The ORF3a protein of SARS-CoV-2 induces apoptosis in cells

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused the ongoing pandemic of Coronavirus Disease 2019. SARS-CoV-2 belongs to the genus Betacoronavirus of the Coronaviridae family, which includes SARS-CoV and Middle East respiratory syndrome coronavirus. Coronavirus-encoded accessory proteins play critical roles in virus–host interactions and the modulation of host immune responses, thereby contributing to coronaviral pathogenicity via different strategies. However, the functions of SARS-CoV-2-encoded accessory proteins are not well understood. Apoptosis is a predominant type of programmed cell death, and has been recognized as an important host antiviral defense mechanism that controls viral infection and regulates the inflammatory response. Previous studies have reported that the SARS-CoV-encoded accessory protein ORF3a can induce apoptosis in cells, leading to the question of whether SARS-CoV-2 ORF3a also has pro-apoptotic activity. Here, we investigated the potential apoptosis-inducing activity of SARS-CoV-2 ORF3a in different cell lines and compared the pro-apoptotic activities of SARS-CoV-2 ORF3a with those of SARS-CoV ORF3a using the same system. We sought to determine whether SARS-CoV-2 ORF3a can induce apoptosis using annexin V-fluorescein 5-isothiocyanate(FITC)/propidium iodide (PI) double staining in cultured HEK293T, HepG2, and Vero E6 cells. We found that annexin V and PI staining was significantly increased in cells expressing SARS-CoV-2 ORF3a compared with that in control cells (Fig. 1a). Moreover, the quantified data based on measuring the apoptosis rate also confirmed the pro-apoptotic activity of ORF3a in different cell lines (Fig. 1b). Furthermore, we examined activated caspase-3, a marker of caspase-dependent apoptosis, by flow cytometry and found that the percentage of cells with activated caspase-3 was significantly elevated in the presence of ORF3a (Fig. 1c). These results show that SARS-CoV-2 ORF3a can efficiently induce apoptosis in cells. To determine the mechanism through which SARS-CoV-2 ORF3a induces apoptosis, activation of the apoptosis cascade in HEK293T cells expressing ORF3a was examined by western blotting, probing for some apoptosis pathway components at 24 and 48 h post transfection. Cells treated with staurosporine, an apoptosis inducer, were used as a positive control. SARS-CoV-2 ORF3a induced the cleavage/activation of caspase-8, whereas Bcl-2 expression levels were not affected (Fig. 1d). The cleavage/activation of caspase-8 is recognized as a hallmark of the extrinsic apoptotic pathway, whereas Bcl-2 plays an important role in initiation of the intrinsic pathway. Moreover, we found that the levels of truncated Bid (tBid), cleaved caspase-9, and cytochrome c were elevated in the presence of SARS-CoV-2 ORF3a (Fig. 1e), and either a caspase-8 or caspase-9 inhibitor significantly suppressed SARS-CoV-2 ORF3a-induced apoptosis (Fig. 1f, g). Thus, our results imply that SARS-CoV-2 ORF3a can induce apoptosis via the extrinsic pathway, in which activated caspase-8 cleaves Bid to tBid and in turn induces the release of mitochondrial cytochrome c, resulting in apoptosome formation and caspase-9 cleavage/activation. We next sought to examine the relationship between the membrane association and pro-apoptotic activity of SARS-CoV-2 ORF3a. As previously reported, SARS-CoV ORF3a is a transmembrane protein that contains several conserved motifs including a cysteine-rich motif (a.a.127–133), tyrosine-based sorting motif (YXXΦ; a.a.160–163), and diacidic EXD motif (a.a. 171–173), and these domains regulate the subcellular location of SARS-CoV ORF3a and play important roles in SARS-CoV ORF3a infection, inducing apoptosis. SARS-CoV-2 ORF3a shares 73% amino acid homology with its counterpart in SARS-CoV, and the cysteine-rich and YXXΦ motifs are conserved but the EXD motif was found to be changed to SGD in SARS-CoV-2 ORF3a (Fig. S1a). Thus, we constructed two mutant ORF3a proteins by mutating C130/133 of the cysteine-rich motif to S (SARS-CoV-2 ORF3a-CS) or Y160 of the YXXΦ motif to A (SARS-CoV-2 ORF3a-YA). The immunofluorescence assays showed that wild-type ORF3a of SARS-CoV-2 (ORF3a-WT) localized to the plasma membrane with punctate cytoplasmic staining, whereas ORF3a-CS and ORF3a-YA exhibited more cytoplasmic localization (Figs. 1h and S1b). The results of cytosol-membrane fractionation assays showed that whereas ORF3a-WT was present in both cytosol and membrane fractions, either ORF3a-CS or ORF3a-YA was absent in the membrane fraction (Figs. 1i and S1c). Moreover, we found that ORF3a-CS or ORF3a-YA showed minimal apoptosis-inducing and caspase-3activiting activity in cells in the presence or absence of z-VAD-fmk, a general caspase inhibitor (Figs. 1j and S1d). In addition, ORF3aCS or ORF3a-YA failed to induce the cleavage of Bid, caspase-8, and caspase-9 or the release of cytochrome c (Fig.1k, l). These results indicate that membrane association is required for the proapoptotic activity of SARS-CoV-2 ORF3a. To investigate if there is any difference between the proapoptotic activities of ORF3a proteins of SARS-CoV-2 and SARS-CoV, we examined the membrane association and apoptosis-induction ability of SARS-CoV ORF3a. SARS-CoV ORF3a variants were generated by mutating C127/130/133 to S (SARS-CoV ORF3a-CS)

removed into a new centrifuge tube and then centrifuged to precipitate cell membrane fragments; the supernatant is the plasma protein.

Mitochondria fractionation assays
At the indicated times post-transfection, cells were washed with PBS followed by dousing 20 times in 1 mL homogenization buffer (ApplyGen) by 1 mL injector. The homogenate was centrifuged at 500 g for 10 min. The supernatant was centrifuged at 5000 g for 10 min to precipitate mitochondria.

Immunofluorescence staining assays
Cells seeded on the confocal dish were transfected with 1 μg indicated plasmids. 24 h after transfection, cells were washed three times with PBS and fixed with 3.7% formaldehyde in PBS for 30 min, then permeabilized by 1% Triton X-100 in PBS for 20 min. After blocking with 5% BSA in PBS for 60 min, cells were incubated with mouse anti-FLAG antibody diluted in 1:100 in 5% BSA in PBS at 4℃ overnight.
After washing three times with PBS, cells were incubated with Alexa Fluor 488 goat anti-mouse IgG (H+L) (Abcam) diluted in 1:500 in 5% BSA in PBS for 30 min. After washing cells three times with PBS, cell nuclei were stained with DAPI (Invitrogen) diluted in 1:250 in PBS for 5 min. Fluorescent signals were detected by using a Nikon microscope and images were analyzed with the NIS-Elements Viewer 4.50.

Apoptosis detection assays
Flow cytometry assay was used to detect cell apoptosis and analyze the cell cycle 4 .
Cells seeded on the dish were transfected with designated plasmids or treated with different caspase inhibitors (Targetmol) or caspase inducers (MCE). After 24h, cells were washed three times with PBS and collected by centrifugation at 1500 g for 5 min, following used the "Annexin V-FITC Apoptosis Detection Kit (Beyotime C1062)" to detect cell apoptosis. The operation steps are briefly described as follows: Add 195 μL Annexin V-FITC binding solution to resuspend cells gently; Then add 5 μL Annexin V-FITC and 10 μL propidium iodide staining solution and mix gently; Incubate at room temperature (20-25°C) in the dark for 10-20 minutes, then place in an ice bath; Flow cytometry analysis within one hour.

Caspase-3/7 activity detection assays
Cells seeded on the dish were transfected with designated plasmid. 24 h post-transfection, cells were washed three times with PBS and collected by centrifugation at 1500 g for 5 min. Then use "Caspase-3/7 Green Flow Cytometry (Invitrogen)" to detect cells displayed caspase-3/7 activation. The operation steps are briefly described as follows: Prepare flow cytometry tubes each containing 1 mL of cell suspension; Add 1 μL of CellEvent ® Caspase-3/7 Green Detection Reagent (Component A) to samples; incubate for 25 minutes at 37°C; Add 1 μL of the 1 mM SYTOX ® AADvanced™ dead cell stain solution to samples; incubate for 5 minutes at 37°C; Analyze the samples using 488-nm excitation and apply for standard fluorescence compensation.