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.SignificanceThe ongoing SARS-CoV-2 pandemic has claimed over 5 million lives with more than 250 million people infected world-wide. While vaccines are effective, the emergence of new viral variants could jeopardize vaccine protection. Antiviral drugs provide an alternative to battle against COVID-19. Our goal was to identify viral therapeutic targets that can be used in antiviral drug discovery. Utilizing a genome-wide functional analysis in a fission yeast cell-based system, we identified twelve viral candidates, including ORF3a, which cause cellular oxidative stress, inflammation and apoptosis and necrosis that contribute to COVID-19. Our findings indicate that antiviral agents targeting ORF3a could greatly impact COVID-19.

A novel diG motif in ORF3a protein of SARS-Cov-2 for intracellular transport

“…Based on the ORF3a protein structural analysis ( Kern et al, 2021 ), we found a unique diG motif between the anti-parallel β4 and β5 sheets. These two glycine residues are part of a type II β-turn that could potentially be critical in maintaining the protein structure ( Zhang et al, 2022a ). Since the dCR1 deletion covers the diG residues, and we previously showed that deletion of the G188 residue (dG188) significantly enhanced the cytopathic effect of ORF3a ( Zhang et al, 2022a ; Zhang et al, 2022b ), we decided to focus on characterizing the possible role of the diG residues in intracellular transport.…”

“…Based on the current literature, the ORF3a (Open-Reading Frame 3a) protein of SARS-CoV-2 could be one of the viral proteins that contribute to COVID-19, as it is well-known for its role in viral pathogenesis and its activities have been linked to cell death and tissue damages ( Ren et al, 2020 ; Silvas et al, 2021 ; Zhang et al, 2022a ; McGrath et al, 2022 ), induction of cytokine storm that is a major cause of COVID-19-related death ( Siu et al, 2019 ; Xu et al, 2022 ) and the severity of COVID-19 ( Issa et al, 2020 ; Majumdar and Niyogi, 2020 ; Lednicky et al, 2021 ; Nagy et al, 2021 ). Furthermore, ORF3a is uniquely shared by SARS-CoV and SARS-CoV-2 within the genus of β-coronaviruses ( Kern et al, 2021 ).…”

“…Even though a number of well conserved functional motifs of ORF3a are linked to various functionalities ( Tan et al, 2004 ; Lu et al, 2006 ; Minakshi and Padhan, 2014 ; Siu et al, 2019 ; Jin et al, 2021 ; Kern et al, 2021 ), the functional relationship of some ORF3a activities with the overall structure of ORF3a protein remains unknown. For instance, we recently showed that ORF3a induces apoptosis and necrosis through the induction of host cellular oxidative stress-mediated reactive oxygen species (ROS) production and NF-κB-mediated pro-inflammatory cytokine productions including TNFα and IL-6 ( Zhang et al, 2022a ), which are two strong and independent survival predictors of the patient with COVID-19 ( Del Valle et al, 2020 ; Sayah et al, 2021 ). Interestingly, a single amino acid deletion at a residue G188 (∆G188) resulted in a marked increase in the ORF3a-induced cytopathic effects ( Zhang et al, 2022a ).…”

“…For instance, we recently showed that ORF3a induces apoptosis and necrosis through the induction of host cellular oxidative stress-mediated reactive oxygen species (ROS) production and NF-κB-mediated pro-inflammatory cytokine productions including TNFα and IL-6 ( Zhang et al, 2022a ), which are two strong and independent survival predictors of the patient with COVID-19 ( Del Valle et al, 2020 ; Sayah et al, 2021 ). Interestingly, a single amino acid deletion at a residue G188 (∆G188) resulted in a marked increase in the ORF3a-induced cytopathic effects ( Zhang et al, 2022a ). The region where G188 resides is not in any one of the known functional domains.…”

Pathogenic Mechanism and Multi-omics Analysis of Oral Manifestations in COVID-19

“…Its RNA encodes four major structural proteins, which include spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N) (41) [Figure 1]. Besides, 16 nonstructural proteins (NSPs) and 9 accessory proteins are included in the 29 proteins encoded (42).…”