Severe Acute Respiratory Syndrome Coronavirus Triggers Apoptosis via Protein Kinase R but Is Resistant to Its Antiviral Activity

293 low-passage cells (PD-02-01; Microbix Bisosystems Inc.) and 293/ACE2 cells (33) (kindly provided by Shinji Makino, UTMB, Galveston, Texas) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), penicillin (50 units/ml), and streptomycin (50 μg/ml). Selection of 293/ACE2 cells constitutively expressing human angiotensin-converting enzyme 2 (ACE2) was performed by addition of blasticidin at a concentration of 12 μg per ml medium. The FFM-1 isolate of SARS-CoV (GenBank accession number AY310120) and vesicular stomatitis virus (VSV) strain Indiana were propagated in Vero E6 cells. Virus titers of SARS-CoV and VSV were determined by 50% tissue culture infectious dose (TCID₅₀) assays. Sendai virus (SeV) strain Cantell (kindly provided by C. F. Basler, Mount Sinai School of Medicine, New York) was propagated in 11-day-old embryonated chicken eggs. SeV hemagglutinating units/ml were determined by a standard hemagglutination test. All work with SARS-CoV was performed in the biosafety level 4 facility of the University of Marburg.

Vero E6 cells were cultured in 96-well plates to 50% confluence and infected with 10-fold serial dilutions of supernatants from cells that were infected and/or treated with peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) and/or IFN-β. At 3 to 8 days post infection (p.i.), when the cytopathic effect had stabilized to a constant rate, cells were analyzed by light microscopy. The TCID₅₀/ml was calculated using the Spearman-Kärber method (28).

At the indicated times p.i., infected cells were fixed with 4% paraformaldehyde in DMEM for at least 12 h. Thereafter, cells were permeabilized with 0.1% Triton X-100 for 10 min, treated with 0.1 M glycine for 10 min, and subsequently incubated in blocking reagent (2% bovine serum albumin, 0.2% Tween 20, 3% glycerin, and 0.05% NaN₃ in phosphate-buffered saline deficient in Mg²⁺ and Ca²⁺) for 15 min. Immunofluorescence analyses were performed as described elsewhere (7) using a rabbit antiserum directed against the nucleoprotein of SARS-CoV (dilution, 1:1,000; kindly provided by L. Martínez-Sobrido and A. Garcia-Sastre, Mount Sinai School of Medicine, New York) or an antibody directed against SeV (dilution, 1:500; kindly provided by W. J. Neubert, Max Planck Institute, Martinsried, Germany). Infected cells were additionally analyzed for the presence of dsRNA by using J2 monoclonal antibody (English & Scientific Consulting; dilution, 1:100). Bound antibodies were detected with either rhodamine-labeled goat anti-rabbit immunoglobulin G or rhodamine-labeled goat anti-mouse immunoglobulin G (dilution, 1:100; Dianova).

Whole-cell extracts were prepared by incubating cells in cell lysis buffer (10 mM Tris [pH 7.4], 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na₄P₂O₇, 2 mM Na₃VO₄, 1% Triton, 10% glycerol, 0.1% sodium dodecyl sulfate [SDS], 0.5% deoxycholate) for 20 min on ice. Protease inhibitor mixture (1× Complete tablets; Roche) and the serine/threonine phosphatase inhibitor Calyculin A (Cell Signaling; 0.1 μM) were added to cell lysis buffer prior to incubation. To analyze cleavage of procaspase-3 and -8 cells were lysed in 50 μl of 1× CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate} buffer (Cell Signaling) containing 1 mM protease inhibitor mix (1× Complete tablets; Roche) and 5 mM dithiothreitol, followed by three freeze-and-thaw cycles. The extracts from both lysing methods were then centrifuged and supernatants transferred to fresh tubes containing 2× SDS sample buffer (25% glycerol, 2.5% SDS, 125 mM Tris [pH 6.8], 125 mM dithiothreitol, 0.25% bromophenol blue). Proteins were separated on 8 to 15% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes. Immunostaining was performed with an appropriate dilution of primary antibody in phosphate-buffered saline or Tris-buffered saline containing either 5% (wt/vol) skim milk or 5% (wt/vol) bovine serum albumin according to the manufacturer's instructions. Rabbit (unless otherwise stated) polyclonal antibodies were used to detect human PARP (Cell Signaling; 1:1,000), phosphorylated eIF2α (Ser51) (Biosource; 1:30,000), PKR (mouse, BD Bioscience; 1:30,000), PERK (Santa Cruz Biotechnology; 1:1,000), phospho-PERK (Thr980) (BioLegend; 1:1,000), GCN2 (Cell Signaling; 1:5,000), phospho-GCN2 (Cell Signaling; 1:1,000), and caspase-3 (monoclonal, Cell Signaling; 1:1,000); mouse (unless otherwise stated) monoclonal antibodies were used to detect eIF2α (Biosource; 1:1,000), phospho-PKR (pT446) (rabbit, Epitomics; 1:1,000), caspase-8 (Cell Signaling; 1:1,000), and β-actin (ab8226, Abcam; 1:40,000); and a rabbit antiserum was used to detect the nucleoprotein of SARS-CoV (dilution, 1:10,000). Western blot detection was done with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G or anti-mouse immunoglobulin G secondary antibody using an enhanced chemiluminescence detection reagent kit (Pierce) according to the manufacturer's protocol. Immunoreactive bands were visualized using an Optimax 2010 imaging system (Protec processor technology) with high-performance chemiluminescence films (GE Healthcare).

Phosphorodiamidate morpholino oligomers (PMO) are single-stranded antisense agents that possess DNA purine and pyrimidine bases attached to a backbone consisting of morpholine rings joined by phosphorodiamidate linkages and were synthesized as previously described (68). To enhance uptake into cells, all PMO were conjugated at the 5′end through a noncleavable linker to the cell-penetrating peptide (RXR)₄XB (R is arginine, X is 6-aminohexanoic acid, and B is beta-alanine) to produce PPMO by methods previously described (2). Four different PKR-specific PPMO were designed to target human PKR mRNA (GenBank accession number BC057805). The sequences and exact target locations of the PPMO are defined in Table 1. PPMO 5′ED and “AUG” target sequences near the 5′ terminus and the AUG translation initiation site, respectively, of the PKR mRNA. PPMO ex-7 and ex-8 were designed against exonic sequences near the intron/exon splice junctions for exons 7 and 8, respectively, of the pre-mRNA (derived from the sequence under GenBank accession number BC057805), in an effort to disrupt mRNA processing. A PPMO of random sequence (scramble) was synthesized to serve as a control for off-target effects.

293 or 293/ACE2 cells were seeded into six-well culture plates at a concentration of 4 × 10⁴ cells per well and allowed to adhere overnight. For detection of PKR mRNA after PPMO treatment, cells were treated with 20 μM PPMO in 1 ml DMEM containing penicillin (50 units/ml), streptomycin (50 μg/ml), and 2% FCS (DMEM+) or with DMEM+ alone; 48 h later, total cellular RNA was isolated using the RNeasy minikit (Qiagen). For detection of PKR, GCN2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs in SARS-CoV-infected cells, cells were infected with SARS-CoV (multiplicity of infection [MOI] of 0.01) or mock infected, and total cellular RNA was isolated at 8, 24, and 48 h p.i. One-eighth of the volume of eluted RNA (5 μl) was used for reverse transcription-PCR (RT-PCR) using the OneStep RT-PCR kit (Qiagen) and PKR-specific primers (forward, 5′-GGTTTCTTCATGGAGGAACTTAATAC; reverse, 5′-TAGAGGTCCACTTCCTTTCCA) designed to amplify nt 457 to 1850 of human PKR mRNA, GCN2-specific primers (forward, 5′-GAAGGCACCGTCAAGATTACG; reverse, 5′-GACTCTGTACCACACCTTGATG), or GAPDH-specific primers (forward, 5′-TGAAGGTCGGAGTCAACGGA; reverse, 5′-CATGTGGGCCATGAGGTCCA). Ten percent of each RT-PCR mixture was resolved on a 1.5% agarose gel and stained with ethidium bromide. RT-PCR products produced from RNA isolated from PPMO-treated cells were sequenced. To analyze cellular RNA levels, 1 μl of extracted RNA was resolved on a 1.5% agarose gel and stained with ethidium bromide.

Transfection of 293 cells was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. A total of 4 × 10⁴ cells were transfected with 0.8 μg of the IFN-stimulated response element-driven firefly luciferase reporter plasmid pHISG-54-Luc (a kind gift of D. Levy, New York University School of Medicine, New York) along with 0.2 μg of the constitutive Renilla luciferase expression plasmid pRL-SV40 (Promega). At 24 h posttransfection, cells were either infected with SeV or treated with 20 μM PPMO; 48 h later, cells were harvested and lysed in passive lysis buffer (Promega) for luciferase assays. Luciferase assays were performed by using the Promega dual luciferase assay system according to the manufacturer's instructions. Relative Renilla luciferase production was used to normalize for transfection efficiency.

Unless otherwise stated, 4 × 10⁴ 293/ACE2 cells were seeded in six-well culture plates and treated the next day with a final concentration of 20 μM PPMO in DMEM+, or DMEM+ alone, for 72 h. Subsequently, PPMO-containing medium was removed and the cells then either infected with SARS-CoV or further treated with 10,000 international units (IU) of IFN-β for 24 h prior to infection with SARS-CoV at an MOI of 0.01 as indicated. After an infection period of 1 h, the supernatant was removed and replaced by DMEM+ or by DMEM+ containing 20 μM of appropriate PPMO, and the cells were then further incubated for 24 to 48 h.

Cell culture demonstrations that SARS-CoV induces apoptosis have typically been conducted with Vero E6 cells, an African green monkey kidney cell line (8, 47, 55, 70, 80). However, Vero cells are less than optimal for investigating human host-virus interactions, in part because many of their cellular proteins are not recognized by available antibodies. We therefore sought to determine if human 293/ACE2 cells stably expressing the SARS-CoV receptor (33) undergo apoptosis after infection with SARS-CoV, as Vero cells do. 293/ACE2 cells were inoculated with SeV, VSV, or SARS-CoV, and cell lysates were analyzed for cleavage of PARP, a reliable indicator for late-stage apoptosis and a generally accepted hallmark of apoptosis. PARP, a 113-kDa nuclear zinc finger protein, is a DNA nick sensor that functions in the DNA repair processes. During apoptosis, PARP is cleaved by caspases into two inactive fragments (89 kDa and 24 kDa) (31). VSV and SeV both are known to be potent inducers of apoptosis (6, 41). In 293/ACE2 cells infected with either virus, PARP cleavage was detectable at 24 h p.i. and robust at 48 h p.i., indicating that these cells undergo apoptosis in response to infection (Fig. 1A). In 293/ACE2 cells infected with SARS-CoV, cleavage of PARP was not detectable at 24 h, although almost all cells were infected (Fig. 1A and E). However, PARP cleavage was clearly evident at 48 h p.i. and was prominent at 72 h p.i. In noninfected cells PARP cleavage was nondetectable at 48 h but prominent at 72 h, suggesting that the high density of cells that had accumulated at this late time point caused induction of apoptosis (Fig. 1A). To confirm the finding that SARS-CoV induces apoptosis in 293/ACE2 cells, cleavage of effector caspase-3 and initiator caspase-8 was analyzed. As shown in Fig. 1B, caspase-3 cleavage was observed at 48 h and 72 h p.i., thus temporally coinciding with PARP cleavage. Cleavage of caspase-3 was also observed in noninfected cells at 72 h p.i., but the proportion of uncleaved to cleaved caspase-3 was much higher in noninfected than in infected cells (Fig. 1B). The total amount of (uncleaved) procaspase-8 was significantly lower in infected cells than in noninfected cells at all three time points tested. At 24 or 48 h p.i., the cleavage product of caspase-8 could not be detected in either infected or noninfected cells. However, at 72 h p.i., a weak band corresponding to cleaved caspase-8 was detected in the infected but not in the noninfected cells (Fig. 1B), providing a further indication of virus-induced apoptosis. Finally, chromatin condensation, considered an indicator for late-stage apoptosis, was observed in SARS-CoV-infected cells at 48 h and 72 h p.i. (Fig. 1C). SARS-CoV N protein was detectable as early as 8 h p.i., reached a robust level by 24 h p.i., and remained at a high level through 72 h p.i. (Fig. 1D). It has previously been shown that SARS-CoV is a fast-replicating virus. Stertz et al. (65) observed budding of SARS-CoV at intracellular membranes at 3 h p.i., and Ng et al. (51) observed virus particle release from infected cells at 5 h p.i. The results shown in Fig. 1A to D suggest that the induction of apoptosis in SARS-CoV-infected 293/ACE2 cells occurred during the later stages of infection.

Apoptosis is a complex cellular process which can be induced in many ways. Since it is known that virus-induced phosphorylation of eIF2α eventually results in the induction of apoptosis (34, 86), we analyzed whether eIF2α becomes phosphorylated in SARS-CoV-infected cells. 293/ACE2 cells were infected with SARS-CoV, harvested at various times p.i., and subjected to Western blot analysis. eIF2α phosphorylation was observed at 8 and 24 h p.i. in SARS-CoV-infected cells (Fig. 2A, upper panel).

Currently, three kinases (PKR, PERK, and GCN2) are known to phosphorylate eIF2α in response to viral stimuli. Since PKR is activated by dsRNA and since dsRNA reportedly becomes abundant in SARS-CoV-infected cells (76), we initially focused on PKR as the kinase most likely to be responsible for eIF2α phosphorylation in SARS-CoV-infected 293/ACE2 cells. We observed that dsRNA was indeed present in large amounts in SARS-CoV-infected 293/ACE2 cells (Fig. 2B) and that PKR was phosphorylated at 8 and 24 h p.i., demonstrating that the processes of PKR activation and eIF2α phosphorylation temporally coincide in SARS-CoV-infected cells (Fig. 2A). The observed eIF2α phosphorylation in noninfected cells at 24 h p.i. is most likely due to the activity of cellular eIF2α kinases other than PKR (see below).

To investigate the effect of PKR activity on apoptosis and SARS-CoV replication, we employed a knockdown strategy using sequence-specific PPMO. PPMO enter cells readily under standard culturing conditions (2, 17, 39) and are relatively stable in human cells and serum (82). A number of evaluations have indicated that incubation with 10 to 20 μM PPMO was minimally cytotoxic to noninfected cells under conditions similar to those used in our study (19, 39).

Four different PPMO, targeted to different regions of PKR mRNA, were tested for their ability to reduce PKR expression in 293 cells (for PPMO sequences and target locations, see Table 1). As controls for off-target effects, actin levels were monitored and a PPMO of nonsense sequence (“scramble”) was included. Initially, 293 cells were incubated with 10 or 20 μM PPMO for 48 h, lysates made, and protein levels visualized by Western blot analysis. As shown in Fig. 3A, treatment with 10 μM of the ex-7 or ex-8 PPMO significantly and specifically reduced the amount of PKR protein in the cells. The reduction was even more pronounced when 20 μM PPMO ex-7 or -8 was used. The “AUG,” 5′ED, and “scramble” PPMO had no effect on PKR expression, and actin levels were generally unaffected (Fig. 3A).

RT-PCR was conducted with cellular RNA isolated from PPMO-treated 293 cells to amplify nt 457 to 1850 of human PKR mRNA and thereby produce a PCR fragment of 1,394 nt in length. While treatment of cells with PPMO ex-7 or ex-8 led to synthesis of appropriately shortened PCR fragments, PPMO “scramble” did not affect the PCR fragment size (Fig. 3B). Sequence analysis of the different PCR fragments of human PKR mRNA revealed a deletion of nt 952 to 1028 in PCR fragment “ex-7” and a deletion of nt 950 to 1116 in PCR fragment “ex-8.” PCR fragment “scramble” was identical in sequence to the corresponding region of the GenBank-annotated PKR mRNA. These results confirm that the ex-7 and ex-8 PPMO interfered with splicing events at the intended locations, presumably by disrupting spliceosome assembly on the PKR pre-mRNA through steric interference (1). PPMO targeted to protein-coding sequence located well downstream from the AUG initiator are unlikely to be effective at interfering with translation (67). However, because the ex-7 and -8 PPMO target sequences are present in the mature mRNA, it is possible that these PPMO also reduced the translation of PKR mRNA.

Gene expression knockdown using small interfering RNAs can induce a type I IFN response (36, 56). To explore whether treatment of cells with PPMO would similarly lead to IFN induction, reporter gene assays were performed. Cells were transfected with the pHISG-54 reporter plasmid, which contains the firefly luciferase gene under the control of the IFN-stimulated response element region of the human IFN-stimulated gene ISG-54, along with the expression plasmid pRL-SV40, which constitutively expresses Renilla luciferase. Twenty-four hours later, the cells were either treated with 20 μM PPMO or infected with SeV to induce ISG-54-activity for 48 h before reporter gene assays. The results show that PPMO 5′ED, ex-7, and ex-8 did not induce a type I IFN response (Fig. 3C). PPMO “AUG” treatment produced a slight increase in reporter gene activity, but since this PPMO did not produce significant downregulation of PKR expression anyway, it was not used further in this study.

Next, we investigated the kinetics of PPMO-mediated inhibition of PKR expression. 293 cells were treated with various PPMO and harvested at different time points posttreatment. PKR expression was determined by analysis of Western blots and quantified by normalizing PKR bands to corresponding actin bands. Figure 3D shows that 72 h of treatment with PPMO ex-8 caused a profound reduction in PKR expression. PPMO ex-7 treatment likewise reduced PKR expression markedly; however, the somewhat reduced actin signal indicated that it may have also caused cytopathic effects (Fig. 3A and D). Therefore, only PPMO ex-8 was used for further studies.

The experiment described above was repeated using 293/ACE2 cells instead of 293 cells, and similar profiles of PPMO-mediated downregulation of PKR and actin expression were obtained (see below). Attempts to inhibit PKR expression in Vero cells failed, probably due to sequence differences between human and simian PKR genes (data not shown). In summary, treatment with PPMO ex-8 effectively and specifically inhibited PKR expression in human 293 and 293/ACE2 cells.

We sought to explore the impact of PKR activation on SARS-CoV growth. 293/ACE2 cells were treated with 20 μM of various PPMO for 72 h and then infected with SARS-CoV at a low MOI. Supernatants were harvested 48 h later, and virus titers were determined by TCID₅₀ assays. Surprisingly, neither SARS-CoV nucleoprotein expression nor replication efficiency was affected by ex-8 PPMO treatment compared to that in infected cells that were mock treated, indicating that SARS-CoV is not sensitive to the antiviral effects of activated PKR in 293/ACE2 cells (Fig. 4A and B). Although the viral titer of ex-8- or mock-treated cells was about 1 log₁₀ unit higher than titers of 5′ED- or “scramble”-treated cells, this difference was calculated to be not significant (Student's t test, P > 0.100).

To determine if SARS-CoV-induced apoptosis was potentially mediated by PKR, infected-cell lysates receiving various treatments were harvested at 48 h p.i. and analyzed for PKR expression and PARP cleavage. As shown in Fig. 4B, cells treated with the ex-8 PPMO displayed no PKR expression, as expected from the above results. PARP cleavage occurred in SARS-CoV-infected cells that were either mock treated or treated with the “scramble” or 5′ED PPMO. However, in SARS-CoV-infected cells treated with the ex-8 PPMO, PARP cleavage was strongly reduced compared to controls, indicating that the induction of apoptosis was impaired in these cells. The amount of phosphorylated eIF2α was also analyzed and found to be high in all samples, irrespective of whether the cells were infected or not and without any correlation to PKR expression (Fig. 4B). We assume that the high background phosphorylation of eIF2α at 48 h p.i. was due to the activity of eIF2α kinases other than PKR, which were induced by the high cell density characteristic of this time point (59).

Together, the results shown in Fig. 4 indicate that although PKR mediates the induction of apoptosis in SARS-CoV-infected cells, neither PKR nor apoptosis has a major effect on virus growth for up to 72 h p.i. in cultured 293/ACE2 cells.

SARS-CoV is known to be sensitive to IFN-β treatment (62). Since PKR is a major antiviral factor known to be upregulated by type I IFNs, we investigated the contribution of PKR to the antiviral effects of IFN-β against SARS-CoV. 293/ACE2 cells were first treated with PPMO and then incubated with IFN-β and subsequently infected with SARS-CoV. At 24 h p.i. lysates of cells were analyzed for total and phosphorylated PKR and eIF2α. Figure 5A shows that PKR expression was strongly upregulated in cells which were both IFN-β treated and SARS-CoV infected. Upregulation of PKR was less pronounced in IFN-β-treated cells which were not infected. Importantly, however, PPMO ex-8 treatment reduced PKR expression, even when IFN-β was simultaneously present. Phosphorylated PKR was barely detectable in either noninfected or SARS-CoV-infected cells treated with ex-8 PPMO (Fig. 5A, top panel). Moderate PKR phosphorylation was observed in noninfected cells treated with IFN-β. In SARS-CoV-infected cells that were not treated with ex-8 PPMO, PKR phosphorylation was strongly induced irrespective of whether the cells were treated with IFN-β or not. Interestingly, the phosphorylation pattern of eIF2α was not consistent with that of PKR phosphorylation (Fig. 5A). Compared to that in nontreated cells, eIF2α phosphorylation was reduced in cells treated with IFN-β and did not correlate with PKR phosphorylation. These data clearly indicate that kinases other than PKR are involved in SARS-CoV-induced eIF2α phosphorylation. We therefore analyzed the phosphorylation state of PERK and GCN2, the two other kinases known to phosphorylate eIF2α in response to viral infections. Indeed, PERK was phosphorylated in SARS-CoV-infected cells, but only if the cells were not additionally treated with IFN-β. Moreover, the total amount of PERK was reduced after IFN-β treatment (Fig. 5A). In contrast to PERK, GCN2 was not phosphorylated in SARS-CoV-infected cells (Fig. 5A). These data indicate that eIF2α phosphorylation correlates with activated PERK in infected cells, suggesting that PERK mediates phosphorylation of eIF2α in SARS-CoV-infected cells.

TCID₅₀ assays revealed that IFN-β treatment of infected cells reduced viral titers by about 2 log₁₀ units (Fig. 5B). Downregulation of PKR by the ex-8 PPMO, however, did not significantly affect viral titers (Student's t test, P > 0.100), confirming that active PKR has no important effect on SARS-CoV replication. As expected, expression of SARS-CoV N protein was dramatically diminished in IFN-β-treated cells (Fig. 5A).

We also examined the expression and phosphorylation state of GCN2 and PERK late in infection, i.e., at a time when apoptosis is observable. 293/ACE2 cells were infected with SARS-CoV, and GCN2 and PERK were then detected by Western blot analysis in samples taken at 24 h or 48 h pi. (Fig. 6A). In parallel, the efficiency of virus infection was determined by immunofluorescence analysis (Fig. 6B). Increased PERK phosphorylation was observed in infected cells at 24 h and 48 h p.i. (Fig. 6A). In contrast, GCN2 was not phosphorylated in SARS-CoV-infected cells late in infection (Fig. 6A). Oddly, GCN2 was found to be phosphorylated in noninfected cells at 48 h p.i., perhaps due to amino acid deprivation in the cells. Interestingly, the amount of GCN2 was drastically reduced in SARS-CoV-infected cells at 48 h p.i., whereas the levels of actin and PKR were only slightly affected (Fig. 6A). Thus, we conclude that the level of GCN2 protein is actively downregulated in SARS-CoV-infected cells. To analyze whether reduced GCN2 protein levels were due to decreased transcription or stability of GCN2 mRNA, RT-PCR analyses of GCN2, PKR, and GAPDH mRNAs were performed. Our results revealed that reduction of the GCN2 protein level did not correlate with degradation of GCN2 mRNA in infected cells. The reduction of GCN2, PKR, and GAPDH mRNAs after 24 and 48 h p.i. likely resulted from the reduced amount of total cellular RNA in SARS-CoV-infected cells, as shown by examination of total RNA by agarose gel electrophoresis (Fig. 6C).