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. 2022 Jun 22;96(12):e0041222.
doi: 10.1128/jvi.00412-22. Epub 2022 Jun 2.

SARS-CoV-2 N Protein Antagonizes Stress Granule Assembly and IFN Production by Interacting with G3BPs to Facilitate Viral Replication

Affiliations

SARS-CoV-2 N Protein Antagonizes Stress Granule Assembly and IFN Production by Interacting with G3BPs to Facilitate Viral Replication

Hainan Liu et al. J Virol. .

Abstract

SARS-CoV-2 is the causative agent of the ongoing pandemic of coronavirus disease 2019 (COVID-19) and poses a significant threat to global health. N protein (NP), which is a major pathogenic protein among betacoronaviruses, binds to the viral RNA genome to allow viral genome packaging and viral particle release. Recent studies showed that NP antagonizes interferon (IFN) induction and mediates phase separation. Using live SARS-CoV-2 viruses, this study provides solid evidence showing that SARS-CoV-2 NP associates with G3BP1 and G3BP2 in vitro and in vivo. NPSARS-CoV-2 could efficiently suppress G3BP-mediated SG formation and potentiate viral infection by overcoming G3BP1-mediated antiviral innate immunity. G3BP1 conditional knockout mice (g3bp1fl/fL, Sftpc-Cre) exhibit significantly higher lung viral loads after SARS-CoV-2 infection than wild-type mice. Our findings contribute to the growing body of knowledge regarding the pathogenicity of NPSARS-CoV-2 and provide insight into new therapeutics targeting NPSARS-CoV-2. IMPORTANCE In this study, by in vitro assay and live SARS-CoV-2 virus infection, we provide solid evidence that the SARS-CoV-2 NP associates with G3BP1 and G3BP2 in vitro and in vivo. NPSARS-CoV-2 could efficiently suppress G3BP-mediated SG formation and potentiate viral infection by overcoming antiviral innate immunity mediated by G3BP1 in A549 cell lines and G3BP1 conditional knockout mice (g3bp1-cKO) mice, which provide in-depth evidence showing the mechanism underlying NP-related SARS-CoV-2 pathogenesis through G3BPs.

Keywords: G3BP1; SARS-CoV-2; nucleocapsid protein; stress granule.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
G3BPs interact directly with NPs of betacoronaviruses. (A) Lysates of 293T cells transfected with Flag-tagged NPSARS-CoV were immunoprecipitated with anti-Flag antibody and then subjected to LC-MS/MS analysis to identify the interacting proteins. The protein scores, exclusive spectrum count, and detected protein coverage were analyzed using MaxQuant software. (B to G) Lysates of 293T cells expressing the indicated plasmids were subjected to immunoprecipitation and immunoblot analysis with the indicated antibodies. (H) Lysates of 293T cells transfected with the indicated plasmids were subjected to SDS-PAGE and blotted onto a PVDF membrane. The PVDF membrane was incubated with purified recombinant His-tagged NPSARS-CoV-2 and then detected with anti-His antibody. (I) SPR assays were performed with the indicated purified proteins to detect the direct interactions between G3BP1/G3BP2 and NPSARS-CoV-2. KD values were calculated using software.
FIG 2
FIG 2
Analysis of interactions between G3BPs and NPSARS-CoV-2 using live SARS-CoV-2 virus. (A and C) In situ PLAs were performed using the indicated antibodies to evaluate lung sections from mice (A) and patients (C). The Duolink signals were subsequently visualized in red, and the nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole; blue). (B and D) The relative fluorescence intensity per cell shown in panels A and C was analyzed using ImageJ software (at least 20 cells). All quantitative data are shown as the means ± SDs. **, P < 0.05.
FIG 3
FIG 3
Domains involved in the G3BPs-NPSARS-CoV-2 association. (A) Schematic diagram of the domains of G3BP1 and SARS-CoV-2 NP. IDR, intrinsically disordered region; RBD, RNA-binding domain. (B) 293T cells were cotransfected with NP (SARS-CoV-2) and G3BP1 or truncated mutants, and immunoprecipitation and immunoblotting were then performed with the indicated antibodies. (C and D) 293T cells coexpressing G3BP1 and NPSARS-CoV-2 or truncated mutants were subjected to immunoprecipitation and immunoblotting with the indicated antibodies.
FIG 4
FIG 4
The binding of NPSARS-CoV-2 to G3BPs is partially dependent on the presence of RNA. (A to C) 293T cells were transfected with the indicated plasmids. After RNase A (100 μg/mL) digestion for 30 min at 37°C, the lysates were subjected to immunoprecipitation and immunoblotting using the indicated antibodies.
FIG 5
FIG 5
SARS-CoV-2 NP suppresses G3BP-mediated SG formation. (A) A549 cells were treated with 500 μM sodium arsenite (SA) for 1 h to induce SG assembly, and saline was used as a control. G3BP1 (green) and G3BP2 (red) were labeled to visualize SGs by immunofluorescence staining. The nuclei were stained with DAPI (blue). (B) A549 cells expressing GFP-tagged NPSARS-CoV-2 or GFP-vector were treated with 500 μM SA for 1 h to induce SG assembly. TIA1 (red) and G3BP1 (violet) were labeled to visualize SGs by immunofluorescence staining. The nuclei were stained with DAPI (blue). (C) The average SG number per cell (≥10) in panel B was calculated using ImageJ software. All quantitative data are shown as the means ± SDs. **, P < 0.05. (D) A549 cells transfected with or without a G3BP1/G3BP2 plasmid were infected with SARS-CoV-2 for 24 h and treated with or without 500 μM SA during the last hour. NPSARS-CoV-2 (green) and the SG marker G3BP1 (red) were detected via immunofluorescence staining. The nuclei were stained with DAPI (blue). (E) The average SG number per cell (≥10) in panel D was calculated using ImageJ software. All quantitative data are shown as the means ± SDs. **, P < 0.05.
FIG 6
FIG 6
SARS-CoV-2 NP suppresses innate immunity by inhibiting G3BP1. (A) A549 cells cotransfected with the indicated plasmids were treated with or without 2 μg/mL poly(I·C) for 18 h, and immunoblot analysis was then performed with the indicated antibodies. (B) A549 cells cotransfected with the indicated plasmids were treated with or without 2.5 μg/mL poly(I·C) for 18 h. mRNA from the indicated cells was extracted to determine the mRNA transcript level of IFN-β via RT-PCR. All quantitative data are shown as the means ± SDs of three independent experiments. **, P < 0.05. (C and D) The subcellular localization of endogenous G3BP1 (green) and SARS-CoV-2 dsRNA (red) in wild-type G3BP1-knockdown A549 cells was detected by immunofluorescence staining using the indicated antibodies. The nuclei were stained with DAPI (blue). (E) The number of red spots in at least 10 cells shown in panel D was calculated, and the G3BP1-knockdown efficiency is shown. (F) RNA levels of SARS-CoV-2 in wild-type and G3BP1-knockdown A549 cells were analyzed by RT-PCR after 48 h of SARS-CoV-2 infection. All quantitative data are shown as the means ± SDs from three experiments. **, P < 0.05. (G) The RNA levels of SARS-CoV-2 in wild-type, G3BP1-knockout, and G3BP1-rescued A549 cells (right) were analyzed by RT-PCR after 48 h of SARS-CoV-2 infection. The G3BP1-knockout efficiency is shown. All quantitative data are shown as the means ± SDs from three experiments. **, P < 0.05. (H) WT mice (aged 8 to 10 weeks) and c-g3bp1-cKO mice (4 in each group) were intranasally infected with SARS-CoV-2 at 0.1 50% lethal dose (LD50), and the viral loads in the lungs of the infected mice were detected via RT-PCR. All quantitative data are shown as the means ± SDs (unpaired Student's t test). **, P < 0.01.

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