARS-CoV-2 might be available if it cannot affect the translation and expression of the N protein, which is a structural protein needed for virion assembly. However, whether this ORF9b-null SARS-CoV-2 virus is accessible needs experimental validation. Once this mutant virus is available, the results from real viral infection studies should contribute to our understanding of the role of ORF9b in IFN antagonization.

Although the administration of exogenous IFNs has been shown to be valid for SARS-CoV2 clearance in both SARS-CoV-2 patients and cell models 24, 27, 35, 45 full evaluation of these treatments requires extensive studies on the relative importance of all IFN-antagonizing viral proteins encoded by SAR-CoV-2. Thus, our finding that SARS-COV-2 ORF9b suppresses type I and III IFN production contributes to our understanding of the pathogenesis of COVID-19, and the identification of multiple protein targets might provide more precise targets for COVID-19 treatment.

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SARS-CoV-2 ORF9b antagonizes type I and III interferons by targeting multiple components of the RIG-I/MDA-5-MAVS, TLR3-TRIF, and cGAS-STING signaling pathways

This study was supported by grants from the COVID-19 emergency tackling research project of Shandong University (Grant No. 2020XGB03 to Pei-Hui Wang), grants from the Natural Science 

The authors declare that there are no conflict of interests.

Chengjiang Gao and Pei-Hui Wang conceptualized the study. Lulu 

The peer review history for this article is available at https://publons. com/publon/10.1002/jmv.27050

The data that support the findings of this study are available from the corresponding author upon reasonable request.

http://orcid.org/0000-0002-9365-4497Pei-Hui Wang https://orcid.org/0000-0001-6853-2423