SARS-CoV-2 Nucleocapsid Protein Targets RIG-I-Like Receptor Pathways to Inhibit the Induction of Interferon Response

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) that has resulted in the current pandemic. The lack of highly efficacious antiviral drugs that can manage this ongoing global emergency gives urgency to establishing a comprehensive understanding of the molecular pathogenesis of SARS-CoV-2. We characterized the role of the nucleocapsid protein (N) of SARS-CoV-2 in modulating antiviral immunity. Overexpression of SARS-CoV-2 N resulted in the attenuation of retinoic acid inducible gene-I (RIG-I)-like receptor-mediated interferon (IFN) production and IFN-induced gene expression. Similar to the SARS-CoV-1 N protein, SARS-CoV-2 N suppressed the interaction between tripartate motif protein 25 (TRIM25) and RIG-I. Furthermore, SARS-CoV-2 N inhibited polyinosinic: polycytidylic acid [poly(I:C)]-mediated IFN signaling at the level of Tank-binding kinase 1 (TBK1) and interfered with the association between TBK1 and interferon regulatory factor 3 (IRF3), subsequently preventing the nuclear translocation of IRF3. We further found that both type I and III IFN production induced by either the influenza virus lacking the nonstructural protein 1 or the Zika virus were suppressed by the SARS-CoV-2 N protein. Our findings provide insights into the molecular function of the SARS-CoV-2 N protein with respect to counteracting the host antiviral immune response.

Keywords: RIG-I like receptors; SARS-CoV-2 N protein; antiviral immune response; coronavirus disease 2019; interferon.

Figure 1

SARS-CoV-2 N is a potent interferon antagonist. (A) Schematic view of SARS-CoV-2 genome is shown. (B) HeLa cells were transfected with SARS-CoV-2 N-GFP-encoding and mito-dsRED plasmids. Scale bar = 10 μm. (C) HeLa and A549 cells transfected with increasing amounts of SARS-CoV-2 N-GFP-encoding plasmids were immunoblotted with with the indicated antibodies to determine transfection efficiency. (D,E) HEK293T cells expressing RIG-IC, or IRF3 (5D) were co-transfected with firefly luciferase reporter plasmids encoding IFN-β, IFN-λ1, ISRE, IP-10, and NF-κB promoter together with increasing amount of SARS-CoV-2 N. The EV-GFP plasmid was used to ensure that each sample contains the same amount of total DNA plasmids. * p < 0.05; ** p < 0.01; *** p < 0.001, non-significant (n.s) versus EV-GFP-transfected cells (F) Recombinant IFN-α (50 ng/mL) or IFN-β (50 ng/mL) protein was added to the cells following the transfection with 100 ng of EV-GFP (EV), SARS-CoV-2 N (N) or ZIKV NS5 (NS5) encoding plasmids. Mean ± SD of three technical replicates are shown. *** p < 0.001 versus EV-GFP-transfected cells. (G) Immunoblot results of IFN-β-induced phosphorylation of STAT1 are shown. The images are representative of three independent experiments. Numbers indicate quantitative densitometric analyses of blot using ImageJ software.

Figure 2

SARS-CoV-2 N interacts with genes involved in the RLR signaling pathway and interferes RIG-I interaction with TRIM25. (A) HeLa cells were transiently transfected with the following plasmids: RIG-I-FLAG, RIG-IC-FLAG, MDA5-HIS and MAVS-MYC along with SARS-CoV-2 N-GFP-encoding plasmids. Cells were fixed in paraformaldehyde and permeabilized with 0.1% Triton X-100. The images are representative of three independent experiments. Scale bar = 20 μm. (B) HEK293T cells in 6-well plates were transfected with RIG-I-FLAG or MAVS-MYC plasmids along with empty vector with GFP tag (EV-GFP) or SARS-CoV-2 N-GFP (N-GFP) plasmids. After 24 h, cells were lysed and immunoprecipitated with indicated antibodies. Whole cell lysates (WCL) and immunoprecipitates were analyzed by immunoblot analysis. (C) HEK293T cells transfected with plasmids encoding RIG-I-FLAG or TRIM25-HA along with EV-GFP or N-GFP-encoding plasmids were lysed and subjected to immunoprecipitation with anti-FLAG tag antibody. The cell lysates and immunoprecipitates were analyzed by immunoblot analysis using indicated antibodies.

Figure 3

SARS-CoV-2 N inhibits the activation of IFN production and signaling in response to poly I:C treatment. (A) Luciferase activities of A549 cells transfected with 100 ng of EV-GFP (EV) or SARS-CoV-2 N-GFP (N) plasmids along with IFN-β, IFN-λ1, IFN-λ2, ISRE, and IP-10 promoter luciferase plasmids for 24 h and treated with poly (I:C) (10 µg/mL) for 6 h (mean ± SD, n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001 versus EV-GFP-transfected cells. (B) A549 cells expressing either EV or N were transfected with poly (I:C) (10 µg/mL) for 6 h. RT-qPCR analysis of ISG15, OAS1, and IFN-β mRNAs is shown (mean ± SD, n = 3). * p < 0.05; ** p < 0.01 versus EV-GFP-transfected cells.(C) A549 cells were co-transfected with IRF3-FLAG and EV-GFP or N-GFP plasmids. After 24 h, cells were treated with poly I:C (10 µg/mL) for 2 h and fixed to observe the subcellular localization of IRF3. Scale bar = 5 μm. (D) A549 cells were transiently transfected with EV-GFP or N-GFP plasmids. After 24 h, cells were transfected with poly (I:C) (10 µg/mL) for 2 h. Cell lysates were collected for immunoblotting with the indicated antibodies. Numbers indicate quantitative densitometric analyses of blot using ImageJ software. (E) HEK293T cells were transfected with plasmids encoding IRF3-FLAG and TBK1-MYC along with EV-GFP or N-GFP for 24 h. Cell lysates were subjected to immunoprecipitation with an anti-FLAG tag antibody, followed by immunoblot analysis with indicated antibodies.

Figure 4

SARS-CoV-2 N markedly attenuates RNA virus-triggered IFN production. (A,B) Luciferase activities of A549 cells transfected with IFN-β, IFN-λ1, IFN-λ2, ISRE, and IP-10 promoter luciferase plasmids together with expression vector for RIG-I, along with 100 ng of EV-GFP (EV) or SARS-CoV-2 N-GFP (N) expression plasmids. At 24 h after transfection, the cells were infected with IFV-delNS1 (IFV-dNS1) (A) or ZIKV (MR766) (B) at multiplicity of infection (MOI) = 1 for 6 h (mean ± SD, n = 3). ** p < 0.01; *** p < 0.001 versus EV-GFP-transfected cells. (C) A549 cells were transiently transfected with EV-GFP or SARS-CoV-2 N-GFP expression plasmids. After 24 h, cells were infected with mock (m), ZIKV (MR766), or IFV-delNS1 (MOI = 1). (D) RT-qPCR reveals mRNA levels of MxA, IFN-β, ISG15, and OAS1 following viral infection. * p < 0.05; ** p < 0.01; *** p < 0.001 versus EV-GFP-transfected cells. (E) IFN-λ1/3 secretion levels were measured by ELISA assays. ** p < 0.01 versus EV-GFP-transfected cells. (F) Schematic representation of how SARS-CoV-2 N protein interferes with IFN induction and signaling.