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. 2023 Jun 15;11(3):e0099423.
doi: 10.1128/spectrum.00994-23. Epub 2023 May 8.

The Nucleocapsid Proteins of SARS-CoV-2 and Its Close Relative Bat Coronavirus RaTG13 Are Capable of Inhibiting PKR- and RNase L-Mediated Antiviral Pathways

Kyle LeBlanc #  1 , Jessie Lynch #  1 , Christine Layne  1 , Robert Vendramelli  2 , Angela Sloan  2 , Nikesh Tailor  2 , Yvon Deschambault  2 , Fushun Zhang  3 , Darwyn Kobasa  2 , David Safronetz  2 , Yan Xiang  3 , Jingxin Cao  1
Affiliations

The Nucleocapsid Proteins of SARS-CoV-2 and Its Close Relative Bat Coronavirus RaTG13 Are Capable of Inhibiting PKR- and RNase L-Mediated Antiviral Pathways

Kyle LeBlanc et al. Microbiol Spectr. .

Abstract

Coronaviruses (CoVs), including severe acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2, produce double-stranded RNA (dsRNA) that activates antiviral pathways such as PKR and OAS/RNase L. To successfully replicate in hosts, viruses must evade such antiviral pathways. Currently, the mechanism of how SARS-CoV-2 antagonizes dsRNA-activated antiviral pathways is unknown. In this study, we demonstrate that the SARS-CoV-2 nucleocapsid (N) protein, the most abundant viral structural protein, is capable of binding to dsRNA and phosphorylated PKR, inhibiting both the PKR and OAS/RNase L pathways. The N protein of the bat coronavirus (bat-CoV) RaTG13, the closest relative of SARS-CoV-2, has a similar ability to inhibit the human PKR and RNase L antiviral pathways. Via mutagenic analysis, we found that the C-terminal domain (CTD) of the N protein is sufficient for binding dsRNA and inhibiting RNase L activity. Interestingly, while the CTD is also sufficient for binding phosphorylated PKR, the inhibition of PKR antiviral activity requires not only the CTD but also the central linker region (LKR). Thus, our findings demonstrate that the SARS-CoV-2 N protein is capable of antagonizing the two critical antiviral pathways activated by viral dsRNA and that its inhibition of PKR activities requires more than dsRNA binding mediated by the CTD. IMPORTANCE The high transmissibility of SARS-CoV-2 is an important viral factor defining the coronavirus disease 2019 (COVID-19) pandemic. To transmit efficiently, SARS-CoV-2 must be capable of disarming the innate immune response of its host efficiently. Here, we describe that the nucleocapsid protein of SARS-CoV-2 is capable of inhibiting two critical innate antiviral pathways, PKR and OAS/RNase L. Moreover, the counterpart of the closest animal coronavirus relative of SARS-CoV-2, bat-CoV RaTG13, can also inhibit human PKR and OAS/RNase L antiviral activities. Thus, the importance of our discovery for understanding the COVID-19 pandemic is 2-fold. First, the ability of SARS-CoV-2 N to inhibit innate antiviral activity is likely a factor contributing to the transmissibility and pathogenicity of the virus. Second, the bat relative of SARS-CoV-2 has the capacity to inhibit human innate immunity, which thus likely contributed to the establishment of infection in humans. The findings described in this study are valuable for developing novel antivirals and vaccines.

Keywords: G3BP1; PKR; RNase L; RaTG13; SARS-CoV-2 nucleocapsid; double-stranded RNA virus; vaccinia virus E3.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
SARS-CoV-2 replication produces dsRNA and does not activate PKR and OAS/RNase L pathways. (A) Immunostaining of dsRNA in SARS-CoV-infected A549/huACE2 cells. Cells were infected with SARS-CoV-2 at an MOI of 0.5, incubated for 24 h, fixed with PBS-buffered formalin, and stained with a dsRNA-specific antibody (J2), antibody for the N protein, and DAPI for the nucleus. The images were taken at a ×40 magnification. The data are representative of results from two independent experiments. (B) Status of PKR and eIF2α phosphorylation. A549/huACE2 cells were infected with SARS-COV-2 at an MOI of 0.5 or VACVwt/VACVΔE3ΔK3 at an MOI of 5. Cell lysates were collected at 24 hpi for SARS-CoV-2-infected cells and 10 hpi for VACV-infected cells. SARS-CoV-2 N and VACV D12 Abs were used as controls for infection. The data are representative of results from three independent experiments. (C) RNA electrophoresis. Cells were infected as described above for panel B. The total RNA was collected for electrophoresis. The integrity of the rRNA is shown as an indicator of RNA degradation. The data are representative of results from two independent experiments. (D) SARS-CoV-2 yield. A549/huACE2 cells were infected with SARS-CoV-2 at an MOI of 0.5, and the supernatant was collected at 1 and 24 hpi for quantification of the virus by plaque assays on Vero cells. The data are representative of results from two independent experiments.
FIG 2
FIG 2
SARS-CoV-2 N binds to dsRNA and inhibits PKR and OAS/RNase L. (A) Schematic illustration of the construction of recombinant VACVΔE3ΔK3 expressing SARS-CoV-2 N. The SARS-CoV-2 N gene tagged at the C terminus with FLAG and driven by the VACV early/late promoter mH5 was inserted into the K3L locus to replace the red fluorescent protein marker of VACVΔE3ΔK3. Infection with VACVΔE3ΔK3 and transfection with the recombinant vector were done in A549/dko cells, and the selection and purification of the recombinant were done in HeLa cells. (B) Confirmation of the expression of SARS-CoV-2 N. Western blot analysis was used to confirm the expression of the SARS-CoV-2 N protein using a FLAG Ab. The cell lysate was prepared using A549/dko cells. D12 is a VACV early protein and was used as an infection control. VACVwt, wild-type VACV; ΔE3ΔK3, VACVΔE3ΔK3; E3, VACVΔK3 expressing VACV E3; N, VACVΔE3ΔK3 expressing SARS-CoV-2 N. (C) Replication of VACVΔE3ΔK3 expressing SARS-CoV-2 N. A549/PKRko, A549/RNaseLko, and wild-type A549 cells were infected with the viruses (shown in panel B) at an MOI of 5. The differences in the virus titers at 48 hpi and 5 hpi indicate the virus yields. The data are representative of results from three independent experiments. (D) dsRNA binding assay by poly(I·C) bead pulldown. BHK21 cells were infected with VACVΔE3 and transfected with the recombinant vectors shown in panel A. E3, VACV E3-expressing vector; E3-167, E3/K167A mutant; N, SARS-CoV-2 N. The protein complex precipitated with poly(I·C) beads was analyzed using Western blotting with FLAG and E3 antibodies. The data are representative of results from three independent experiments. (E) Status of PKR phosphorylation. A549/RNaseLko cells were infected with wild-type VACV (VACVwt), VACVΔE3ΔK3 (ΔE3ΔK3), VACVΔK3 expressing VACV E3 (E3), and VACVΔE3ΔK3 expressing SARS-CoV-2 N (N) at an MOI of 5 for 8 h. The cell lysate was analyzed using Western blotting with Abs for PKR (both phosphorylated and nonphosphorylated), p-PKR (phosphorylated PKR), p-eIF2α (eIF2α phosphorylated at S51), the VACV early/late protein D12, and actin. The data are representative of results from three independent experiments. (F) RNA electrophoresis. A549/PKRko cells were infected with wild-type VACV, VACVΔE3ΔK3, VACVΔK3 expressing VACV E3, and VACVΔE3ΔK3 expressing SARS-CoV-2 N at an MOI of 5. The total RNA collected at 12 hpi was analyzed by gel electrophoresis. The data are representative of results from two independent experiments.
FIG 3
FIG 3
dsRNA binding and inhibition of PKR and OAS/RNase L by SARS-CoV-2 truncated mutants. (A) Schematic illustration of the construction of recombinant VACVΔE3ΔK3 expressing SARS-CoV-2 N truncated mutants. All of the truncated mutants were tagged with FLAG at their C termini and inserted into VACVΔE3ΔK3 at the A45R locus with the swinepox virus K3L ortholog SPV010 as a selection marker. The numbers under full-length N indicate the amino acids. Infection with VACVΔE3ΔK3 and transfection with the recombinant vector were done in A549/dko cells, and the selection and purification of the recombinant were done in PK15 pig cells. (B) Confirmation of the expression of the SARS-CoV-2 N truncated mutants. Western blotting was used to confirm the expression of all truncated N proteins as described in the legend of Fig. 2B. (C) Replication of VACVΔE3ΔK3 expressing the truncated SARS-CoV-2 N mutants. Infection and titration were performed as described in the legend of Fig. 2C. (D) dsRNA binding of the truncated N proteins by poly(I·C) bead pulldown. The assay was performed as described in the legend of Fig. 2D. (E) The status of PKR phosphorylation in cells infected by VACVΔE3ΔK3 expressing the N truncated mutants was examined as described in the legend of Fig. 2E. (F) RNA electrophoresis. The OAS/RNase L activity in cells infected by the N truncated mutants was examined as described in the legend of Fig. 2F.
FIG 4
FIG 4
dsRNA binding and inhibition of PKR and OAS/RNase L by SARS-CoV-2 N domain mutants. (A) Schematic illustration of the construction of recombinant VACVΔE3ΔK3 expressing the SARS-CoV-2 N domain mutants. Only the NTD, LKR+CTD, and CTD mutants are shown since no protein was detected for the other domain mutants (C/IDR, LKR, and N/IDR). The construction of the recombinants expressing the N domain mutants was the same as the procedure described in the legend of Fig. 3A. (B) Confirmation of the expression of the SARS-CoV-2 N domain mutants. Western blotting was used to confirm the expression of the N domain mutant proteins as described in the legend of Fig. 2B. (C) Replication of VACVΔE3ΔK3 expressing the SARS-CoV-2 N domain mutants. Infection and titration were done as described in the legend of Fig. 2C. (D) dsRNA binding of the N domain mutant proteins by poly(I·C) bead pulldown. The assay was performed as described in the legend of Fig. 2D. (E) The status of PKR phosphorylation in cells infected by VACVΔE3ΔK3 expressing the N domain mutants was examined as described in the legend of Fig. 2E. (F) RNA electrophoresis. The OAS/RNase L activity in cells infected by the N domain mutants was examined as described in the legend of Fig. 2F.
FIG 5
FIG 5
dsRNA binding and inhibition of PKR and OAS/RNase L by bat-CoV RaTG13 N domain mutants. (A) Schematic illustration of the construction of recombinant VACVΔE3ΔK3 expressing the bat-CoV RaTG13 N protein. The bat-CoV RaTG13 N gene tagged with a FLAG tag at the C terminus and driven by the VACV early/late promoter mH5 was inserted into VACVΔE3ΔK3 as shown in Fig. 3A. (B) Confirmation of the expression of the bat-CoV RaTG13 N protein using Western blotting as described in the legend of Fig. 2B. (C) Replication of VACVΔE3ΔK3 expressing the bat-CoV RaTG13 N protein. Infection and titration were done as described in the legend of Fig. 2C. (D) dsRNA binding of the bat-CoV RaTG13 N protein by poly(I·C) bead pulldown. The assay was performed as described in the legend of Fig. 2D. (E) The status of PKR phosphorylation in cells infected by VACVΔE3ΔK3 expressing the bat-CoV RaTG13 N protein was examined as described in the legend of Fig. 2E. (F) RNA electrophoresis. The OAS/RNase L activity in cells infected by bat-CoV RaTG13 N was examined as described in the legend of Fig. 2F.
FIG 6
FIG 6
Coimmunoprecipitation of the SARS-CoV-2 N protein and its truncated and domain mutants with PKR and G3BP1. (A) Total PKR and phosphorylated PKR in the cell lysates from uninfected and VACVΔE3ΔK3-infected A549 cells. The cell lysates from uninfected and VACVΔE3ΔK3-infected A549 cells were checked with antibodies for total PKR (left) and PKR phosphorylated at T446 (right). (B) Immunoprecipitation of SARS-CoV N and its truncated mutant proteins. Cell lysates containing SARS-CoV-2 N and its truncated mutant protein were incubated with cell lysates from uninfected or VACVΔE3ΔK3-infected A549 cells (shown in panel A) and precipitated with FLAG antibody and protein G beads. The protein complexes were analyzed using Western blotting with antibodies for the FLAG tag, total PKR, phosphorylated PKR (p-PKR), and G3BP1. The data are representative of results from three independent experiments; CC, cell control. (C) Immunoprecipitation of SARS-CoV N and its domain mutant proteins. The protein complexes containing SARS-CoV N, its domain mutant proteins, and their binding partners were prepared and analyzed as described above for panel B. The data are representative of results from three independent experiments.
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References

    1. Wu F, Zhao S, Yu B, Chen Y-M, Wang W, Song Z-G, Hu Y, Tao Z-W, Tian J-H, Pei Y-Y, Yuan M-L, Zhang Y-L, Dai F-H, Liu Y, Wang Q-M, Zheng J-J, Xu L, Holmes EC, Zhang Y-Z. 2020. A new coronavirus associated with human respiratory disease in China. Nature 579:265–269. doi:10.1038/s41586-020-2008-3. - DOI - PMC - PubMed
    1. Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. 2020. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 5:536–544. doi:10.1038/s41564-020-0695-z. - DOI - PMC - PubMed
    1. Fung TS, Liu DX. 2019. Human coronavirus: host-pathogen interaction. Annu Rev Microbiol 73:529–557. doi:10.1146/annurev-micro-020518-115759. - DOI - PubMed
    1. Holmes EC, Goldstein SA, Rasmussen AL, Robertson DL, Crits-Christoph A, Wertheim JO, Anthony SJ, Barclay WS, Boni MF, Doherty PC, Farrar J, Geoghegan JL, Jiang X, Leibowitz JL, Neil SJD, Skern T, Weiss SR, Worobey M, Andersen KG, Garry RF, Rambaut A. 2021. The origins of SARS-CoV-2: a critical review. Cell 184:4848–4856. doi:10.1016/j.cell.2021.08.017. - DOI - PMC - PubMed
    1. Lytras S, Xia W, Hughes J, Jiang X, Robertson DL. 2021. The animal origin of SARS-CoV-2. Science 373:968–970. doi:10.1126/science.abh0117. - DOI - PubMed

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