Genome-wide characterization of SARS-CoV-2 cytopathogenic proteins in the search of antiviral targets

Abstract

Therapeutic inhibition of critical viral functions is important for curtailing coronavirus disease-2019 (COVID-19). We sought to identify antiviral targets through genome-wide characterization of SARS-CoV-2 proteins that are crucial for viral pathogenesis and that cause harmful cytopathic effects. All twenty-nine viral proteins were tested in a fission yeast cell-based system using inducible gene expression. Twelve proteins including eight non-structural proteins (NSP1, NSP3, NSP4, NSP5, NSP6, NSP13, NSP14 and NSP15) and four accessory proteins (ORF3a, ORF6, ORF7a and ORF7b) were identified that altered cellular proliferation and integrity, and induced cell death. Cell death correlated with the activation of cellular oxidative stress. Of the twelve proteins, ORF3a was chosen for further study in mammalian cells. In human pulmonary and kidney epithelial cells, ORF3a induced cellular oxidative stress associated with apoptosis and necrosis, and caused activation of pro-inflammatory response with production of the cytokines TNF-α, IL-6, and IFN-β1, possibly through the activation of NF-κB. To further characterize the mechanism, we tested a natural ORF3a Beta variant, Q57H, and a mutant with deletion of the highly conserved residue, ΔG188. Compared to wild type ORF3a, the ΔG188 variant yielded more robust activation of cellular oxidative stress, cell death, and innate immune response. Since cellular oxidative stress and inflammation contribute to cell death and tissue damage linked to the severity of COVID-19, our findings suggest that ORF3a is a promising, novel therapeutic target against COVID-19.

Keywords: ORF3a; SARS-CoV-2; Schizosaccharomyces pombe; apoptosis and necrosis; fission yeast; oxidative stress; pro-inflammatory response; viral therapeutic target.

Figure 1 |. The effect of SARS-CoV-2 protein on cell proliferation.

Effect of SARS-CoV-2 expression on fission yeast colony formation. (A), cellular growth (B) and summary of the relative cellular growth (15%, red; 25% blue) of each of the SARS-CoV-2 protein-expressing cells (C). The name of each SARS-CoV-2 protein is labeled above each agar plate. An empty pYZ1N vector (Ctr) was used as a control. Gene-off, no SARS-CoV-2 protein production; gene-on, the specific SARS-CoV-2 protein production was induced by triggering the nmt1 promoter-mediated gene transcription. Fission yeast colony formation was measured by growing SARS-CoV-2 protein-expressing fission yeast cells on the selective EMM agar plates and incubated at 30°C for 3–5 days before the pictures were taken. Cell proliferation analysis was carried out by comparing cellular growth between the SARS-CoV-2 protein-producing cells and the SARS-CoV-2 protein-suppressing cells over time. Cell growth was measured by spectrophotometry (OD₆₅₀). Only the effect of those SARS-CoV-2 proteins that showed complete (NSP1, NSP4, NSP6, NSP13, NSP14, ORF3a, ORF6 and ORF7a) or nearly complete (NSP3, NSP5 and NSP15) inhibition of yeast colony formation is shown in (A) and thereafter. Complete data on cell proliferation are included in Figure S2. Each experiment was repeated at least three times and the standard errors of each time point were calculated.

Figure 2 |. The effect of SARS-CoV-2 protein on fission yeast cellular morphology.

Only those SARS-CoV-2 proteins that affected cell proliferation presented in Figure 1 are shown here. Complete SARS-CoV-2 genome-wide data on fission yeast cellular morphology are included in Fig. S3. (A) shows the effect of individual SARS-CoV-2 proteins on fission yeast cell morphology. Each image was taken 48 h agi using bright field microscopy. Scale bar = 10 μM. (B) Overall cell morphology as shown by the forward scattered analysis. Ten thousand cells were measured 48 h agi. The forward-scatter (FSC) measures the distribution of all cell sizes. The side-scatter (SSC) determines intracellular complexity. Gene-off, no SARS-CoV-2 protein production; gene-on, SARS-CoV-2 protein produced.

Figure 3 |. Correlation of SARS-CoV-2 protein-mediated cell death with induction of oxidative stress in fission yeast.

(A) SARS-CoV-2 protein induces oxidative stress, as indicated by the DHE staining showing the production of ROS. Images were taken 48 h agi. Scale bar = 10 μM. BF, bright field; ROS, reactive oxidative species. DHE, an oxidative stress-specific dye (5, 58). (B) SARS-CoV-2 protein-induced cell death was measured 48 h agi by the trypan blue staining. (C) Quantitative correlation of SARS-CoV-2 protein-induced cell death (blue bars) and the production of ROS (red bars). Data represents mean ± SE from three independent experiments. Complete SARS-CoV-2 genome-wide data on fission yeast cell death and ROS production are included in Fig. S4.

Figure 4 |. SARS-CoV-2 ORF3a-induced apoptosis and necrosis are correlated with the induction of cellular oxidative stress and innate immune pro-inflammatory responses in mammalian cells.

Expression of SARS-CoV-2 ORF3a induces cellular growth reduction and cell death 72 hpt in human lung epithelial A549 cells (A) and human kidney epithelial 293T cells (B). (C) ORF3a induces apoptosis and necrosis 48 hpt measured by Annexin V (a), necrosis (b) and caspase-3 cleavage (c). (D) ORF3a triggers the induction of oxidative stress 48 hpt measured by the DHE straining. The ORF3a was cloned in a lentiviral constitutive expression vector (3). Scale bar = 20 μM. (E) ORF3a triggers elevated production of TNF-α, IL-6 and NF-κB in Calu-3 (a) and 293T (b) cells. Data are presented as mean ± SE from three independent experiments. Statistical differences between ORF3a and mock (indicated with #) or empty vector control (indicated with *) were evaluated. * or #, p < 0.05; ** or ##, p < 0.01; *** or ###, p < 0.001 (Pair-wise t-test).

Figure 5 |. ORF3a-induced apoptosis by natural and artificial mutant variants correlate with cellular oxidative stress and innate immune pro-inflammatory responses.

293T cells were transfected with plasmids harboring ORF3a wild type (WT), ΔG188 or Q57H mutant variant. Effects of ORF3a mutant variants on cytopathic effects (A), as measured by cellular growth (a), cell viability (b) and cell death (c) at the indicated times. (B) Effects of ORF3a mutant variants on apoptosis (a) and necrosis (b). (C) Induction of oxidative stress. The images were taken at 72 hpt. Scale bar = 20 μM. (D) Activation of cellular innate immune pro-inflammatory responses, as measured by qRT-PCR. Data are presented as mean ± SE from three independent experiments. Statistical differences between control and ORF3a WT or mutants (indicated with #) or between WT and mutants (indicated with *) were evaluated. * or #, p < 0.05; ** or ##, p < 0.01; *** or ###, p < 0.001 (one-way ANOVA).