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

SARS-CoV-2 suppresses dsRNA-activated antiviral activity.

A previous study reported that SARS-CoV-2 infection leads to the activation of PKR and RNase L in A549 cells expressing human ACE2 (A549/huACE2) at 48 h postinfection (hpi). However, virus replication was not affected at either 24 or 48 h postinfection (12). It is not clear if the unaffected virus replication was due to the lack of dsRNA and/or activated PKR/RNase L at an earlier time point after infection. Here, we examined dsRNA production, the activation of PKR, and virus replication in A549/huACE2 cells at 24 h postinfection. The presence of dsRNA was examined using an immunofluorescence assay (IFA) with a J2 monoclonal antibody (Ab) that specifically recognizes dsRNA species of >40 bp in length (9, 19). As shown in Fig. 1A, dsRNA was detected extensively 24 h after SARS-CoV-2 infection, while no dsRNA was observed in uninfected cells. Interestingly, the N protein colocalized with dsRNA. To investigate if the dsRNA produced during SARS-CoV-2 replication activated PKR, the cell lysate collected at 24 hpi was examined for PKR phosphorylation at Thr446. As shown in Fig. 1B, only a trace amount of phosphorylated PKR (p-PKR) was detected in the SARS-CoV-2-infected cells. To confirm if the PKR- and OAS/RNase L-mediated antiviral pathways are functional in the cells, PKR in the cells infected with VACV with its two PKR inhibitor proteins deleted (VACVΔE3ΔK3) was almost completely phosphorylated, as indicated by the reduced mobility of the PKR protein in the cells infected by the mutant VACV. Similarly, the OAS/RNase L activity was confirmed by examining the degradation of 28S/18S rRNA following virus infection. No degradation of rRNA was observed in the SARS-CoV-2-infected cells at 24 hpi (Fig. 1C). In contrast, the majority of the rRNA was cleaved in the cells infected with VACVΔE3ΔK3. The replication of SARS-CoV-2 was examined by comparing the virus titers at 1 hpi and 24 hpi, and we found that the virus titers were increased by more than 100-fold at 24 hpi (Fig. 1D). Thus, SARS-CoV-2 replication produces a significant level of dsRNA, but it does not activate PKR and OAS/RNase L. It is known that early coronavirus replication occurs within the endoplasmic reticulum-associated double-membrane vesicles (20). It is likely that the double-membrane vesicles may contribute to shield the dsRNA produced during early replication from being detected by host pattern recognition receptors such as PKR and OAS/RNase L. However, it was previously reported that the ns2 protein of mouse hepatitis virus (MHV) plays a role in inhibiting OAS/RNase L activity as early as 9 h postinfection (11). Therefore, our observations indicated that SARS-CoV-2 might have another mechanism, in addition to the double-membrane-associated vesicles, to suppress dsRNA-activated antiviral activities to facilitate its replication.

SARS-CoV-2 N protein binds dsRNA and inhibits PKR/RNase L activities.

As shown in Fig. 1A, the SARS-CoV-2 N protein colocalized with dsRNA in the infected cells. Therefore, we speculated that the N protein might have the ability to inhibit dsRNA-activated antiviral activities in a manner similar to that of the VACV E3 protein (21–23). To test this hypothesis, we expressed the N protein in VACVΔE3ΔK3, which can replicate only in cells defective in PKR and OAS/RNase L (18; Cao, unpublished observation). Thus, if SARS-CoV-2 N has the function of inhibiting PKR and RNase L, recombinant VACVΔE3ΔK3 expressing the N protein (VACVΔE3ΔK3/N) would be able to replicate in cells with functional PKR and RNase L (e.g., HeLa and A549 cells), which are not permissive for VACVΔE3ΔK3.

The SARS-CoV-2 N gene driven by a vaccinia virus early/late promoter, mH5 (24), was inserted into VACVΔE3ΔK3, as we described previously (Fig. 2A) (24, 25). As we speculated, recombinant VACVΔE3ΔK3 expressing the N protein can replicate in HeLa cells and thus could be easily selected from the cells. The expression of the N protein by the recombinant viruses was confirmed using a FLAG Ab (Fig. 2B). The replication capacity of VACVΔE3ΔK3/N was further examined and compared to those of VACVΔE3ΔK3 and VACVE3 (expressing E3) in A549 cells (which have functional PKR and RNase L pathways), PKR knockout A549 cells (A549/PKRko), and RNase L knockout A549 cells (A549/RNaseLko) (26). The cells were infected with the viruses at a multiplicity of infection (MOI) of 5, and the virus was collected at 5 and 48 hpi. The virus titers were determined using a plaque assay in A549 PKR/RNase L double-knockout cells (A549/dko), which are permissive for all of the viruses, including VACVΔE3ΔK3 (data not shown). The increase in the virus titer at 48 hpi in comparison to that of the sample collected at 5 hpi indicates the virus yield. As shown in Fig. 2C, VACVΔE3ΔK3/N replicated to a level similar to that of VACVE3 in all three A549 cell lines. In contrast, VACVΔE3ΔK3 was not able to replicate in A549 wild-type cells (A549/wt), and only low virus yields (less than 0.2 logs) were observed in A549/PKRko and A549/RNaseLko cells. Thus, SARS-CoV-2 N can supplement VACV E3 to restore VACVΔE3ΔK3 replication in cells with functional PKR and RNase L pathways.

Next, we examined the dsRNA binding capacity of SARS-CoV-2 N. Previously, we examined VACV E3 and poxvirus E3 orthologs for their dsRNA binding using poly(I·C)-conjugated Sepharose beads (27). Here, wild-type VACV E3 (VACVwt) was used as the positive control, while an E3 mutant, E3/K167A, which was defective in dsRNA binding (28), was used as the negative control. Similar to VACV E3, the SARS-CoV-2 N protein was pulled down with poly(I·C) beads, while the E3/K167A mutant was not (Fig. 2D). Thus, similar to VACV E3, the SARS-CoV-2 N protein is a dsRNA binding protein.

The VACV E3 protein is a potent inhibitor of PKR and OAS/RNase L antiviral activities. When the gene encoding the E3 protein is deleted, the VACV deletion mutant virus induces PKR phosphorylation at threonine 446 and activates OAS/RNase L, as shown by the degradation of rRNAs. As shown in Fig. 2E and F, VACVΔE3ΔK3 induced significant PKR phosphorylation (A549/RNaseLko cells) and degradation of rRNAs (A549/PKRko cells). In contrast, the PKR phosphorylation and RNase L activities in VACVΔE3ΔK3/N-infected cells were similar to those in cells infected with VACV expressing E3 (VACVwt and VACVE3). Thus, the SARS-CoV-2 N protein functions similarly to VACV E3 in inhibiting the activation of the PKR and RNase L pathways.

The LKR and CTD of SARS-CoV-2 N are the determinants of dsRNA binding and inhibition of PKR/RNase L antiviral pathways.

Coronavirus N proteins have a modular organization consisting of two structural domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), flanked by three intrinsically disordered regions (IDRs), the N-terminal IDR (N/IDR), the linker region (LKR), and the C-terminal IDR (C/IDR) (29, 30). We constructed recombinant VACVΔE3ΔK3 expressing mutants with truncations of each domain of the SARS-CoV-2 N protein and examined their roles in dsRNA binding and the inhibition of PKR and RNase L (Fig. 3). Additionally, we constructed a mutant in which a stretch of 6 serine amino acids (aa) (aa 183 to 190, which also includes 185R and 189R) in the serine-rich motif of the LKR was deleted (LKRΔ6S). The mutant genes, containing a FLAG tag at the C terminus and driven by the vaccinia virus early/late promoter mH5, were inserted into the nonessential A45R gene of VACVΔE3ΔK3 (31). To facilitate the selection of recombinant VACVΔE3ΔK3 expressing the truncated mutants, the swinepox virus K3 ortholog SPV010, which is capable of rescuing the virus only in pig cells (18), was used as a selection marker to select and purify the recombinant viruses in pig cells.

The proteins of all of the truncated SARS-CoV-2 N mutants were expressed from VACVΔE3ΔK3-infected A549/dko cells at comparable levels (Fig. 3A and B). In comparison to the full-length N protein, the deletions of the N/IDR, NTD, LKR6S, and C/IDR (ΔN/IDR, ΔNTD, LKRΔ6S, and ΔC/IDR) had little effect on the ability to rescue VACVΔE3ΔK3 replication in wild-type A549, A549/PKRko, and A549/RNaseLko cells. In contrast, the mutants with a deletion of the LKR or the CTD (ΔLKR and ΔCTD) were not able to rescue VACVΔE3ΔK3 replication in A549 and A549/RNaseLko cells (Fig. 3C), indicating that both the LKR and CTD are required for the inhibition of PKR activities. In contrast, in A549/PKRko cells (which still express RNase L), while the ΔCTD virus was not able to grow, the ΔLKR virus was able to grow with a reduced efficiency (1 log versus 3 logs).

The dsRNA binding capacity and the inhibition of the PKR activation and RNase L activity of the truncated SARS-CoV-2 N mutants were analyzed as described above. As shown in Fig. 3D, the deletion of the N/IDR, NTD, and C/IDR had no effect on the dsRNA binding activity. In contrast, the dsRNA binding of the ΔCTD virus was significantly reduced, while it was slightly reduced by the deletion of LKR (ΔLKR). Correspondingly, the ΔLKR virus inhibited RNA degradation more than the ΔCTD virus in A549/PKRko cells (Fig. 3F). However, PKR was phosphorylated to similar degrees in A549/RNaseLko cells infected with the ΔLKR and ΔCTD viruses (Fig. 3E). These data correlate well with the virus yield data shown in Fig. 3C.

The LKR and CTD are sufficient for inhibition of PKR and RNase L antiviral activities.

Next, we examined if the LKR and CTD themselves could perform the function of inhibiting PKR and OAS/RNase L. The individual domains (N/IDR, NTD, LKR, CTD, and C/IDR) and a dual LKR-CTD (LKR+CTD), which were driven by the vaccinia virus early/late promoter mH5, were inserted into VACVΔE3ΔK3 as described above (Fig. 3A). Although transcribed, no protein was detected for the three IDR constructs (N/IDR, LKR, and C/IDR) (data not shown). The protein expression levels of the NTD, LKR+CTD, and CTD constructs are shown in Fig. 4B.

Similar to the full-length N protein, the LKR+CTD was fully functional in rescuing the replication of VACVΔE3ΔK3 in A549/wt, A549/PKRko, and A549/RNaseLko cells (Fig. 4C). Furthermore, the CTD was similar to the full-length N protein and the LKR+CTD in rescuing the replication of VACVΔE3ΔK3 in A549/PKRko cells. However, the virus expressing the CTD showed significantly less replication in A549/wt and almost no replication in A549/RNaseLko cells in comparison to A549/PKRko cells. This indicates that the CTD by itself can inhibit RNase L activity but is not able to inhibit PKR. VACVΔE3ΔK3 expressing the NTD was not able to replicate in A549/wt, A549/PKRko, or A549/RNaseLko cells.

The CTD and the LKR+CTD bound to poly(I·C), while the NTD could not (Fig. 4D). In A549/RNaseLko cells (expressing PKR), the LKR+CTD demonstrated a potency similar to that of the full-length N protein in inhibiting the phosphorylation of PKR and eIF2α, while the CTD and NTD did not (Fig. 4E). The LKR+CTD and the CTD completely inhibited RNase L activity in A549/PKRko cells, while the NTD did not (Fig. 4F).

Bat-CoV RaTG13 nucleocapsid protein is capable of inhibiting human PKR and RNase L.

The closest coronavirus to SARS-CoV-2 is a bat coronavirus (bat-CoV), RaTG13, with 96.2% genome sequence identity (32). For the N protein, there are only 4 amino acid residues (S37P, G215S, G243S, and A267Q) that vary between the two coronaviruses. Interestingly, 3 out of the 4 residues (G215S, G243S, and A267Q) are in the LKR and CTD regions (Fig. 3A), which are important for the function of inhibiting PKR and/or RNase L (Fig. 3 and 4). Here, we mutated these residues in SARS-CoV-2 N to the residues of RaTG13 N. The RaTG13 N protein was expressed in VACVΔE3ΔK3 and analyzed as described above (Fig. 5A and B).

VACVΔE3ΔK3 expressing the bat-CoV RaTG13 N protein replicated at a level comparable to that of VACVΔE3ΔK3 expressing SARS-CoV-2 N in A549/PKRko, A549/RNaseLko, and A549/wt cells, while VACVΔE3ΔK3 did not grow (Fig. 5C). Similar to SARS-CoV-2 N, bat-CoV RaTG13 N bound to poly(I·C) (Fig. 5D) and inhibited PKR phosphorylation and RNase L-mediated RNA degradation (Fig. 5E and F). Thus, the N protein from the bat-CoV is fully capable of inhibiting human PKR- and OAS/RNase L-mediated innate antiviral activities.

SARS-CoV-2 N specifically binds to phosphorylated PKR.

A previous study reported that SARS-CoV-2 N could bind to PKR and G3BP1 using an ectopic-overexpression assay (14). Here, we performed a coimmunoprecipitation assay to examine if the SARS-CoV-2 N protein binds to PKR and G3BP1 in the context of virus infection. Since the VACVΔE3ΔK3 recombinant viruses expressing the N protein and the N mutants can induce different levels of phosphorylated PKR, we prepared cell lysates containing nonphosphorylated and phosphorylated forms of PKR separately. Nonphosphorylated PKR was prepared using uninfected A549 cells, and there was little of the phosphorylated form of PKR (Fig. 6A). Phosphorylated PKR was prepared using A549 cells infected with VACVΔE3ΔK3, as almost all of the PKR becomes phosphorylated (Fig. 6A). Concurrently, cell lysates containing SARS-CoV-2 N and its mutants were prepared using A549/dko cells, as all of the N proteins are comparably expressed in these cells. The N protein lysate was then incubated with the cell lysates containing nonphosphorylated or phosphorylated PKR. The immune complex was precipitated using a FLAG antibody to the FLAG tag fused to the C termini of the N protein and its mutants.

As shown in Fig. 6B, the N protein and its truncated mutants were precipitated comparably with the FLAG antibody. No PKR was detected by precipitation from the mixture of N and its mutants with the uninfected cell lysate, as detected by either total PKR- or p-PKR-specific antibodies. In contrast, PKR was precipitated in the lysate of VACVΔE3ΔK3-infected cells incubated with N and its truncated mutants that can rescue VACVΔE3ΔK3 replication in cells with functional PKR and OAS/RNase L (i.e., ΔN/IDR, ΔNTD, LKRΔ6S, and ΔC/IDR) but not in the precipitation complex from the ΔLKR and ΔCTD mutants. Similarly, the LKR+CTD and CTD coprecipitated with PKR from the lysate of VACVΔE3ΔK3-infected cells but not in the lysate of uninfected cells (Fig. 6C). The NTD was not precipitated with PKR after incubation with either cell lysate. Thus, the CTD is sufficient for the interaction between SARS-CoV-2 N and the phosphorylated form of PKR.

Previous studies have shown that SARS-CoV-2 N binds to G3BP1 and that the N/IDR and CTD are required for binding (13). In our experiment, we confirmed the results of that previous report, and additionally, we found that the C/IDR is also required for binding to G3BP1, although it is not required for binding to dsRNA and the inhibition of PKR and OAS/RNase L (Fig. 6B). Interestingly, the NTD, LKR+CTD, and CTD mutants were not capable of binding to G3BP1, although the CTD is essential for binding (Fig. 6B and C).

Cells and viruses.

A549, A549 PKR knockout (A549/PKRko), A549 RNase L knockout (A549/RNaseLko), and A549 PKR/RNase L double-knockout (A549/dko) cells were provided by B. Moss (NIH, USA) (26). A549 cells expressing human ACE2 and TMPRSS2 were purchased from InvivoGen (San Diego, CA, USA). PK15, BHK21, and HeLa cells were obtained from the ATCC. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), glutamine, and penicillin-streptomycin at 37°C with 5% CO₂. The vaccinia virus (VACV) Western Reserve (WR) strain and the E3L and K3L double-deletion mutant (VACVΔE3ΔK3) were described previously (18), and SARS-CoV-2 was an ancestral Canadian isolate (hCoV/Canada/ON-VIDO-01/2020) described previously (61). The VACV-related experiments were performed in our biosafety level 2 (BSL-2) laboratory, and SARS-CoV-2 infections were done in our BSL-3/4 containment laboratory.

Construction of recombinant VACV.

VACVΔE3ΔK3, described previously (18), was used as the parental virus to construct recombinant viruses expressing SARS-CoV-2 nucleocapsid (N) and N mutants. The N and N mutant sequences were based on the SARS-CoV-2 Wuhan isolate (GenBank accession number MN908947). The modular structure of the SARS-CoV-2 N protein was based on an alignment with a SARS virus: the N-terminal intrinsically disordered region (N/IDR) (amino acids 1 to 44), the N-terminal domain (NTD) (amino acids 45 to 180), the linker region (LKR) (amino acids 181 to 247), the C-terminal domain (CTD) (amino acids 248 to 364), and the C-terminal IDR (C/IDR) (amino acids 365 to 419) (29, 30). The full-length N protein, the N truncated mutants (ΔN/IDR, ΔNTD, ΔLKR, ΔCTD, and ΔC/IDR), and the domain mutants (N/IDR, NTD, LKR, CTD, and C/IDR) with a FLAG tag added to their C termini and driven by a VACV early/late promoter, mH5 (24), were synthesized by GenScript (NJ, USA). Full-length N was inserted into the K3L locus of VACVΔE3ΔK3 using a method that we described previously (25). The N mutants were inserted into the A45R locus of VACVΔE3ΔK3, which is nonessential for VACV both in vivo and in vitro (31), as we previously described (18). Since we expected that some of the N mutants would lose the function of rescuing the replication of VACVΔE3ΔK3 in human cells, the swinepox virus K3L ortholog SPV010, which is capable of rescuing the replication of VACVΔE3ΔK3 only in swine cells, was used as a selection marker to select and purify recombinant VACVΔE3ΔK3 in a pig cell line, PK15 (18). The working stocks of all of the VACVΔE3ΔK3 recombinants expressing N and the N mutants were prepared in A549/dko cells.

Confocal microscopy.

Cell monolayers in a four-chambered slide (Thermo Fisher) were infected with the virus at a multiplicity of infection (MOI) of 0.5 and incubated at 37°C. At 24 h postinfection (hpi), cells were washed twice with phosphate-buffered saline (PBS) and fixed using a 3% paraformaldehyde–PBS solution for 10 min at room temperature. The fixed cells were washed 3 times with PBS for 3 min and then permeabilized using a 0.2% Triton X-100–PBS solution for 5 min at room temperature, with gentle rotation. Following another 3 washes with PBS (1 min each wash), the cells were incubated with a blocking buffer (2% FBS and 2% bovine serum albumin [BSA] in PBS) at 4°C for 1 h. The cells were then incubated with the primary antibody (SARS-CoV-2 N Ab from Sino Biological or dsRNA J2 Ab from Scicons), diluted 1/200 in blocking buffer, at 4°C for 2 h. The cells were washed 3 times with PBS for 3 min each wash and incubated with a fluorescence-conjugated secondary antibody specific for the primary antibody (Alexa Fluor secondary antibodies; Thermo Fisher), diluted 1/1,000 in blocking buffer, at 4°C for 1 h. The cells were washed 3 more times with PBS for 3 min each. The chambers were removed from the slides and rinsed with water. Prolong Diamond antifade mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen) was added, and the slides were covered with a coverslip and incubated at room temperature in the dark overnight. Slides were imaged using a confocal microscope (Observer.Z1; Zeiss) at a ×40 magnification.

RNA electrophoresis.

Cell monolayers were infected with the viruses listed in the related figure legends at an MOI of 5 and incubated at 37°C for 12 h (VACV) or 24 or 48 h (SARS-CoV-2) after infection. The total RNA was extracted using the RNeasy minikit (Qiagen) according to the manufacturer’s instructions. RNA concentrations were determined using the Qubit 4 system (Thermo Fisher) and diluted to a final concentration of 5 ng/μL. Ten nanograms of the RNA samples was analyzed by RNA electrophoresis using the high-sensitivity RNA ScreenTape system (Agilent) according to the manufacturer’s instructions, and rRNA was visualized using the 2200 TapeStation system (Agilent).

Poly(I·C) pulldown.

Poly(I·C) (Sigma-Aldrich)-conjugated Sepharose beads (GE Healthcare) were prepared as previously described (28). BHK21 cells were infected with VACVΔE3 at an MOI of 1 and transfected with the recombinant plasmids constructed as described above using Attractene transfection reagent (Qiagen) according to the manufacturer’s protocol. Twenty-four hours after infection and transfection, the cells were washed twice with cold PBS and then lysed using a nondenaturing lysis buffer (20 mM Tris-HCl, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, and a Roche protease inhibitor cocktail) plus 1 round of freezing and thawing. Cell debris was removed by centrifugation at 5,400 × g for 10 min, and an aliquot was removed and used as a prepulldown control. The rest of the supernatant was incubated with the poly(I·C)-conjugated Sepharose beads at room temperature for 2 h with end-over-end rotation. The beads were washed 3 times with nondenaturing lysis buffer to remove unbound proteins. Bound proteins were released from the poly(I·C) beads using an SDS-PAGE loading buffer (50 mM Tris-HCl, 2% SDS, 0.1% bromophenol blue, 10% glycerol, and 100 mM 2-mercaptoethanol) with boiling at 95°C for 10 min. Samples were analyzed using Western blotting as described previously (18).

Immunoprecipitation.

For the preparation of the SARS-CoV-2 N and mutant protein cell lysates, A549/dko cells were infected with the corresponding viruses for 8 h. Following one wash with PBS, the cell lysate was collected using the nondenaturing lysis buffer described above, and nonsoluble cell debris was removed by centrifugation at 2,000 × g at 4°C for 5 min. To prepare the cell lysate containing nonphosphorylated or phosphorylated PKR, wild-type A549 cells were mock infected (for nonphosphorylated PKR) or infected with VACVΔE3ΔK3 (for phosphorylated PKR) for 8 h, and the cell lysate was then prepared as described above for the N and mutant cell lysates. Coimmunoprecipitation was performed using the Dynabeads-protein G immunoprecipitation kit (Invitrogen) according to the manufacturer’s instructions. A mouse anti-FLAG antibody (Sigma-Aldrich) was used to bind to SARS-CoV-2 N and its mutants. The final precipitated protein complex was eluted from the Dynabeads-protein G using SDS sample buffer as described above and analyzed using Western blotting as described previously (18).

Western blotting.

Confluent cell monolayers in a 12-well tissue culture plate were infected with the viruses and collected at the time points indicated in the related figure legends. SDS-PAGE, membrane transfer, antibody blotting, and protein band detection were performed as described previously (18). Total PKR, p-PKR, p-EIF2α, and actin antibodies were purchased from Abcam. FLAG tag antibodies were purchased from Sigma-Aldrich. VACV D12 protein antibody was described previously (27). SARS-CoV-2 N protein antibody was purchased from Sino Biological.