Translational shutdown and evasion of the innate immune response by SARS-CoV-2 NSP14 protein

Figures

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  Fig. 1.. SARS-CoV-2 infection inhibits cellular translation. (A) Vero E6 cells were infected with SARS-CoV-2 at the indicated MOIs. After 24 h of infection, cells were pulse labeled with puromycin for 15 min. Puromycin incorporation was determined by immunoblotting using anti-puromycin antibody (Puro). Expression of SARS-CoV-2 viral protein was determined using anti-SARS-CoV ORF3a antibody (ORF3a). (B) Confocal images of Vero E6 cells infected with recombinant SARS-CoV-2 expressing mNeonGreen (CoV-2-mNG) (22) at an MOI of 0.5. After 24 h of infection, cells were pulse labeled with OP-Puro for 1 h, fixed, fluorescently labeled by the Click chemistry reaction, and stained by Hoechst. (Scale bars, 10 µm.)

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  Fig. 2.. SARS-CoV-2 NSP14 inhibits cellular translation. (A) The 293T cells were transfected with plasmids encoding SARS-CoV-2 viral proteins. After 24 h of transfection, cells were pulse labeled with OP-Puro for 1 h, fixed, fluorescently labeled by the Click chemistry reaction, and analyzed by fluorescence-activated cell sorter (FACS). EV, empty vector. (B) The 293T cells were transfected with plasmids encoding indicated viral proteins for 24 h and puromycin labeled for 15 min. Puromycin incorporation was determined by immunoblotting using anti-puromycin antibody. (C) The 293T cells transfected and OP-Puro labeled as shown in A. Cells were stained by Hoechst and analyzed by confocal microscopy. (Scale bars, 50 µm.) (D) The 293T cells were transfected with plasmids encoding indicated viral proteins for 24 h. Cell lysates were cleared by centrifugation, loaded onto a 15 to 50% sucrose gradient, and subjected to ultracentrifugation. Absorbance was monitored at 254 nm to record the polysome profile. The monosome and polysome pools are indicated. (E) Quantification of polysome profile assay. Data are represented as the polysome-to-monosome (P/M) ratio. For A, B, and E, data are shown as mean…

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  Fig. 3.. Human coronavirus NSP14 proteins inhibit cellular translation. (A) Comparison of the amino acid sequences of coronavirus NSP14. Sequence similarity of NSP14 between avian gammacoronavirus (IBV), human alphacoronavirus (HCoV-229E; 229E), and human betacoronaviruses (MERS-CoV; MERS, SARS-CoV; CoV-1, SARS-CoV-2; CoV-2). (B) The 293T cells were transfected with plasmids encoding different HA-tagged NSP14. After 24 h of transfection, cells were pulse labeled with OP-Puro for 1 h, fixed, fluorescently labeled by the Click chemistry reaction, and analyzed by FACS. (C) The 293T cells were transfected for 24 h and puromycin labeled for 15 min. Puromycin incorporation was determined by immunoblotting using anti-puromycin antibody (Puro). HA-tagged NSP14 proteins were detected by anti-HA antibody (HA). (D) Quantification of puromycin incorporation assay shown in C. For B and D, data are shown as mean ± SD of three biological repeats. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student’s t test; ns, not significant.

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  Fig. 4.. ExoN and N7-MTase are required for translation inhibition activity of NSP14. (A) Crystal structure of SARS-CoV NSP10−NSP14 complex (Protein Data Bank ID code 5NFY). NSP10 is shown in gray. N-terminal ExoN and C-terminal N7-MTase domains are in blue and green, respectively. NSP14 mutants are described below, in the table. The mutation residues are highlighted in the structure. (B) The 293T cells were transfected with plasmids encoding WT or NSP14 mutants for 24 h and puromycin labeled for 15 min. Puromycin incorporation was determined by immunoblotting using anti-puromycin antibody (Puro). HA-tagged NSP14 proteins were detected by anti-HA antibody (HA). (C) Quantification of puromycin incorporation assay shown in B. (D) The 293T cells were transfected with plasmids encoding WT or NSP14 mutants. After 24 h of transfection, cells were pulse labeled with OP-Puro for 1 h, fixed, fluorescently labeled, and analyzed by FACS. (E) The 293T cells were transfected with plasmids encoding NSP14 or M2 mutant for 24 h. Cell lysates were cleared by centrifugation, loaded onto a 15 to 50% sucrose gradient, and subjected to ultracentrifugation. Absorbance was monitored at 254 nm to record the…

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  Fig. 5.. NSP14−NSP10 complex formation enhances the translation inhibition activity of NSP14. (A) The 293T cells were transfected with plasmids encoding indicated proteins for 24 h. Cell lysates were subjected to IP using mouse anti-HA antibody and followed by immunoblotting with rabbit anti-HA and anti-FLAG antibodies. WCE, whole cell extract. (B) The 293T cells were transfected with plasmids encoding NSP10 or NSP14 for 24 h and puromycin labeled for 15 min. Puromycin incorporation was determined by immunoblotting using anti-puromycin antibody (Puro). HA-tagged NSP14 and FLAG-tagged NSP10 proteins were detected by anti-HA and anti-FLAG antibodies, respectively. (C) Quantification of puromycin incorporation assay shown in B. (D) The 293T cells were transfected for 24 h and puromycin labeled for 15 min. Puromycin incorporation was determined by immunoblotting. (E) The 293T cells were cotransfected with HA-tagged NSP14 and FLAG-tagged NSP10 or its mutants for 24 h. Cell lysates were subjected to IP using mouse anti-HA antibody and followed by immunoblotting with rabbit anti-HA and anti-FLAG antibodies. (F) The 293T cells were transfected for 24 h and puromycin labeled for 15 min. Puromycin…

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  Fig. 6.. NSP14 inhibits the protein expression of ISGs. (A) Vero E6 cells were transfected with plasmids encoding NSP1 or NSP14 for 24 h and treated with IFN-I for 18 h. The expression of IFN-stimulated proteins was determined by immunoblotting with antibodies against depicted proteins. (B) The induction of ISGs on mRNA level from A were determined by qRT-PCR with primers against genes encoding RIG-I and viperin proteins (RSAD2). (C) The 293T cells were transfected with HA-tagged NSP14 for 24 h and treated with IFN-I for 18 h. The induction of ISGs was determined by immunoblotting with antibodies against depicted proteins. Asterisk (*) indicates nonspecific protein bands. (D) The 293T cells were transfected with plasmids encoding NSP1 or NSP14 for 24 h and treated with IFN-I for 18 h. The expression of IFN-stimulated proteins of ISGs was determined by immunoblotting with antibodies against depicted proteins. For B, data are shown as mean ± SD of six biological repeats by unpaired Student’s t test.