SARS-CoV-2 Nonstructural Protein 1 Inhibits the Interferon Response by Causing Depletion of Key Host Signaling Factors

To understand how the host cell responds to SARS-CoV-2 during infection, we assessed the transcriptional induction of Ifnb mRNA and secretion of IFN-β from ACE2-expressing HEK 293T (HEK 293T-ACE2) cells using quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. While robust replication of the virus was observed in HEK 293T-ACE2 cells, similar to that in Vero E6 cells, by 24 and 48 hpi (Fig. 1A), no significant increase in Ifnb mRNA (Fig. 1B) or secreted IFN-β (Fig. 1C) was observed in response to infection. Conversely, infection of these cells with Sendai virus (SeV) resulted in increased secretion of IFN-β into culture medium. In contrast, cells which were first infected with SARS-CoV-2 secreted very low levels of IFN-β in response to SeV infection (Fig. 1C). Similarly, no induction of the ISG IFIT1 was observed in SARS-CoV-2-infected cells, and significantly lower levels were induced upon challenge with SeV or poly(I·C) (Fig. 1D and E). These results are in agreement with a recent study describing human airway epithelial cell cultures infected with SARS-CoV-2 (23). Next, we examined the ability of SARS-CoV-2 to infect cells in which type I IFN had been induced by SeV either 8 h preinfection or 16 h postinfection (hpi). Induction of IFNs by SeV pretreatment significantly reduced SARS-CoV-2 replication (Fig. 1F). In contrast, addition of SeV to cells already infected with SARS-CoV-2 had little effect, indicating that this coronavirus actively blocks IFN induction (Fig. 1F). Of note, similar amounts of intracellular SeV RNA were detected from mock- and SARS-CoV-2-infected cells after 16 h of SeV infection (Fig. 1G), indicating that SARS-CoV-2-infected cells are as permissive for SeV infection as mock-infected cells. The suppression of IFN induction during SARS-CoV-2 infection was further evident from the lack of phosphorylation of IRF3 (Fig. 1H) and absence of SeV-induced-IRF3 transport into the nucleus (Fig. 1I).

To determine which viral proteins were responsible for antagonizing type I IFN induction, we constructed expression plasmids for all known proteins of SARS-CoV-2 with C-terminal 3× FLAG tags using cDNA generated from infected-cell lysates. NSP10 was tagged with 3× FLAG on its N terminus due to the instability of the C-terminally tagged version. The expression and molecular weights of most sequence-verified constructs were determined by immunoblotting (Fig. 2A). Expression of NSP6 and ORF10 was verified by immunoprecipitation coupled with immunoblotting (Fig. 2B), whereas expression of NSP3 (>250 kDa) was confirmed by indirect immunofluorescence (IF) (Fig. 2C).

The effect of SARS-CoV-2 proteins on SeV-induced IFN induction was assessed using an IFN-β luciferase reporter assay and ELISA. Expression of the N and NSP1 proteins reduced IFN-β reporter activity by 70% and 30%, respectively (Fig. 3A). Secretion of IFN-β was also significantly impaired in cells expressing these viral proteins (Fig. 3B). As the antiviral transcription factors IRF3 and NF-κB are activated during the induction of IFNs (6), we assessed whether N or NSP1 affected their activities. Whereas both viral proteins inhibited IRF3-dependent transcription by >50%, NF-κB reporter activity was more strongly affected by NSP1 (64% reduction) (Fig. 3C and D). We next examined whether localization of IRF3 was affected in SeV-infected cells expressing N protein or NSP1. IRF3 did not translocate to nuclei of NSP1-expressing cells, which is consistent with the observation that this viral protein inhibits IFN induction (Fig. 3E and F). Conversely, nuclear accumulation of IRF3 was not significantly affected in cells expressing N protein or the control protein NS3A (Fig. 3E and F). Mapping studies using overexpression of IFN induction pathway components suggest that one critical step targeted by NSP1 is the phosphorylation of IRF3 (Fig. 3G). Specifically, NSP1-mediated reduction in IRF3 signaling was rescued by expression of a constitutively active form of IRF3 (IRF3-5D). We tested how expression of an NSP1 mutant (KH164AA) that is unable to suppress translation (24) affected induction of type I IFNs. The KH164AA mutant was unable to abrogate IFN-β reporter activity (Fig. 4A and B), and this phenotype corresponded to its inability to block IRF3 nuclear accumulation (Fig. 4C and D). These data strongly suggest that NSP1-mediated suppression of IFN production is linked to its ability to shut down host translational machinery and nuclear transport of IRF3.

To further understand how N protein affected IFN induction, we probed for an interaction between SARS-CoV-2 N protein and TRIM25. Previously it was reported that the N protein of the closely related coronavirus SARS-CoV inhibits TRIM25-mediated ubiquitination of RIG-I (25), an essential step in RIG-I activation. Stable interaction between N protein and TRIM25 was observed by coimmunoprecipitation (Fig. 4E), and the importance of TRIM25 in restricting SARS-CoV-2 replication was demonstrated by overexpression of TRIM25 (Fig. 4F and G). However, N protein did not impair RIG-I ubiquitination by TRIM25 (Fig. 4H), and thus, the mechanism of N-mediated suppression of IFN-β induction remains to be elucidated.

To investigate how SARS-CoV-2 affects the signaling arm of the IFN response, we first assessed the induction of ISGs in SARS-CoV-2-infected HEK 293T-ACE2 cells. While robust viral replication was observed in these cells (Fig. 1A), no significant induction of the ISG Ifit1 mRNA was detected (Fig. 5A). Conversely, treatment of HEK 293T-ACE2 cells with IFN-α strongly upregulated Ifit1 expression, indicating the presence of a functional IFN signaling pathway in these cells. Next, we examined the sensitivity of SARS-CoV-2 to pre- and postinfection treatment with IFN-α, IFN-γ, and IFN-λ. Consistent with recent reports (20, 23, 26, 27), replication of SARS-CoV-2 was strongly inhibited by pretreatment with IFN-α, whereas posttreatment had only a moderate effect (Fig. 5B). Interestingly, neither pre- nor posttreatment of cells with IFN-γ reduced viral replication, whereas IFN-λ inhibited virus replication but not to the same degree as IFN-α. These results were further confirmed by IFN-stimulated response element (ISRE) promoter reporter assays, in which strong inhibition was observed in SARS-CoV-2-infected cells (Fig. 5C). In contrast, IFN-γ activation site (GAS) promoter activity was not inhibited in SARS-CoV-2-infected cells treated with IFN-γ (Fig. 5D). We next examined how SARS-CoV-2 affected levels of cellular proteins involved in type I IFN signaling. Immunoblot analysis revealed that levels of STAT2 and Tyk2 were significantly reduced during infection, whereas IFNAR1, Jak1, and STAT1 levels were largely unaffected (Fig. 5E and F).

Next, infected cells were treated with IFN-α or IFN-γ for 2 h, after which localization of STAT1 and STAT2 was examined by confocal microscopy. Compared to uninfected cells, STAT2 fluorescence was greatly diminished in infected cells, and nuclear translocation of the protein in response to IFN treatment was not evident (Fig. 6A). In contrast, neither the STAT1 fluorescent signal nor its nuclear translocation was noticeably affected by infection (Fig. 6B). To determine how SARS-CoV-2-mediated STAT2 depletion occurred, we treated infected cells with inhibitors of proteasome (epoxomicin)- and lysosome (bafilomycin A1)-dependent degradation. Epoxomicin treatment partially rescued STAT2 levels, suggesting that depletion of this transcription factor during SARS-CoV-2 infection is mediated at least in part by proteasomal degradation (Fig. 6C and D). As SARS-CoV-2 is known to suppress host cell translation (28), we assessed whether the loss of STAT2 and potentially other antiviral host factors was due to this effect. When green fluorescent protein (GFP)-expressing cells were infected with SARS-CoV-2, there was no reduction in GFP mRNA but a 30% decrease in protein levels was observed (Fig. 6E to G). Treatment of uninfected cells with cycloheximide resulted in reduction of STAT2 and Tyk2 protein levels comparable to those seen during infection (Fig. 6H and I), suggesting that virus-mediated suppression of host cell translation is important for blocking IFN signaling.

To identify the viral proteins that inhibit IFN signaling, we performed ISRE reporter assays in cells expressing individual SARS-CoV-2 proteins. NSP1 was the only viral protein that significantly suppressed ISRE reporter activity (Fig. 7A). To examine the mechanism behind this phenomenon, we again employed the NSP1 mutant KH164AA, which is unable to inhibit host cell translation. The mutant NSP1 did not block ISRE-dependent transcription (Fig. 7B), which is consistent with a role for viral translational suppression in preventing IFN induction (12, 24, 29). Levels of STAT2 in cells expressing NSP1 were also examined by confocal microscopy. Compared to signal in cells expressing SARS-CoV-2 NS7B or the NSP1 KH164AA mutant, STAT2 signal was significantly reduced in cells expressing wild-type NSP1 (Fig. 7C and D). These results were consistent with immunoblotting data, in which levels of ectopically expressed STAT2-GFP were reduced by wild-type NSP1 but not by the KH164AA mutant (Fig. 8A and B). Furthermore, in cells coexpressing Renilla luciferase and wild-type NSP1, a 40% reduction in luciferase reporter activity was observed (Fig. 8C). Similarly, in cells expressing GFP and NSP1, there was no significant reduction in mRNA level but a 40 to 45% reduction in protein level of GFP was observed (Fig. 8D to F), demonstrating an inhibitory effect of NSP1 on overall protein production. NSP1-mediated depletion of STAT2 could be partially rescued by treatment with epoxomicin (Fig. 8G and H), indicating that proteasome-mediated degradation plays a role in STAT2 depletion. However, although the difference was statistically insignificant, epoxomicin treatment also increased STAT2 protein levels in control groups (Fig. 8G and H), indicating that STAT2 is normally turned over via the proteasomal machinery. Our data are consistent with a scenario where virus-induced global translational shutdown leads to depletion of critical host antiviral factors with short half-lives, such as STAT2 and Tyk2, that are needed for induction of ISGs.

A549, Vero E6, HEK 293T, and Huh7 cells from the American Type Culture Collection (Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 100 U/ml penicillin and streptomycin, 1 mM HEPES (Gibco), 2 mM glutamine (Gibco), and 10% heat-inactivated fetal bovine serum (FBS) at 37°C in 5% CO₂. SARS-CoV-2 (hCoV-19/Canada/ON-VIDO-01/2020; GISAID accession no. EPI_ISL_425177) was kindly provided by Darryl Falzarano (Vaccine and Infectious Disease Organization, Saskatoon, Canada). HEK 293T-ACE2 cells were developed by electroporating a plasmid encoding human ACE2 (32) (Addgene plasmid no. 1786; a gift from Hyeryun Choe) into HEK 293T cells. The cells were passaged six times in culture, surface-stained for ACE2 (goat anti-ACE2; AF933-SP; R&D Systems), and the highest 2% of cells expressing ACE2 flow were sorted from the bulk population. Virus culture and experiments were performed according to level 3 containment procedures. Virus stocks were generated and titrated (by plaque assay) in Vero E6 cells. Sendai virus (Cantell strain no. 10100774) was purchased from Charles River.

SARS-CoV-2 protein plasmids were generated from cDNA template prepared from RNA isolated from SARS-CoV-2-infected Vero E6 cells by reverse transcription and PCR. Viral-gene-specific cDNAs, including C-terminal FLAG tag cassettes, were cloned between NheI and FseI restriction sites in pCDNA 3.1(−) 3× FLAG plasmid. Due to the instability of the C-terminally tagged NSP10 construct in bacteria, a 3× FLAG sequence was added in frame to the N-terminal region of the protein. The primers used for cloning are listed in Table 1. All constructs were verified by Sanger sequencing.

For indirect immunofluorescence analysis, the appropriate expression plasmids were transfected into A549 cells or Huh7 cells using TransIT-LT1 (Mirus). For luciferase reporter assays in A549 or HEK 293T cells, plasmid transfection was performed using Lipofectamine 2000 (Invitrogen).

The following antibodies were purchased from the indicated sources: mouse anti-SARS-CoV/SARS-CoV-2 spike antibody (1A9) (GTX632604) from GeneTex; rabbit anti-STAT2 (human, sc‐476), rabbit anti-STAT1 (sc‐346), and mouse anti-Jak1 (sc-1677) from Santa Cruz; mouse anti‐FLAG (F3165), rabbit anti-glutathione S-transferase (GST) (G7781), and mouse anti‐β‐actin (A3853) from Sigma-Aldrich; goat anti-GFP (ab6673), rabbit anti-Tyk2 (ab223733), rabbit anti-IFNAR1 (ab124764), and rabbit anti-TRIM25 (ab167154) from Abcam; rabbit anti-IRF-3 (no. 11904) and rabbit anti-phospho-IRF-3 (no. 4947) from Cell Signaling; mouse horseradish peroxidase (HRP)-conjugated anti-hemagglutinin (HA) (A01244) from GenScript; and mouse anti-V5 (R96025) from Invitrogen. The proteasome inhibitor epoxomicin, the lysosome inhibitor bafilomycin A1, and the translation inhibitor cycloheximide were purchased from Sigma‐Aldrich. High-molecular-weight (HMW) poly(I·C) (no. tlrl-pic) was purchased from InvivoGen.

HEK 293T-ACE2 cells (3 × 10⁵) were seeded into 12-well plates and were infected the next day with SARS-CoV-2 or transfected with appropriate expression plasmids. At designated time points postinfection or posttransfection, cells were washed twice with phosphate-buffered saline (PBS) before being lysed with 2× SDS sample buffer with β-mercaptoethanol (2%). The samples were incubated at 98°C for 10 min to denature proteins, which were then separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes for immunoblotting. The membranes were incubated with blocking solution (5% bovine serum albumin [BSA; Sigma-Aldrich] in PBS–0.05% Tween 20) for 30 min before exposure to primary antibodies diluted in blocking solution for 90 min. Following three washes with PBS–0.05% Tween 20 for 10 min each, the blots were incubated with secondary antibodies in blocking solution for 60 min. The blots were washed three times with PBS–0.05% Tween 20 and once with PBS and then imaged with an Odyssey infrared imaging system. Quantification of proteins was performed using Odyssey Image Studio Lite software version 5.2.

HEK 293T cells were transfected with indicated plasmids using Lipofectamine 2000 (Life Technologies) for 24 h according to the manufacturer’s protocol. The cells were pelleted and resuspended in immunoprecipitation (IP) buffer (150 mM NaCl, 50 mM Tris [pH 7.5], 1% Triton X-100, 1 mM NaF, 1 mM dithiothreitol [DTT], and protease inhibitor cocktail [Roche]). The supernatant was clarified by centrifugation at 16,000 × g for 15 min. Aliquots of the cell lysate were incubated with anti-FLAG M2 magnetic beads (Millipore) or glutathione Sepharose 4B resin (GE Healthcare) at 4°C for 2 h. After washes with IP buffer, SDS sample buffer was added to the beads and boiled, and the proteins were resolved by SDS-PAGE.

Huh7 or A549 cells on coverslips were fixed for 10 min at room temperature with 4% electron microscopy-grade paraformaldehyde (Electron Microscope Sciences) in PBS and permeabilized with 0.5% Triton X-100 in PBS. Following three washes with PBS, the samples were incubated in blocking buffer at room temperature for 30 min. Primary antibodies were added in blocking buffer and incubated at room temperature for 45 min. After three washes in PBS, samples were incubated with secondary antibodies (1:1,000; Invitrogen) in blocking buffer and DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich) (1 μg/ml) for 45 min at room temperature. Coverslips were washed three times with PBS and once with deionized water and mounted on microscope slides using Prolong Gold antifade mounting reagent (Life Technologies). Samples were imaged using an Olympus IX-81 spinning-disk confocal microscope equipped with a 60× PlanApo N oil objective. Images were analyzed using Volocity 6.2.1 software (PerkinElmer).

Total RNA was isolated from cells using the RNA NucleoSpin kit (Macherey Nagel) and reverse transcribed using random primers (Invitrogen) and Improm-II reverse transcriptase (Promega) at 42°C for 1.5 h to generate cDNAs. The cDNAs were diluted 1:5 with water, and 5% volume was mixed with the appropriate primers (Integrated DNA Technologies) and PerfecTa SYBR green SuperMix with low ROX (Quanta Biosciences) and amplified for 40 cycles (30 s at 94°C, 40 s at 55°C, and 20 s at 68°C) in a Bio-Rad CFX96 qRT-PCR machine. The gene targets and primer sequences are listed in Table 2. The cycle threshold (C_T) values were normalized using ACTB mRNA as the internal control. The ΔΔC_T values were determined using control samples as the reference value. Relative levels of mRNAs were calculated using the formula 2^−ΔΔCT.

HEK 293T cells seeded in 12-well plates were transfected with the following promoter reporter (firefly luciferase) constructs: ISG56:pGL3B/561 (gift from Ganes Sen, Lerner Research Institute, Cleveland, OH, USA), IFN‐β:p125‐luc and IRF3:p55‐CIB‐Luc (provided by T. Taniguchi, University of Tokyo, Japan), ISRE:pGL4 ISRE (Promega), NF‐κB:pNF‐kB‐Luc (Stratagene), or GAS:pGAS‐Luc plasmid (Stratagene) together with the Renilla luciferase:pRL‐TK (Promega) construct as a transfection control. At indicated time points, cells were washed once with PBS and then lysed in 200 μl luciferase lysis buffer (0.1% [vol/vol] Triton X-100, 25 mM glycylglycine [pH 7.8], 15 mM MgSO₄, 4 mM EGTA, and 1 mM dithiothreitol), after which the samples were used immediately or stored at −80°C until use. For luciferase assays, the samples were thawed, and 50 μl of each was aliquoted into white 96-well microplates (Greiner Bio-one) in duplicates for both firefly and Renilla luciferase assays. The firefly luciferase substrate d-luciferin (Gold Biotechnology USA) was prepared at a final concentration of 70 μM in luciferase assay buffer (25 mM glycylglycine [pH 7.8], 15 mM K₂PO₄, [pH 7.8], 15 mM MgSO₄, 4 mM EGTA, 1 mM DTT, and 2 mM ATP), and 50 μl was added to each well and incubated in the absence of light for 5 min. Luciferase activity measured using a Synergy HTX plate reader (Biotek). For Renilla luciferase measurements, the substrate coelenterazine (Gold Biotechnology USA) was prepared at a final concentration of 1.4 μM in luciferase assay buffer (25 mM glycylglycine [pH 7.8], 15 mM K₂PO₄, [pH 7.8], 15 mM MgSO₄, and 4 mM EGTA). Fifty microliters was added to each well, and luciferase activity was measured using a Synergy HTX plate reader (Biotek).

Levels of human IFN-β in the cell culture supernatant were measured using a Quantikine human IFN-β immunoassay kit (R&D Systems, Inc.) according to the manufacturer’s instructions. The total fluorescence was measured using a Synergy HTX plate reader (Biotek).

All statistical analyses were performed using GraphPad Prism software. Paired Student's t test was performed for pairwise statistical comparison, while one-way analysis of variance (ANOVA) was used for comparison of multiple samples. The means and standard errors of the means are shown in all bar and line graphs.