SARS-CoV-2 nucleocapsid protein impairs stress granule formation to promote viral replication

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  Fig. 1. SARS-CoV-2 RNA but not SARS-CoV-2 infection induces SG formation. a Induction of SGs by arsenite, transfection of poly(I:C) and SARS-CoV-2 RNA. HeLa cells were treated with sodium arsenite (1 mM) for 1 h or transfected with poly(I:C) (2 μg) or SARS-CoV-2 RNA (1 μg) for 10 h. Cells were immunostained for TIA-1 (green) and G3BP1 (red). Nuclei were stained with DAPI (blue). The cells were observed with a Nikon confocal microscope under a 60× oil objective. b SGs were not induced by SARS-CoV-2 infection. HeLa-ACE2 cells were infected with SARS-CoV-2 (MOI = 1) for indicated times, and then untreated or treated with sodium arsenite (1 mM) for 1 h. Cells were immunostained for TIA-1 (green) and G3BP1 (red). Nuclei were stained with DAPI (blue). The cells were observed with a Nikon confocal microscope under a 60× oil objective. c Transfection of poly(I:C) and SARS-CoV-2 RNA and arsenite treatment induced PKR and eIF2α phosphorylation. HeLa cells were treated with sodium arsenite (1 mM) for 1 h or transfected with poly(I:C) (3 μg) or SARS-CoV-2 RNA (2 μg) for 10 h before immunoblot analysis with the indicated antibodies. d SARS-CoV-2 infection did not induce PKR and eIF2α phosphorylation.…

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  Fig. 2. Knockdown of PKR and G3BP1 facilitates viral replication. a, b Deficiency of PKR or G3BP1 promotes the transcription of SARS-CoV-2 genes. HeLa-ACE2 cells were transfected with siRNAs specific for PKR (siPKR) or G3BP1 (siG3BP1) or control siRNA (siNC) for 48 h. The cells were then infected with SARS-CoV-2 (MOI = 0.01) for 24 h before qPCR analysis with the indicated primers. c, d Knockdown of PKR or G3BP1 promotes SARS-CoV-2 replication. HeLa-ACE2 cells were transfected with siRNAs specific for PKR (siPKR) or G3BP1 (siG3BP1) or control siRNA (siNC) for 48 h. The cells were then infected with SARS-CoV-2 (MOI = 0.01) for 24 h. Virus yield in the infected cell supernatants was quantified by qPCR analysis with primers based on RBD of SARS-CoV-2 S gene (c), and NP expression in infected cells was analyzed by immunoblot with the indicated antibodies (d). Graphs show means ± SD, n = 3. *P < 0.05, **P < 0.01 (Student’s t-test).

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  Fig. 3. SARS-CoV-2 NP inhibits SG formation. a Effects of SARS-CoV-2 proteins on poly(I:C)-induced SG assembly. HeLa cells were transfected with plasmids encoding the indicated Flag-tagged SARS-CoV-2 proteins for 12 h, then transfected with poly(I:C) (2 μg) for 10 h. Cells were immunostained for Flag (green) and G3BP1 (red). Nuclei were stained with DAPI (blue). The cells were observed with a Nikon confocal microscope under a 60× oil objective. b Quantitative analysis of the cells (in a) with G3BP1 foci. The percentage of cells containing SGs was quantified and at least 100 cells were counted each time. c SARS-CoV-2 NP impairs poly(I:C)-, SARS-CoV-2 RNA-, and sodium arsenite-induced SG assembly. HeLa cells were transfected with SARS-CoV-2 NP-Flag plasmid for 12 h, then transfected with poly(I:C) (2 μg) or SARS-CoV-2 RNA (1 μg) for 10 h or treated with sodium arsenite (1 mM) for 1 h. Cells were immunostained for Flag (green) and G3BP1 (red). Nuclei were stained with DAPI (blue). The cells were observed with a Nikon confocal microscope under a 60× oil objective. “+” indicates the cells expressing NP. Graphs show means ± SD, n = 3. *P < 0.05 (Student’s t-test).

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  Fig. 4. SARS-CoV-2 NP inhibits SG formation by targeting PKR and G3BP1. a SARS-CoV-2 NP interacts with PKR in mammalian overexpression system. HEK293T cells (5 × 106) were transfected with the indicated plasmids for 24 h. The cell lysates were added with poly(I:C) (8 μg) or left untreated. Co-immunoprecipitation and immunoblot analysis were performed with the indicated antibodies. b Association of SARS-CoV-2 NP with endogenous PKR. HEK293T cells (5 × 106) were transfected with SARS-CoV-2 NP-HA plasmid (10 μg) for 24 h. Cell lysates were left untreated or mixed with poly(I:C) (8 μg) or treated by RNase A. Co-immunoprecipitation and immunoblot analysis were performed with the indicated antibodies. c Effects of SARS-CoV-2 NP on poly(I:C)-induced PKR oligomerization and phosphorylation of PKR and eIF2α. HEK293T cells (5 × 106) were transfected with control or SARS-CoV-2 NP-Flag plasmid (10 μg) for 24 h, and then further transfected with poly(I:C) (8 μg) for another 4 h. Cell lysates were separated by native- or SDS–PAGE as indicated and analyzed by immunoblot with the indicated antibodies. d Effects of SARS-CoV-2 NP on sodium arsenite-induced phosphorylation of eIF2α. HEK293T cells were…

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  Fig. 5. Inhibition of SG formation by NPs of three coronaviruses. a The amino acid sequences of NPs of SARS-CoV, SARS-CoV-2, and MERS-CoV were aligned by MAFFT (version 7.4.71) and visualized in ESPrit (version 3.0)52. The conserved amino acids are highlighted. Residues highlighted in red are fully conserved and those in yellow are not conserved. Residues highlighted in blue are conserved between SARS-CoV-2 and SARS-CoV, but not MERS-CoV. b The NPs of SARS-CoV-2, SARS-CoV, and MERS-CoV associate with endogenous PKR. HEK293T cells (5 × 106) were transfected with the indicated plasmids for 24 h. The lysates were left untreated or added with poly(I:C) (8 μg) or treated by RNase A. Co-immunoprecipitation and immunoblot analysis were performed with the indicated antibodies. c The NPs of SARS-CoV-2, SARS-CoV, and MERS-CoV inhibit poly(I:C)-induced phosphorylation of PKR and eIF2α. HEK293T cells were transfected with the indicated plasmids for 12 h and then further transfected with poly(I:C) (2 μg) for 10 h before immunoblotting analysis with the indicated antibodies. d The NPs of SARS-CoV-2 and SARS-CoV, but not MERS-CoV associate with endogenous G3BP1. HEK293T cells (5 × 106) were transfected…

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  Fig. 6. A model on inhibition of SG formation by the NPs of coronaviruses. After coronavirus infection, the viral dsRNA binds to PKR, facilitating dimerization/oligomerization and autophosphorylation of PKR. The activated PKR catalyzes the phosphorylation of eIF2α, leading to the recruitment of SG-nucleating proteins, such as TIA-1 and G3BP1, and eventually SGs assembly. The NPs of SARS-CoV-2, SARS-CoV, and MERS-CoV can inhibit PKR phosphorylation, leading to the impairment of SG formation. The NPs of SARS-CoV-2 and SARS-CoV, but not MERS-CoV can also sequester G3BP1 to impair G3BP1-mediated SG assembly.