SARS-CoV-2-ORF3a variant Q57H reduces its pro-apoptotic activity in host cells

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Cypress" }, { "@type": "Person", "name": "Isabel Chaput" }, { "@type": "Person", "name": "Bridget Nieto" }, { "@type": "Person", "name": "Bong Sook Jhun" }, { "@type": "Person", "name": "Jin O-Uchi" } ], "publisher": { "@type": "Organization", "name": "F1000Research", "logo": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 480, "width": 60 } }, "image": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 1200, "width": 150 }, "description": " Background Mutations in the viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can enhance its pathogenicity by affecting its transmissibility, disease severity, and overall mortality in human populations. In addition to mutations within the coding region of SARS-CoV-2 structural proteins, there have been reports of mutations in other SARS-CoV-2 proteins that affect virulence, such as open reading frame 3a (ORF3a), which is involved in viral replication. The expression of ORF3a in host cells activates cell death signaling, leading to tissue damage, which affects the severity of COVID-19. The ORF3a-Q57H variant is the most frequent and recurrent variant of ORF3a and is likely associated with increased transmissibility but lower mortality in the 4th epidemic wave of COVID-19 in Hong Kong. Computational structural modeling predicted that the Q57H variant destabilizes the protein structure of ORF3a, which may result in reduced protein expression in human cells. However, it is still unknown how this mutation affects ORF3a protein function and, if so, whether it can change the severity of host cell damage. Methods Plasmids carrying SARS-CoV-2-ORF3a from Wuhan-Hu-1 strain (i.e., wild-type; WT) and its variant Q57H were transiently transfected into HEK293T cells and used for biochemical and cell biological assays. Results SARS-CoV-2-ORF3a-Q57H variant exhibits higher protein expression than WT, but ORF3a-Q57H expression results in less apoptosis in host cells compared to WT via lower activation of the extrinsic apoptotic pathway. Conclusion The relatively mild phenotype of the SARS-CoV-2-ORF3a-Q57H variant may result from alterations to ORF3a function by this mutation, rather than its protein expression levels in host cells. 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F1000Research 2024, 13 :331 ( https://doi.org/10.12688/f1000research.146123.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Brief Report SARS-CoV-2-ORF3a variant Q57H reduces its pro-apoptotic activity in host cells [version 1; peer review: 1 approved with reservations, 1 not approved] Maria Landherr 1 , Iuliia Polina 1 , Michael W. Cypress 1 , [...] Isabel Chaput https://orcid.org/0000-0003-1890-3518 1 , Bridget Nieto https://orcid.org/0009-0005-0751-8157 1 , Bong Sook Jhun https://orcid.org/0000-0001-9916-9962 1 , Jin O-Uchi https://orcid.org/0000-0001-7566-5400 1 Maria Landherr 1 , Iuliia Polina 1 , [...] Michael W. Cypress 1 , Isabel Chaput https://orcid.org/0000-0003-1890-3518 1 , Bridget Nieto https://orcid.org/0009-0005-0751-8157 1 , Bong Sook Jhun https://orcid.org/0000-0001-9916-9962 1 , Jin O-Uchi https://orcid.org/0000-0001-7566-5400 1 PUBLISHED 23 Apr 2024 Author details Author details 1 Medicine, University of Minnesota Twin Cities, Minneapolis, Minnesota, 55455, USA Maria Landherr Roles: Formal Analysis, Investigation, Writing – Original Draft Preparation, Writing – Review & Editing Iuliia Polina Roles: Conceptualization, Formal Analysis, Funding Acquisition, Investigation, Supervision, Validation, Writing – Review & Editing Michael W. Cypress Roles: Investigation, Writing – Review & Editing Isabel Chaput Roles: Investigation, Writing – Review & Editing Bridget Nieto Roles: Investigation, Writing – Review & Editing Bong Sook Jhun Roles: Funding Acquisition, Investigation, Validation, Writing – Review & Editing Jin O-Uchi Roles: Conceptualization, Formal Analysis, Funding Acquisition, Investigation, Methodology, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Cell & Molecular Biology gateway. This article is included in the Coronavirus (COVID-19) collection. Abstract Background Mutations in the viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can enhance its pathogenicity by affecting its transmissibility, disease severity, and overall mortality in human populations. In addition to mutations within the coding region of SARS-CoV-2 structural proteins, there have been reports of mutations in other SARS-CoV-2 proteins that affect virulence, such as open reading frame 3a (ORF3a), which is involved in viral replication. The expression of ORF3a in host cells activates cell death signaling, leading to tissue damage, which affects the severity of COVID-19. The ORF3a-Q57H variant is the most frequent and recurrent variant of ORF3a and is likely associated with increased transmissibility but lower mortality in the 4th epidemic wave of COVID-19 in Hong Kong. Computational structural modeling predicted that the Q57H variant destabilizes the protein structure of ORF3a, which may result in reduced protein expression in human cells. However, it is still unknown how this mutation affects ORF3a protein function and, if so, whether it can change the severity of host cell damage. Methods Plasmids carrying SARS-CoV-2-ORF3a from Wuhan-Hu-1 strain (i.e., wild-type; WT) and its variant Q57H were transiently transfected into HEK293T cells and used for biochemical and cell biological assays. Results SARS-CoV-2-ORF3a-Q57H variant exhibits higher protein expression than WT, but ORF3a-Q57H expression results in less apoptosis in host cells compared to WT via lower activation of the extrinsic apoptotic pathway. Conclusion The relatively mild phenotype of the SARS-CoV-2-ORF3a-Q57H variant may result from alterations to ORF3a function by this mutation, rather than its protein expression levels in host cells. READ ALL READ LESS Keywords mitochondria, apoptosis, cell death, cell signaling Corresponding Author(s) Jin O-Uchi ( [email protected] ) Close Corresponding author: Jin O-Uchi Competing interests: No competing interests were disclosed. Grant information: • National Heart, Lung, and Blood Institute R01HL136757 • National Heart, Lung, and Blood Institute R01HL160699 • Institute of Engineering in Medicine at University of Minnesota COVID19 Response Grant • Office of Academic Clinical Affairs at University of Minnesota COVID19 Response Grants • Institute of Engineering in Medicine at University of Minnesota IEM Annual Conference Pilot Project Grant • American Heart Association 18CDA34110091 The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2024 Landherr M et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Landherr M, Polina I, Cypress MW et al. SARS-CoV-2-ORF3a variant Q57H reduces its pro-apoptotic activity in host cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :331 ( https://doi.org/10.12688/f1000research.146123.1 ) First published: 23 Apr 2024, 13 :331 ( https://doi.org/10.12688/f1000research.146123.1 ) Latest published: 22 Nov 2025, 13 :331 ( https://doi.org/10.12688/f1000research.146123.2 )  There is a newer version of this article available. Suppress this message for one day. Introduction COVID-19 is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the global pandemic that began in 2020. 1 SARS-CoV-2 can produce 29 proteins, including 9 accessory proteins encoded by open reading frames. 2 , 3 These proteins were originally identified as critical factors for viral entry, viral genome production and replication, virion morphogenesis, and viral release from the host cells. 2 , 4 Mutations in the SARS-CoV-2 genome can alter its pathogenic potential, ultimately affecting the severity and transmissivity of COVID-19 in humans. 5 Since 2020, the World Health Organization has been identifying, tracking, characterizing, and labeling some SARS-CoV-2 variants as “variants of interest” and “variants of concern” to prioritize global monitoring and research. 6 Thirty-six non-synonymous and 78 synonymous mutations have been reported in open reading frame 3a (ORF3a), which is the largest accessory protein in the SARS-CoV-2 genome. 7 The 25563G>T-(Q57H) variant is the most common ORF3a variant (30-40%) reported in COVID-19 patients in the US, and the next most frequent ORF3a variant is 10 times less prevalent than Q57H. 8 Q57 is located near the end of the first transmembrane domain of ORF3a, facing the hydrophobic lipid interface, 9 , 10 which changes the amino acid glutamine (Q), which has a non-charged polar side chain, into the positively charged amino acid histidine (H). ORF3a-Q57H was first identified in Singapore in 2020 and has since been observed in the COVID-19 Beta, epsilon, and Mu variants. 3 Q57H was the only mutant consistently reported with a high frequency in the entire period of 2020, whereas the frequency of the other ORF3a mutations fluctuated. 10 In the fourth epidemic wave of COVID-19 in Hong Kong, this variant was associated with increased transmission and decreased mortality rates. 11 Viral samples isolated from patients during this wave did not exhibit enhanced replication kinetics or cytokine/chemokine induction in the host cells. A recent study using computational modeling 9 predicted that the Q57H mutation may decrease protein stability and increase the rigidity of the ORF3a protein compared to the original Wuhan-Hu-1 strain ( i.e. , wild-type; WT), which likely affects downstream signaling in host cells. However, it has not been established whether the Q57H mutant affects the function of ORF3, its role in host cell damage during SARS-CoV-2 infection, and ultimately the severity of COVID-19 phenotypes in patients. Based on the computational prediction of negative folding stability in SARS-CoV-2-Q57H by Wang et al., 9 we hypothesized that the SARS-CoV-2-ORF3a-Q57H variant produces less protein expression in host cells than WT-ORF3a, thus exhibiting less oxidative stress and apoptosis, which may contribute to decreased mortality in COVID-19 patients. Here, we report that the SARS-CoV-2-ORF3a-Q57H variant does not exhibit lower protein expression, but rather exhibits a relatively higher expression compared to the WT. Moreover, this variant expression causes less activation of the extrinsic apoptotic pathway in the host cells. Our findings may support the potential molecular linkage between this major mutation and a mild phenotype, but higher transmissibility, in COVID-19 patients. Methods Plasmids, antibodies, and reagents The antibodies and plasmids used in the experiments are listed in Tables 1 and 2 , respectively. All the cells, chemicals and reagents were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA) unless otherwise listed in Table 3 . Table 1. List of commercial primary antibodies used in this study. Targeted Protein Type Company Catalog number, RRID Immunogen GFP Mouse monoclonal Sigma-Aldrich, St. Louis, MO, USA 1181446000, AB_390913 Recombinant Aequorea victoria GFP GFP Rabbit monoclonal Cell Signaling Technology, Danvers, MA, USA 2956, AB_1196615 A synthetic peptide corresponding to the amino terminus of GFP Tubulin Mouse monoclonal Sigma-Aldrich T5168, AB_477579 Sarkosyl-resistant filaments from S. purpuratus (sea urchin) sperm axonemes Mitofusin 2 (Mfn2) Rabbit polyclonal Sigma-Aldrich M6319, AB_477221 Synthetic peptide corresponding to amino acid residues 38-55 of human Mfn2 with C-terminal added cysteine, conjugated to KLH Cleaved caspase 3 (Asp175) Rabbit polyclonal Cell Signaling Technology 9661T, AB_2341188 N-terminal residues adjacent to (Asp175) in human caspase-3 HtrA2/Omi Rabbit monoclonal Cell Signaling Technology 9745, AB_11220423 Peptide corresponding to residues surrounding Phe341 of human HtrA2/Omi protein Optic atrophy-1 (OPA1) Mouse monoclonal BD Bioscience, Franklin Lakes, NJ, USA 612607, AB_399889 Amino acids 708-830 from human OPA1 Cyclophilin D (CyD) Mouse monoclonal Thermo Fisher Scientific, Waltham, MA, USA 455900, AB_2533820 Recombinant rat Cyclophilin F Caspase 1 (Cleaved Asp210) Rabbit polyclonal Thermo Fisher Scientific PA599390, AB_2818323 Synthetic peptide corresponding to amino acid residues I280-V299 from human CASP1 (Accession P29466). LC3 A/B Microtubule-associated protein 1A/1B-light chain 3 Rabbit monoclonal Cell Signaling Technology 12741, AB_2617131 Peptide corresponding to residues surrounding Leu44 of human LC3B protein (conserved in LC3A) IL-1β/IL-1F2 Rabbit polyclonal Novus Biologicals, Centennial, CO, USA NB600-633, AB_10001060 Recombinant human IL-1β/IL-1F2 produced in E. coli. Glucose-regulated protein with sequence homology to Hsp90 (Grp94) Rabbit monoclonal Cell Signaling Technology 20292, AB_2722657 Peptide corresponding to residues surrounding Leu397 of human Grp94 Glucose-regulated protein with sequence homology to Hsp70 (Grp78/Bip) Rabbit monoclonal Cell Signaling Technology 3177, AB_2119845 Peptide corresponding to residues surrounding Gly584 of human BiP C/EBP-homologous protein (CHOP) Mouse monoclonal Cell Signaling Technology 2895, AB_2089254 Peptide corresponding to the sequence of human CHOP NLR family pyrin domain-containing protein 3 (NLRP3) Rabbit polyclonal Thermo Fisher Scientific PA5-79740, AB_2746855 Synthetic peptide corresponding to a sequence at the N-terminus of human CIAS1 Bid Rabbit polyclonal Novus Biologicals NB100-56106SS, AB_2065641 Full-length recombinant mouse Bid protein Strep tag II Rabbit polyclonal GenScript, Piscataway NJ, USA A00626, AB_915541 Epitope tag peptide NWSHPQFEK conjugated to KLH Argonaute 2 (Argo2) Rabbit monoclonal Cell Signaling Technology 2897S, AB_2096291 Synthetic peptide corresponding to mouse argonaute 2 Cytochrome C Mouse monoclonal BD Bioscience 556433, AB_396417 Synthetic peptides of pigeon Cytochrome C Table 2. List of plasmids used in this study. Inserted gene Vector Backbone Source/Provider/RRID (if available) Company Notes Ref. Mitochondrial matrix-targeted DsRed (mt-RFP) pDsRed1-N1 (Clontech, Mountain View, CA, USA #6921-1) Dr. Yisang Yoon 38 Empty pEGFP-C1 (Clonetech) Clontech SARS-CoV-2-ORF3a- P2A-eGFP pcDNA3.1+P2A-eGFP (GenScript) GenScript ORF3a was tagged with GFP by bridging “self-cleaving” small polypeptides (P2A) SARS-CoV-2-ORF3a-GFP pcDNA3.1+C-eGFP (RRID:Addgene_129020) GenScript SARS-CoV-2-ORF3a Q57H-GFP pcDNA3.1+C-eGFP (RRID:Addgene_129020) GenScript This construct was generated by PCR-based site-directed mutagenesis using the SARS-CoV-2-ORF3a-GFP constructs as a template. SARS-CoV-2-ORF3a-Q57H-P2A-GFP pcDNA3.1+P2A-eGFP (GenScript) GenScript This construct was generated by PCR-based site-directed mutagenesis using the SARS-CoV-2-ORF3a- P2A-eGFP constructs as a template. SARS-CoV-2-orf3a-2xStrep pLVX-EF1alpha-IRES-Puro (Clontech) Dr. Nevan Krogan/RRID: Addgene_141383 Addgene, Watertown, MA, USA Addgene plasmi # 141383 , 39 Mouse MCU-L-GFP pEGFP-N1 (Clontech) Dr. Rosario Rizzuto 40 Empty pLVX-EF1α-IRES-puro ZAGENO, Cambridge, MA, USA PVT2308 Table 3. List of Specific cells, chemicals and regents used in this study. Name of cells, chemical/reagents Supplier Catalog number Notes Ref. HEK293T cells Dr. Keigi Fujiwara, University of Texas, MD Anderson Cancer Center, Houston, TX, USA N/A Used in Figure 1 . 13 H9c2 rat cardiac myoblasts ATCC, Manassas, VA, USA CRL-1446 Used in Figures 2 - 5 . 12 Interleukin 1β (IL-1β)/IL-1F2 recombinant protein R&D Systems, Minneapolis, MN, USA 501-RL Used in Figure 3A . 100 ng of recombinant IL-1β was used for the positive control for the western blotting. Z-LEHD-FMK ApexBio, Houston, TX,USA B3233 Used in Figure 5D . Z-LEHD-FMK (PubChem CID: 10032582) was dissolved in DMSO and used for the final concentration of 20 μM. Caspase-8 Staining Kit (Red) Abnova. Taipei City, Taiwan KA0760 Used in Figure 4A - C . One μL of Red-IETD-FMK (PubChem CID 25108681) was added to 300 μl of cell culture medium and cells were incubated for 30 min at 37°C incubator with 5% CO 2 . The caspase inhibitor Z-VAD-FMK (PubChem SID: 404336810) at 1 μl/ml was added to inhibit caspase activation. 41 FuGENE HD Promega, Madison, WI, USA E2312 Used in Figures 1 - 5 . 0.5-3 μg of plasmids and 7 µl of FuGENE HD was added to 100 μL Opti-mem (Thermo Fisher Scientific) at room temperature. The mixture was incubated for 15 min and added to 2 ml cell culture medium. Cell lysis buffer Cell Signaling Technology 9803S Used in Figures 1 - 5 . Two hundred μl of 1x Cell lysis buffer were used for each 6-cm dish to harvest protein. Fluorescence-conjugated secondary antibodies LI-COR Biosciences, Lincoln, NE, USA 926-32211 and 926-68020 Used in Figures 1 - 5 . Secondary antibodies were added in 0.05% PBST (1;20, 000 dilution). The nitrocellulose membrane was incubated with secondary antibody-containing PBST for 1 hr at room temperature. NucView® 405 substrates Biotium, Fremont, CA, USA 10407 Used in Figure 2C and D . PBS containing 2 µM NucView® 405 substrate was treated to the cells at room temperature for 30 min before observation. 42 Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Red Fluorescence* AAT Bioquest, Pleasanton CA, USA 22817 Used in Figure 5D . Five μL of 200X Ac-LEHD-ProRed™ stock solution was added to 1 mL of Assay Buffer provided from the manufacturer to make caspase 9 substrate working solution. Cells were incubated with the working solution at room temperature for 1 hr, before observation. 43 Cell culture and transfection Study protocol was approved by the Institutional Biosafety Committee at University of Minnesota (#2305-41075H). HEK293T and H9c2 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 4.5 g/L glucose, 1 mM sodium pyruvate, 1% L-glutamine, 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C with 5% CO 2 in a humidified incubator, transfected with plasmids (0.5-3 μg/3.5-cm dish) using Fugene HD, and used for experiments 48 to 72-hr after transfection. 12 , 13 Western blot analysis Mitochondria-enriched fractions were separated from cytosolic fractions by different centrifugation speeds and dissolved with lysis buffer containing protease inhibitor cocktails and 1 mM phenylmethylsulfonyl fluoride, and subjected to western blotting. 12 , 13 The immunoreactive bands were visualized, and the whole blotting images for each figure panel 14 were obtained using a near-infrared fluorescence imaging system (LI-COR Biotechnology, Lincoln, NE, USA). 12 , 13 Live cell imaging Cells stained with cell-permeable dyes ( Table 3 ) were observed by an FV3000 confocal microscope (Olympus, Tokyo, Japan) at room temperature. Localization of GFP-tagged proteins was observed in H9c2 cells stably overexpressing mitochondrial matrix-targeted DsRed (mt-RFP) and the colocalization efficiency was estimated using Pearson’s correlation coefficient. 12 Statistics All data are presented as the mean ± standard error (SEM). Unpaired Student’s t-test and one-way ANOVA followed by Tukey’s post-hoc test were performed for two-group comparisons and multiple comparisons, respectively, with statistical significance defined as a p-value < 0.05. Results We first tested the effect of Q57H on ORF3a protein expression levels in HEK293T cells by transiently expressing non-tagged and GFP-tagged SARS-CoV-2-ORF3a ( Figure 1A ). In the plasmid expressing non-tagged ORF3a, ORF3a is tagged with GFP by bridging “self-cleaving” small polypeptides (P2A), 15 allowing for bicistronic expression of non-tagged ORF3a proteins and GFP. Small amounts of the non-cleaved form also existed, but ORF3a protein expression levels were estimated by the amount of cleaved GFP ( Figure 1B ). Contrary to computational predictions, 9 we did not observe a significant decrease in ORF3a protein expression by Q57H mutations, but rather ORF3a-Q57H exhibited relatively higher protein expression compared to WT-ORF3a ( Figure 1B - D ) as assessed by immunostaining with GFP antibody. Thus, differences in protein expression levels between WT and mutant ORF3a are more likely to occur via post-translational processes rather than transcriptional regulation. Figure 1. ORF3a-Q57H exhibits higher protein expression compared to WT-ORF3a. A. SARS-CoV-2-ORF3a constructs used in this study. B. Expression of WT- and Q57H mutant ORF3a-P2A-GFP in HEK293T cells. The pEGFP-C1 empty plasmid expressing only EGFP was shown as a control. Each construct was transfected with 0.5, 1.0, or 3.0 μg per 3.5 cm dish. Tubulin was used as a loading control. CTR, cells with no transfection. IB, immunoblotting C. Expression of WT- and Q57H-ORF3a-GFP constructs in HEK293T cells. D. Summary data of B and C (n = 4). In each panel, band intensity was normalized to the value from 0.5 μg of WT- ORF3a construct transfection. Using a computational model, Wang et al. predicted that the ORF3a protein structure becomes more rigid and less flexible after the Q57H mutation 9 and may result in less activation of downstream signaling that causes host cell damage. We tested cellular damage by WT-ORF3a and Q57H-ORF3a proteins in H9c2 cardiac myoblasts because this cell line is more vulnerable to oxidative, apoptotic, and inflammatory signaling than cancer cell lines, including HEK293T cells. 12 , 16 We used non-tagged ORF3a constructs ( i.e. , ORF3a-P2A-GFP, Figure 1A ) for this assay to avoid the potential impact of tag modification on ORF3a protein activity. GFP was used as the control. A recent report has shown that SARS-CoV-2-ORF3a expression can activate apoptotic signaling. 17 We found that the expression of WT-ORF3a, but not ORF3a-Q57H, increased caspase-3 activity in H9c2 cells, as assessed by the amount of cleaved caspase-3 ( Figure 2A and B ). Caspase-3 activity was also evaluated by live-cell staining with a fluorogenic DNA dye coupled to the caspase-3/7 DEVD recognition sequence (NucView® substrates). GFP itself produced a population of apoptotic cells as reported, 18 but WT-ORF3a expression significantly increased the number of apoptotic cells compared to GFP ( Figure 2C and D ). The number of apoptotic cells in Q57H cells was similar to that in GFP cells and significantly lower than that in WT-ORF3a cells ( Figure 2C and D ). Figure 2. WT-ORF3a but not Q57H activates apoptotic signaling. A. Cleaved caspase-3 in H9c2 cells overexpressing WT and mutant ORF3a. GFP was transfected as a control. Each construct was transfected with 0.5, 1.0, or 3.0 μg per 3.5-cm dish. B. Summary data of A (n= 5). All values were normalized to the value from 0.5 μg of the transfected GFP control. * p <0.05. C. Detection of caspase-3 activity in live H9c2 cells stained with Nucview 405 Caspase-3. GFP-positive cells were selected as transfected cells, and nuclear staining-positive cells by fluorogenic DNA dye were counted as apoptotic cells under the confocal microscopy. D. Summary data of C from three independent experiments. * p <0.05. N.S., not significant . In addition to apoptotic responses, several groups have shown that the expression of SARS-CoV-2-ORF3a constructs with protein tags activate inflammatory signaling, endoplasmic reticulum (ER) stress, and autophagy flux. 19 – 24 First, non-tagged WT-ORF3a and Q57H did not produce significant inflammatory responses, as assessed by the protein expression levels of IL-1β, NLRP3, and cleaved caspase-1 ( Figure 3A - D ). The expression of ER stress markers, including glucose-regulated protein 94 (Grp94), glucose-regulated protein 78 (Bip/Grp78), and C/EBP-homologous protein (CHOP), 25 did not change after the expression of either WT-ORF3a or -Q57H in our system ( Figure 3E and F ). Finally, both WT-ORF3a and its mutant Q57H showed a similar tendency of increased microtubule-associated protein light chain 3 (LC3)-II/LC3-I ratio, a standard marker indicating the induction of autophagy, but these changes were not significant compared to control cells transfected with GFP ( Figure 3G and H ). In summary, SARS-CoV-2-ORF3a expression induces apoptotic signaling activation rather than modulating inflammation, ER stress, and autophagic signaling cascades. Importantly, Q57H expression was less involved in apoptotic signaling activation than that of WT-ORF3a. Figure 3. ORF3a-WT and -Q57H do not significantly activate inflammatory, endoplasmic reticulum (ER) stress, and autophagy signaling. A. Assessment of inflammatory activity by cleaved caspase-1 and IL-1β in H9c2 cells expressing WT and mutant ORF3a. GFP was transfected as a control. Cell lysates treated with nigericin (15 μm for 60 min) and a recombinant IL-1β protein were used as positive controls. All values were normalized to the value from 0.5 μg of the transfected GFP control. B. Summary data of A (n=3). C. Detection of the NLRP3 inflammasome in H9c2 cells expressing WT and mutant ORF3a. All values were normalized to the value from the cells transfected with 0.5 μg of GFP. D. Summary data of C (n= 4). E. Assessment of the expression of ER stress markers Grp78, Grp94, and CHOP in H9c2 cells transfected with WT and mutant ORF3a. GFP was transfected as a control. F. Summary data of E (n=4, n =3, n=3, respectively). G. Assessment of autophagic flux by the LC3-II/LC3-I ratio in H9c2 cells expressing WT and mutant ORF3a. GFP was transfected as a control. H. Summary data of G (n=5). The ratio of LC3-II (low molecular weight) to LC3-I (high molecular weight) was calculated and normalized to the value from 0.5 ug of the transfected GFP control. A recent report showed that SARS-CoV-2-ORF3a-ORF3a could activate both the intrinsic and extrinsic pathways of apoptosis. 17 Therefore, we examined the activities of signaling molecules from both apoptotic pathways after WT-ORF3a or ORF3a-Q57H expression. The extrinsic pathway caspase-8 was significantly activated by WT-ORF3a expression compared to that in control cells, as assessed by a cell-permeable caspase-8 activity marker, Red-IETD-FMK. However, preincubation with a general caspase inhibitor, Z-VAD-FMK, abolished this change ( Figure 4A and B ). Q57H expression did not show significant caspase-8 activation ( Figure 4A and B ), and this difference between ORF3a-WT and -Q57H was unlikely based on the expression levels of the constructs, as confirmed by the expression levels of bicistronically expressed GFP ( Figure 4B and C ). Caspase-8 is activated by an extrinsic pathway (e.g., cell-surface death receptors) and is known to propagate the apoptotic signal either by directly cleaving and activating downstream caspases (e.g., caspase-3) or by cleaving Bid. 26 Cells expressing WT-ORF3a (but only in the lower transfection conditions) showed a significant increase in the truncated Bid (tBid)/Bid ratio, but not by Q57H ( Figure 4D and E ). These results suggest that the Q57H variant exhibits less activation of the extrinsic apoptotic pathway compared to the WT. Figure 4. ORF3a-Q57H exhibits less activation of extrinsic apoptotic signaling compared to WT. A. Representative confocal images of H9c2 cells transfected with the indicated plasmids and stained with a cell-permeable marker dye for caspase-8 activation, Red-IETD-FMK. Red-IETD-FMK was detected under confocal microscopy with excitation and emission wavelengths of 488 and 570 nm, respectively. ORF3a-WT overexpressed cells pretreated with Z-VAD-FMK for 1 hr were used as a negative control. Scale Bars = 20 μm B. Summary data of A. * p <0.05, compared to GFP-transfected cells. Each fluorescence value was normalized to the average fluorescence calculated from GFP-transfected cells. C. Scatter plots of GFP and Red-IETD-FMK measured from individual cells. D. Representative immunoblot of tBid/Bid in H9c2 cells expressing WT and mutant ORF3a. GFP was transfected as a control. E. Summary data of D. * p <0.05, compared to 0.5 ug of the transfected GFP control. * p <0.05. Next, we investigated the effect of WT-ORF3a and ORF3a-Q57H on the intrinsic apoptotic pathway. Since the SARS-CoV-2-ORF3a protein is predicted to possess three transmembrane domains similar to SARS-CoV-1-ORF3a, and its subcellular localization is likely distributed to several membrane structures, 27 we next tested whether ORF3a can be expressed in the mitochondria. Indeed, ORF3a protein was found in the mitochondria-enriched fraction compared to that in the cytosolic fraction ( Figure 5A ). Both WT-ORF3a and Q57H-ORF3a were partially localized in the mitochondrial area labeled by mt-RFP, and their subcellular distribution patterns were not significantly different, as assessed by the values of Pearson’s correlation coefficient ( Figure 5B and C ). We also found that the Q57H variant was capable of activating caspase-9, and assessed caspase-9 activity, an initiator of intrinsic apoptosis, whose level was comparable to that in WT assessed by Ac-LEHD-ProRed staining ( Figure 5D ). In summary, these results suggest that the different caspase-3 activation levels in WT-ORF3a and Q57H-ORF3a are mainly due to their different effects on the extrinsic apoptotic pathway. Figure 5. Both WT-ORF3a and ORF3a-Q57H variant activate intrinsic apoptotic signaling. A. Expression of WT-ORF3a-Strep in fractionated proteins from H9c2 cells. Cells transfected with pLVX-EF1α-IRES-puro were used as a control (CTR). Argonaute 2 (Argo2) and optic atrophy-1 (OPA1) were used as markers for the cytosolic fraction (C) and mitochondrial fraction (M), respectively. Whole cell lysates (W) were shown for comparison. B. Representative confocal images of the subcellular localization of GFP, WT-ORF3a-GFP, ORF3a-Q57H-GFP, mitochondrial Ca 2+ uniporter (MCU)-GFP (as a positive control) in live H9c2 cells stably expressing mt-RFP. Scale bars = 20 μm. C. Summary data of the mitochondrial localization of GFP constructs estimated by Pearson’s correlation values between the GFP and mt-RFP signals. *p<0.05. N.S., not significant. Cells transfected with a mitochondrial protein MCU-GFP were used as a positive control. D. Assessment of caspase-9 activity in live H9c2 cells transfected with indicated plasmids stained with a cell-permeable caspase-9-specific fluorogenic substrate, Ac-LEHD-ProRed. ORF3a-WT overexpressed cells pretreated for 1 hr with a caspase 9-specific inhibitor, Z-LEHD-FMK, were used as a negative control. ProRed cleaved from Ac-LEHD-ProRed was detected using confocal microscopy with excitation and emission wavelengths of 540 and 620 nm, respectively. The ProRed fluorescence value was normalized to the average fluorescence calculated from GFP-transfected cells. Discussion Although the protein sequences of ORF3a from SARS-CoV-1 and CoV-2 have only moderate homology (72%), 3 the expression of both proteins in mammalian cells promotes apoptosis. 17 Our results showed that Q57H, the most frequent and recurrent variant of SARS-CoV-2-ORF3a, exhibits higher protein expression compared to SARS-CoV-2-ORF3a-WT ( Figure 1 ) but induces less apoptosis in host cells due to a lack of extrinsic apoptotic pathway activation ( Figures 2 - 4 , and 6 ). This property may provide advantages for the SARS-CoV-2-Q57H infection to be relatively mild, thus allowing the virus to have higher transmissivity, as was the case in the fourth epidemic wave of COVID-19 in Hong Kong. 11 Figure 6. Current Model: SARS-CoV-2-ORF3a-Q57H causes less apoptosis via less activation of the extrinsic apoptotic pathway. SARS-CoV-2-ORF3a induces apoptosis in host cells via activating both intrinsic and extrinsic pathways. ORF3a-Q57H shows less apoptotic activity compared to WT via less activation of the extrinsic apoptotic pathway. Although the SARS-CoV-2-ORF3a protein can induce apoptotic signaling activation similar to SARS-CoV-1-ORF3a, 28 Ren et al. recently reported that SARS-CoV-2-ORF3a has a relatively weaker effect on activating apoptotic signaling than SARS-CoV-1. 17 Moreover, they showed that plasma membrane localization of ORF3a is required for activating apoptotic signaling in SARS-CoV-2. Ren et al. suggested that 1) SARS-CoV-2-ORF3a mainly activates the extrinsic apoptotic pathway, and 2) the intrinsic pathway is secondarily activated downstream of the extrinsic apoptotic pathway. Importantly, the location of the Q57H mutation in the ORF3a structure is far from the key motifs for plasma membrane sorting (i.e., cysteine-rich motif C130/133 and/or tyrosine-based sorting motif Y160 17 ). This indicates that the mutation does not likely interfere with the plasma membrane sorting of the RFF3a protein, although neither SARS-CoV-2-ORF3a-WT nor -Q57H showed specific plasma membrane expression localized in multiple cellular compartments, including the cytosol and mitochondria ( Figure 5B ). Another key finding was that SARS-CoV-2-ORF3a-WT was able to activate the extrinsic apoptotic pathway in the absence of death receptor ligands ( Figures 4 and 6 ). This observation 17 suggests that it is likely that ORF3a at the plasma membrane is capable of 1) transactivating death receptors (DRs) by direct or indirect interactions with DRs at the plasma membrane, 2) causing conformational changes in the death-inducing signaling complex (DISC) (i.e., association of the receptor-bound Fas-associated cytoplasmic death domain [FADD] and caspase-8), 29 and/or 3) inhibiting the activity of cellular FADD-like IL-1β-converting enzyme-inhibitory proteins such as c-FLIP. 29 Because Q57H is located near the end of the first transmembrane domain of ORF3a, which is close to the cytoplasmic face, 9 , 10 the Q57H mutation may alter the interaction between ORF3a, DRs, and/or DISC within or beneath the plasma membrane. Further studies are required to identify the detailed molecular mechanisms by which ORF3a activates DRs and/or DISC, and whether the Q57H mutation alters this mechanism. Our data also showed that WT-ORF3a and ORF3a-Q57H both activated intrinsic apoptotic signaling at similar levels, even though Q57H exhibited less activation of extrinsic apoptotic signaling compared to WT ( Figures 4 - 6 ). This result indicates that SARS-CoV-2-ORF3a can initiate intrinsic apoptotic signaling independent of extrinsic apoptotic signaling ( Figure 6 ). In both SARS-CoV-1 and -CoV-2, ORF3a has three predicted transmembrane domains 3 , 10 and has been localized in several cellular membrane structures/organelles in host cells, including the plasma membrane, endoplasmic reticulum, Golgi, and lysosomes. 30 – 34 Our protein fractionation and imaging data showed that ORF3a was also localized in the mitochondria ( Figure 5A - C ), where it likely increased mitochondrial membrane permeability and promoted the release of apoptotic proteins. SARS-CoV-1-ORF3a can form K + -permeable viroporins 28 , 34 that are required to induce ORF3a-mediated cell apoptosis. 28 Although still controversial, 33 SARS-CoV-2-ORF3a might also form K + -permeable channels at the inner mitochondrial membrane (IMM), which can depolarize the mitochondrial membrane potential similar to the opening of endogenous K + channels expressed at the IMM, such as the mitochondrial BK Ca channel. 35 If ORF3a is expressed in the outer mitochondrial membrane (OMM), it is possible that ORF3a may interact with structural proteins that regulate OMM permeability. Lastly, we tested whether SARS-CoV-2-ORF3a can modulate autophagy flux, ER stress, and inflammatory signaling in addition to apoptosis, but these signaling pathways were not significantly activated in our system ( Figure 3 ). The different results may be partly due to the use of different cell types, which may provide different ORF3a expression levels and/or sensitivity to the stress-signaling pathway. In addition, the majority of published data 19 – 24 , 36 were generated from ORF3a constructs with various protein tags, which may alter ORF3a protein function because it is a relatively small protein (~30 kDa). Nevertheless, our results clearly showed a major difference in the activation of apoptotic signaling between ORF3a-WT and Q57H, especially in the extrinsic signaling pathway. In summary, despite its relatively higher protein expression compared to WT, SARS-CoV-2-ORF3a-Q57H variant expression causes less apoptosis in mammalian cells because of lower activation of the extrinsic apoptotic pathway. As our experiments were performed only in cultured cell lines transfected with a part of SARS-CoV-2 (i.e., ORF3a), we still need to consider that our findings cannot be directly applicable to the in vivo situation with SARS-CoV-2 infection. Animal models using SARS-CoV-2 are indispensable for exploring the detailed role of the ORF3a signaling pathway in vivo. Despite these limitations, our results suggest that the relatively mild phenotype of the Q57H variant observed in 4th epidemic wave of COVID-19 in Hong Kong and several COVID-19 variants (i.e., Beta, Epsilon, and Mu) may result from weaker pro-apoptotic signaling. Assessing the cellular effects of ORF3a mutations will improve our understanding of the pathophysiology of COVID-19 and inform the design of new therapeutic strategies to prevent and treat COVID-19 and its long-term symptoms. 37 Data availability statement Figshare: Supplementary materials for manuscript “SARS-CoV-2-ORF3a variant Q57H reduces its pro-apoptotic activity in host cells”. https://doi.org/10.6084/m9.figshare.24803106.v1 . 14 This project contains the following underlying data: Original Western blotting images Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication). Acknowledgements This study was partially supported by NIH/NHLBI R01HL136757 (to J.O.-U.) and R01HL160699 (to B.S.J.), a COVID19 Response Grant (to J.O.-U.) from the Institute of Engineering in Medicine (IEM) at the University of Minnesota (UMN) and COVID-19 Response Grants (to J.O.-U. and B.S.J.) from the Office of Academic Clinical Affairs at UMN, the American Heart Association 18CDA34110091 (to B.S.J), and the IEM Annual Conference Pilot Project Grant (to I.P.) from IEM at UMN. References 1. Al-Awwal N, Dweik F, Mahdi S, et al. : A Review of SARS-CoV-2 Disease (COVID-19): Pandemic in Our Time. Pathogens. 2022; 11 : 368. PubMed Abstract | Publisher Full Text | Free Full Text 2. Yadav R, Chaudhary JK, Jain N, et al. : Role of Structural and Non-Structural Proteins and Therapeutic Targets of SARS-CoV-2 for COVID-19. Cells. 2021; 10 : 821. PubMed Abstract | Publisher Full Text | Free Full Text 3. 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Cancer Cell. 2016; 30 : 925–939. PubMed Abstract | Publisher Full Text | Free Full Text 43. Rath M, Schwefel K, Malinverno M, et al. : Contact-dependent signaling triggers tumor-like proliferation of CCM3 knockout endothelial cells in co-culture with wild-type cells. Cell. Mol. Life Sci. 2022; 79 : 340–346. PubMed Abstract | Publisher Full Text | Free Full Text Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 23 Apr 2024 ADD YOUR COMMENT Comment Author details Author details 1 Medicine, University of Minnesota Twin Cities, Minneapolis, Minnesota, 55455, USA Maria Landherr Roles: Formal Analysis, Investigation, Writing – Original Draft Preparation, Writing – Review & Editing Iuliia Polina Roles: Conceptualization, Formal Analysis, Funding Acquisition, Investigation, Supervision, Validation, Writing – Review & Editing Michael W. Cypress Roles: Investigation, Writing – Review & Editing Isabel Chaput Roles: Investigation, Writing – Review & Editing Bridget Nieto Roles: Investigation, Writing – Review & Editing Bong Sook Jhun Roles: Funding Acquisition, Investigation, Validation, Writing – Review & Editing Jin O-Uchi Roles: Conceptualization, Formal Analysis, Funding Acquisition, Investigation, Methodology, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information • National Heart, Lung, and Blood Institute R01HL136757 • National Heart, Lung, and Blood Institute R01HL160699 • Institute of Engineering in Medicine at University of Minnesota COVID19 Response Grant • Office of Academic Clinical Affairs at University of Minnesota COVID19 Response Grants • Institute of Engineering in Medicine at University of Minnesota IEM Annual Conference Pilot Project Grant • American Heart Association 18CDA34110091 The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (2) version 2 Revised Published: 22 Nov 2025, 13:331 https://doi.org/10.12688/f1000research.146123.2 version 1 Published: 23 Apr 2024, 13:331 https://doi.org/10.12688/f1000research.146123.1 Copyright © 2024 Landherr M et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Landherr M, Polina I, Cypress MW et al. SARS-CoV-2-ORF3a variant Q57H reduces its pro-apoptotic activity in host cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :331 ( https://doi.org/10.12688/f1000research.146123.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 23 Apr 2024 Views 0 Cite How to cite this report: Liu W. Reviewer Report For: SARS-CoV-2-ORF3a variant Q57H reduces its pro-apoptotic activity in host cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :331 ( https://doi.org/10.5256/f1000research.160166.r290352 ) The direct URL for this report is: https://f1000research.com/articles/13-331/v1#referee-response-290352 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 27 Jun 2024 Wei Liu , Henan Agricultural University, Zhengzhou, China Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.160166.r290352 In their manuscript, Landherr et al. showed that SARS-CoV-2-ORF3a variant Q57H reduces its pro-apoptotic activity in host cells. As a consequence, SARS-CoV-2-ORF3a-Q57H variant does not exhibit lower protein expression, but rather exhibits a relatively higher expression compared to the WT. ... Continue reading READ ALL In their manuscript, Landherr et al. showed that SARS-CoV-2-ORF3a variant Q57H reduces its pro-apoptotic activity in host cells. As a consequence, SARS-CoV-2-ORF3a-Q57H variant does not exhibit lower protein expression, but rather exhibits a relatively higher expression compared to the WT. Finally, the authors demonstrated that SARS-CoV-2-ORF3a-Q57H variant expression causes less activation of the extrinsic apoptotic pathway in the host cells. This study adds may support the potential molecular linkage between this major mutation and a mild phenotype. Despite the interesting phenotypic observations, several major issues must be addressed before further consideration. 1) The writing lacks clarity and logical coherence, and these issues must be thoroughly resolved to enhance the manuscript's overall quality and scientific rigor. For instance, the sentence “we hypothesized that the SARS-CoV-2-ORF3a-Q57H variant produces less protein expression in host cells than” in the final part of the Introduction, is more appropriate to move this hypothetical sentence in the Discussion. 2)- In Figure 1B, there are two GFP indicator arrow? In addition, no trend of increasing concentration was observed. The WB-blot need to be repeated. 3)- In Figure 1D, the band intensity should be was normalized to the value from Tubulin. 4)- In Figure 2A, the molecular weight of the caspase-3 should be displayed. 5)- In Figure 3, the molecular weight of the proteins should be displayed. Figure 3A, the “cleaved” IL-1β should be shown in the panel. Figure 3G, although statistical analysis shows no significant difference in LC3-II to LC3-I, the he expression of LC3-II suggests that there is the activation in t ORF3a-WT and -Q57H. Maybe more representative WB-blot need to be displayed. 6)- In Figure 4, the tBid or Bid need to be indicated clearly in panel. 7)- “caspase-8” “Caspase 8” are inconsistent in manuscript. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Virus infection and immunity; Interaction between viruses and host cells I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Liu W. Reviewer Report For: SARS-CoV-2-ORF3a variant Q57H reduces its pro-apoptotic activity in host cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :331 ( https://doi.org/10.5256/f1000research.160166.r290352 ) The direct URL for this report is: https://f1000research.com/articles/13-331/v1#referee-response-290352 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 08 Nov 2025 Jin O-Uchi , Medicine, University of Minnesota Twin Cities, Minneapolis, 55455, USA 08 Nov 2025 Author Response We appreciate the constructive comments from the reviewers and editor. We have addressed all of them in our revisions. 1) The writing lacks clarity and logical coherence, and these ... Continue reading We appreciate the constructive comments from the reviewers and editor. We have addressed all of them in our revisions. 1) The writing lacks clarity and logical coherence, and these issues must be thoroughly resolved to enhance the manuscript's overall quality and scientific rigor. For instance, the sentence “we hypothesized that the SARS-CoV-2-ORF3a-Q57H variant produces less protein expression in host cells than” in the final part of the Introduction, is more appropriate to move this hypothetical sentence in the Discussion. A: Following the reviewer’s suggestion, we moved this sentence to the revised discussion section. 2) In Figure 1B, there are two GFP indicator arrow? In addition, no trend of increasing concentration was observed. The WB-blot need to be repeated. A: The two arrows indicate the cleaved form (corresponding to the size of GFP) and non-cleaved (the size of ORF3a + P2A + GFP) form of ORF3a-P2A-GFP. The reviewer is correct that we could not observe a concentration-dependent increase in GFP expression from the transfection of either ORF3a-P2A-GFP or ORF3a-Q57H-P2A-GFP. Following another reviewer’s suggestion, we changed our assessment method to use an anti-ORF3a antibody. We have now observed a concentration-dependent increase in ORF3a protein levels. We included ths new data set as a revised Figure 1. The original Figure 1 using anti-GFP antibody has been moved to the online Supplementary Materials as a Supplementary Figure (see https://doi.org/10.6084/m9.figshare.30473513.v1). 3) In Figure 1D, the band intensity should be normalized to the value from Tubulin . A: The band intensity was normalized to the value from tubulin The 0.5 µg GFP/tubulin was set as 1 and showed as relative values in each panel (See online Supplementary Figure 1D). We also clarified all the normalization procedures in each panel in the main figure set 4) In Figure 2A, the molecular weight of the caspase-3 should be displayed . A: Added. See also the supplementary materials https://doi.org/10.6084/m9.figshare.30473513.v1 5-1) In Figure 3, the molecular weight of the proteins should be displayed. A: Added. See also the supplementary materials https://doi.org/10.6084/m9.figshare.30473513.v1 5-2) Figure 3A, the “cleaved” IL-1β should be shown in the panel. A: Added “cleaved”. 5-3) Figure 3G, although statistical analysis shows no significant difference in LC3-II to LC3-I, the expression of LC3-II suggests that there is the activation in the ORF3a-WT and -Q57H. Maybe more representative WB-blot need to be displayed. A: We performed three additional independent experiments and analyzed a total of 8 experiments. We confirmed that there is still no significant difference. New representative images were shown. See also the supplementary materials https://doi.org/10.6084/m9.figshare.30473513.v1 6) In Figure 4, the tBid or Bid need to be indicated clearly in panel. A: Added the label for tBid to Figure 4 C. 7) “caspase-8” “Caspase 8” are inconsistent in manuscript. A: Revised and made consistent with caspase-8 in the revised manuscript. We appreciate the constructive comments from the reviewers and editor. We have addressed all of them in our revisions. 1) The writing lacks clarity and logical coherence, and these issues must be thoroughly resolved to enhance the manuscript's overall quality and scientific rigor. For instance, the sentence “we hypothesized that the SARS-CoV-2-ORF3a-Q57H variant produces less protein expression in host cells than” in the final part of the Introduction, is more appropriate to move this hypothetical sentence in the Discussion. A: Following the reviewer’s suggestion, we moved this sentence to the revised discussion section. 2) In Figure 1B, there are two GFP indicator arrow? In addition, no trend of increasing concentration was observed. The WB-blot need to be repeated. A: The two arrows indicate the cleaved form (corresponding to the size of GFP) and non-cleaved (the size of ORF3a + P2A + GFP) form of ORF3a-P2A-GFP. The reviewer is correct that we could not observe a concentration-dependent increase in GFP expression from the transfection of either ORF3a-P2A-GFP or ORF3a-Q57H-P2A-GFP. Following another reviewer’s suggestion, we changed our assessment method to use an anti-ORF3a antibody. We have now observed a concentration-dependent increase in ORF3a protein levels. We included ths new data set as a revised Figure 1. The original Figure 1 using anti-GFP antibody has been moved to the online Supplementary Materials as a Supplementary Figure (see https://doi.org/10.6084/m9.figshare.30473513.v1). 3) In Figure 1D, the band intensity should be normalized to the value from Tubulin . A: The band intensity was normalized to the value from tubulin The 0.5 µg GFP/tubulin was set as 1 and showed as relative values in each panel (See online Supplementary Figure 1D). We also clarified all the normalization procedures in each panel in the main figure set 4) In Figure 2A, the molecular weight of the caspase-3 should be displayed . A: Added. See also the supplementary materials https://doi.org/10.6084/m9.figshare.30473513.v1 5-1) In Figure 3, the molecular weight of the proteins should be displayed. A: Added. See also the supplementary materials https://doi.org/10.6084/m9.figshare.30473513.v1 5-2) Figure 3A, the “cleaved” IL-1β should be shown in the panel. A: Added “cleaved”. 5-3) Figure 3G, although statistical analysis shows no significant difference in LC3-II to LC3-I, the expression of LC3-II suggests that there is the activation in the ORF3a-WT and -Q57H. Maybe more representative WB-blot need to be displayed. A: We performed three additional independent experiments and analyzed a total of 8 experiments. We confirmed that there is still no significant difference. New representative images were shown. See also the supplementary materials https://doi.org/10.6084/m9.figshare.30473513.v1 6) In Figure 4, the tBid or Bid need to be indicated clearly in panel. A: Added the label for tBid to Figure 4 C. 7) “caspase-8” “Caspase 8” are inconsistent in manuscript. A: Revised and made consistent with caspase-8 in the revised manuscript. Competing Interests: none Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 08 Nov 2025 Jin O-Uchi , Medicine, University of Minnesota Twin Cities, Minneapolis, 55455, USA 08 Nov 2025 Author Response We appreciate the constructive comments from the reviewers and editor. We have addressed all of them in our revisions. 1) The writing lacks clarity and logical coherence, and these ... Continue reading We appreciate the constructive comments from the reviewers and editor. We have addressed all of them in our revisions. 1) The writing lacks clarity and logical coherence, and these issues must be thoroughly resolved to enhance the manuscript's overall quality and scientific rigor. For instance, the sentence “we hypothesized that the SARS-CoV-2-ORF3a-Q57H variant produces less protein expression in host cells than” in the final part of the Introduction, is more appropriate to move this hypothetical sentence in the Discussion. A: Following the reviewer’s suggestion, we moved this sentence to the revised discussion section. 2) In Figure 1B, there are two GFP indicator arrow? In addition, no trend of increasing concentration was observed. The WB-blot need to be repeated. A: The two arrows indicate the cleaved form (corresponding to the size of GFP) and non-cleaved (the size of ORF3a + P2A + GFP) form of ORF3a-P2A-GFP. The reviewer is correct that we could not observe a concentration-dependent increase in GFP expression from the transfection of either ORF3a-P2A-GFP or ORF3a-Q57H-P2A-GFP. Following another reviewer’s suggestion, we changed our assessment method to use an anti-ORF3a antibody. We have now observed a concentration-dependent increase in ORF3a protein levels. We included ths new data set as a revised Figure 1. The original Figure 1 using anti-GFP antibody has been moved to the online Supplementary Materials as a Supplementary Figure (see https://doi.org/10.6084/m9.figshare.30473513.v1). 3) In Figure 1D, the band intensity should be normalized to the value from Tubulin . A: The band intensity was normalized to the value from tubulin The 0.5 µg GFP/tubulin was set as 1 and showed as relative values in each panel (See online Supplementary Figure 1D). We also clarified all the normalization procedures in each panel in the main figure set 4) In Figure 2A, the molecular weight of the caspase-3 should be displayed . A: Added. See also the supplementary materials https://doi.org/10.6084/m9.figshare.30473513.v1 5-1) In Figure 3, the molecular weight of the proteins should be displayed. A: Added. See also the supplementary materials https://doi.org/10.6084/m9.figshare.30473513.v1 5-2) Figure 3A, the “cleaved” IL-1β should be shown in the panel. A: Added “cleaved”. 5-3) Figure 3G, although statistical analysis shows no significant difference in LC3-II to LC3-I, the expression of LC3-II suggests that there is the activation in the ORF3a-WT and -Q57H. Maybe more representative WB-blot need to be displayed. A: We performed three additional independent experiments and analyzed a total of 8 experiments. We confirmed that there is still no significant difference. New representative images were shown. See also the supplementary materials https://doi.org/10.6084/m9.figshare.30473513.v1 6) In Figure 4, the tBid or Bid need to be indicated clearly in panel. A: Added the label for tBid to Figure 4 C. 7) “caspase-8” “Caspase 8” are inconsistent in manuscript. A: Revised and made consistent with caspase-8 in the revised manuscript. We appreciate the constructive comments from the reviewers and editor. We have addressed all of them in our revisions. 1) The writing lacks clarity and logical coherence, and these issues must be thoroughly resolved to enhance the manuscript's overall quality and scientific rigor. For instance, the sentence “we hypothesized that the SARS-CoV-2-ORF3a-Q57H variant produces less protein expression in host cells than” in the final part of the Introduction, is more appropriate to move this hypothetical sentence in the Discussion. A: Following the reviewer’s suggestion, we moved this sentence to the revised discussion section. 2) In Figure 1B, there are two GFP indicator arrow? In addition, no trend of increasing concentration was observed. The WB-blot need to be repeated. A: The two arrows indicate the cleaved form (corresponding to the size of GFP) and non-cleaved (the size of ORF3a + P2A + GFP) form of ORF3a-P2A-GFP. The reviewer is correct that we could not observe a concentration-dependent increase in GFP expression from the transfection of either ORF3a-P2A-GFP or ORF3a-Q57H-P2A-GFP. Following another reviewer’s suggestion, we changed our assessment method to use an anti-ORF3a antibody. We have now observed a concentration-dependent increase in ORF3a protein levels. We included ths new data set as a revised Figure 1. The original Figure 1 using anti-GFP antibody has been moved to the online Supplementary Materials as a Supplementary Figure (see https://doi.org/10.6084/m9.figshare.30473513.v1). 3) In Figure 1D, the band intensity should be normalized to the value from Tubulin . A: The band intensity was normalized to the value from tubulin The 0.5 µg GFP/tubulin was set as 1 and showed as relative values in each panel (See online Supplementary Figure 1D). We also clarified all the normalization procedures in each panel in the main figure set 4) In Figure 2A, the molecular weight of the caspase-3 should be displayed . A: Added. See also the supplementary materials https://doi.org/10.6084/m9.figshare.30473513.v1 5-1) In Figure 3, the molecular weight of the proteins should be displayed. A: Added. See also the supplementary materials https://doi.org/10.6084/m9.figshare.30473513.v1 5-2) Figure 3A, the “cleaved” IL-1β should be shown in the panel. A: Added “cleaved”. 5-3) Figure 3G, although statistical analysis shows no significant difference in LC3-II to LC3-I, the expression of LC3-II suggests that there is the activation in the ORF3a-WT and -Q57H. Maybe more representative WB-blot need to be displayed. A: We performed three additional independent experiments and analyzed a total of 8 experiments. We confirmed that there is still no significant difference. New representative images were shown. See also the supplementary materials https://doi.org/10.6084/m9.figshare.30473513.v1 6) In Figure 4, the tBid or Bid need to be indicated clearly in panel. A: Added the label for tBid to Figure 4 C. 7) “caspase-8” “Caspase 8” are inconsistent in manuscript. A: Revised and made consistent with caspase-8 in the revised manuscript. Competing Interests: none Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Leibowitz J and Na Pombejra S. Reviewer Report For: SARS-CoV-2-ORF3a variant Q57H reduces its pro-apoptotic activity in host cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :331 ( https://doi.org/10.5256/f1000research.160166.r290354 ) The direct URL for this report is: https://f1000research.com/articles/13-331/v1#referee-response-290354 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 24 Jun 2024 Julian Leibowitz , Microbial Pathogenesis and Immunology, Texas A&M School of Medicine, Bryan, Texas, USA Sarisa Na Pombejra , Microbial Pathogenesis and Immunology, Texas A&M University School of Medicine, Bryan, Texas, USA Not Approved VIEWS 0 https://doi.org/10.5256/f1000research.160166.r290354 In this manuscript Landherr et al compare the effect of the common SARS-CoV-2 ORF3a Q57H mutation to the parental sequence on host cells using ectopic over expression strategies. The manuscript presents the interesting finding that over expression of the ORF3a ... Continue reading READ ALL In this manuscript Landherr et al compare the effect of the common SARS-CoV-2 ORF3a Q57H mutation to the parental sequence on host cells using ectopic over expression strategies. The manuscript presents the interesting finding that over expression of the ORF3a Q57H protein decreases apoptosis relative to that observed by the parental protein, and that this difference is due to greater activation of the extrinsic apoptosis pathway by the parental protein. These data are the strength of the manuscript. However, there are several critical issues not adequately explained or addressed. Below are specific comments and suggestions to improve the manuscript. Specific Comments 1. Differences in ORF3a Protein Expression: -The expression strategy employed is somewhat unusual, in creating an ORF3a-p2A-GFP fusion construct in which the primary translation product undergoes efficient self-cleavage mediated by its picornavirus p2A protease domain, liberating GFP and ORF3a-p2A polypeptides. This allows tracking transfected cells by GFP fluorescence and the comparison of ORF3a-p2A expression between variant and parental proteins indirectly by performing immunoblots for GFP and quantitating the amount of the fusion proteins and liberated GFP. They report that, contrary to a computational prediction by Wang et al that the Q57H variant will be less stable than the parental protein, that it is somewhat more stable. A weakness of this approach is it ignores the possibility that the p2A-GFP fusion to ORF3a has a stabilizing effect on the fusion protein. Furthermore, by utilizing immunoblots to GFP it makes the assumption that liberated ORF3a-p2A peptides from parental and Q57H variants degrade at equivalent rates and that GFP immunoblots reflect the amount of ORF3a-p2A protein. It would have strengthened the paper to have directly measured the relative amounts of ORF3a-p2A protein with an anti-ORF3a antibody. -The statement "Differences in protein expression levels between WT and mutant ORF3a are more likely to occur via post translational processes rather than transcriptional regulation" (Results, Paragraph 1). It is not clear what the relevance of transcriptional regulation in transfected cells is when expression of the ORF3a mRNA is regulated quite differently in the context of SARS-CoV-2 infected cells than in transfected cells. Even in the context of transfected cells this claim would need experimental evidence, such as qRT-PCR for transcriptional analysis, post-translational modification studies, or protein folding studies. 2. Differing Results From Previous Studies: -The results negative related to ORF3a effects on inflammasome activation, ER stress, and autophagy in this paper differ from other reported studies. The authors should discuss potential reasons for these discrepancies. One obvious difference regarding inflammasome activation is the use of H9c2 cells, a cardiomyoblast cell line, in the current work. It is likely that these cells are not competent for inflammasome activation since Figure 3A shows that pharmacologic activation of inflammasomes by Nigericin fails to activate inflammasomes (IL-beta or caspase-1 cleavage) in these cells. 3. Mechanistic Studies on Q57H Mutation: -The manuscript lacks detailed mechanistic studies on how the Q57H mutation could affect extrinsic apoptosis pathway activation. For example, the authors should have examined the effect of individual death receptor knockdown on ORF3a induction of the extrinsic pathway for apoptosis to both identify the receptor activated and confirm their results. Additionally, the authors should investigate and discuss the potential alterations in ORF3a interaction with death receptors, and any differences in downstream signaling activation between WT and Q57H. Comparative studies between WT and Q57H regarding their interactions and signaling pathways would significantly enhance the manuscript's contribution to the field. Conclusion: The manuscript presents interesting findings but requires substantial improvements in experimental support and detailed mechanistic studies. Addressing the points mentioned above will strengthen the manuscript and provide a more comprehensive understanding of the ORF3a protein's role and its mutations. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Replication and pathogenesis of coronaviruses We confirm that we have read this submission and believe that we have an appropriate level of expertise to state that we do not consider it to be of an acceptable scientific standard, for reasons outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Leibowitz J and Na Pombejra S. Reviewer Report For: SARS-CoV-2-ORF3a variant Q57H reduces its pro-apoptotic activity in host cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :331 ( https://doi.org/10.5256/f1000research.160166.r290354 ) The direct URL for this report is: https://f1000research.com/articles/13-331/v1#referee-response-290354 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 08 Nov 2025 Jin O-Uchi , Medicine, University of Minnesota Twin Cities, Minneapolis, 55455, USA 08 Nov 2025 Author Response We appreciate the constructive comments from the reviewers and editor. We have addressed all of them in our revisions. (It would have strengthened the paper to have directly measured ... Continue reading We appreciate the constructive comments from the reviewers and editor. We have addressed all of them in our revisions. (It would have strengthened the paper to have directly measured the relative amounts of ORF3a-p2A protein with an anti-ORF3a antibody.) A: Following the reviewer’s suggestion, we used an anti-ORF3a antibody instead of an anti-GFP antibody to re-evaluate the expression levels of WT-ORF3a and ORF3a-Q57H mutant, and replaced the previous data obtained using the GFP antibody with this new data (see Revised Figure 1A-C). When the high amounts of plasmid (≥1.0 µg/well ) were transfected, ORF3a-WT and -Q57H constructs produced similar levels of ORF3a protein, as assessed with ORF3a-specific antibody (see revised Figure 1B and C). However, under lower amounts of plasmid transfection (0.5 µg/well), we found a significantly higher protein expression of Q57H compared to WT-ORF3a (see revised Figure 1B and C). Based on this new data, we have revised the abstract, result section, and discussion section. (It is not clear what the relevance of transcriptional regulation in transfected cells is when expression of the ORF3a mRNA is regulated quite differently in the context of SARS-CoV-2 infected cells than in transfected cells. Even in the context of transfected cells this claim would need experimental evidence, such as qRT-PCR for transcriptional analysis, post-translational modification studies, or protein folding studies.) A: The qRT-PCR experiment was conducted, and the results were shown in the revised Figure 1D. We found no significant differences in mRNA levels between ORF3a-WT and -Q57H in all transfection conditions. In response to the reviewer’s suggestion, we removed the statement "Differences in protein expression levels between WT and mutant ORF3a are more likely to occur via post-translational processes rather than transcriptional regulation” from the result section. (2. The results negative related to ORF3a effects on inflammasome activation, ER stress, and autophagy in this paper differ from other reported studies. The authors should discuss potential reasons for these discrepancies. One obvious difference regarding inflammasome activation is the use of H9c2 cells, a cardiomyoblast cell line, in the current work. It is likely that these cells are not competent for inflammasome activation since Figure 3A shows that pharmacologic activation of inflammasomes by Nigericin fails to activate inflammasomes (IL-beta or caspase-1 cleavage) in these cells.) A: We used the established protocol (Using the higher concentration of nigericin (NG) with longer stimulation, see PMID: 34432650) and confirmed that NG can indeed activate inflammatory signaling in H9c2 cells as reported previously (see revised Figure 3A). Nevertheless, following the reviewer’s comment, we added the following sentence to the discussion section: “H9c2 cells may be less competent for inflammasome activation”. (-The manuscript lacks detailed mechanistic studies on how the Q57H mutation could affect extrinsic apoptosis pathway activation. For example, the authors should have examined the effect of individual death receptor knockdown on ORF3a induction of the extrinsic pathway for apoptosis to both identify the receptor activated and confirm their results. Additionally, the authors should investigate and discuss the potential alterations in ORF3a interaction with death receptors, and any differences in downstream signaling activation between WT and Q57H. Comparative studies between WT and Q57H regarding their interactions and signaling pathways would significantly enhance the manuscript's contribution to the field.) A: Following the reviewer’s suggestions, we further investigated the potential molecular mechanism underlying the ORF3a-mediated apoptosis and its alteration by the Q57H mutation. Although we did not screen the ORF3a interaction with all eight types of human death receptors due to the lack of reliable antibodies for co-immunoprecipitations, we found that ORF3a (but not Q57H) increases the protein expression of FADD, a crucial adaptor protein that acts as a bridge between death receptors and caspases to trigger extrinsic apoptotic pathways. The previous report showed that ORF3a expression at the plasma membrane (PM) is required for activating the apoptotic signaling (PMID: 32555321), ORF3a expression at the PM may enhance the stability of FADD protein within the death-inducing signaling complex (DISC) located just beneath the PM. Moreover, we found that the protein expression level of Q57H at the PM is lower thsan that of WT, which may contribute to the reduced activation of the extrinsic apoptotic pathways. We have included this new data in Figure 4D and E and revised the result section, figure legends, and discussion section accordingly. We appreciate the constructive comments from the reviewers and editor. We have addressed all of them in our revisions. (It would have strengthened the paper to have directly measured the relative amounts of ORF3a-p2A protein with an anti-ORF3a antibody.) A: Following the reviewer’s suggestion, we used an anti-ORF3a antibody instead of an anti-GFP antibody to re-evaluate the expression levels of WT-ORF3a and ORF3a-Q57H mutant, and replaced the previous data obtained using the GFP antibody with this new data (see Revised Figure 1A-C). When the high amounts of plasmid (≥1.0 µg/well ) were transfected, ORF3a-WT and -Q57H constructs produced similar levels of ORF3a protein, as assessed with ORF3a-specific antibody (see revised Figure 1B and C). However, under lower amounts of plasmid transfection (0.5 µg/well), we found a significantly higher protein expression of Q57H compared to WT-ORF3a (see revised Figure 1B and C). Based on this new data, we have revised the abstract, result section, and discussion section. (It is not clear what the relevance of transcriptional regulation in transfected cells is when expression of the ORF3a mRNA is regulated quite differently in the context of SARS-CoV-2 infected cells than in transfected cells. Even in the context of transfected cells this claim would need experimental evidence, such as qRT-PCR for transcriptional analysis, post-translational modification studies, or protein folding studies.) A: The qRT-PCR experiment was conducted, and the results were shown in the revised Figure 1D. We found no significant differences in mRNA levels between ORF3a-WT and -Q57H in all transfection conditions. In response to the reviewer’s suggestion, we removed the statement "Differences in protein expression levels between WT and mutant ORF3a are more likely to occur via post-translational processes rather than transcriptional regulation” from the result section. (2. The results negative related to ORF3a effects on inflammasome activation, ER stress, and autophagy in this paper differ from other reported studies. The authors should discuss potential reasons for these discrepancies. One obvious difference regarding inflammasome activation is the use of H9c2 cells, a cardiomyoblast cell line, in the current work. It is likely that these cells are not competent for inflammasome activation since Figure 3A shows that pharmacologic activation of inflammasomes by Nigericin fails to activate inflammasomes (IL-beta or caspase-1 cleavage) in these cells.) A: We used the established protocol (Using the higher concentration of nigericin (NG) with longer stimulation, see PMID: 34432650) and confirmed that NG can indeed activate inflammatory signaling in H9c2 cells as reported previously (see revised Figure 3A). Nevertheless, following the reviewer’s comment, we added the following sentence to the discussion section: “H9c2 cells may be less competent for inflammasome activation”. (-The manuscript lacks detailed mechanistic studies on how the Q57H mutation could affect extrinsic apoptosis pathway activation. For example, the authors should have examined the effect of individual death receptor knockdown on ORF3a induction of the extrinsic pathway for apoptosis to both identify the receptor activated and confirm their results. Additionally, the authors should investigate and discuss the potential alterations in ORF3a interaction with death receptors, and any differences in downstream signaling activation between WT and Q57H. Comparative studies between WT and Q57H regarding their interactions and signaling pathways would significantly enhance the manuscript's contribution to the field.) A: Following the reviewer’s suggestions, we further investigated the potential molecular mechanism underlying the ORF3a-mediated apoptosis and its alteration by the Q57H mutation. Although we did not screen the ORF3a interaction with all eight types of human death receptors due to the lack of reliable antibodies for co-immunoprecipitations, we found that ORF3a (but not Q57H) increases the protein expression of FADD, a crucial adaptor protein that acts as a bridge between death receptors and caspases to trigger extrinsic apoptotic pathways. The previous report showed that ORF3a expression at the plasma membrane (PM) is required for activating the apoptotic signaling (PMID: 32555321), ORF3a expression at the PM may enhance the stability of FADD protein within the death-inducing signaling complex (DISC) located just beneath the PM. Moreover, we found that the protein expression level of Q57H at the PM is lower thsan that of WT, which may contribute to the reduced activation of the extrinsic apoptotic pathways. We have included this new data in Figure 4D and E and revised the result section, figure legends, and discussion section accordingly. Competing Interests: None. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 08 Nov 2025 Jin O-Uchi , Medicine, University of Minnesota Twin Cities, Minneapolis, 55455, USA 08 Nov 2025 Author Response We appreciate the constructive comments from the reviewers and editor. We have addressed all of them in our revisions. (It would have strengthened the paper to have directly measured ... Continue reading We appreciate the constructive comments from the reviewers and editor. We have addressed all of them in our revisions. (It would have strengthened the paper to have directly measured the relative amounts of ORF3a-p2A protein with an anti-ORF3a antibody.) A: Following the reviewer’s suggestion, we used an anti-ORF3a antibody instead of an anti-GFP antibody to re-evaluate the expression levels of WT-ORF3a and ORF3a-Q57H mutant, and replaced the previous data obtained using the GFP antibody with this new data (see Revised Figure 1A-C). When the high amounts of plasmid (≥1.0 µg/well ) were transfected, ORF3a-WT and -Q57H constructs produced similar levels of ORF3a protein, as assessed with ORF3a-specific antibody (see revised Figure 1B and C). However, under lower amounts of plasmid transfection (0.5 µg/well), we found a significantly higher protein expression of Q57H compared to WT-ORF3a (see revised Figure 1B and C). Based on this new data, we have revised the abstract, result section, and discussion section. (It is not clear what the relevance of transcriptional regulation in transfected cells is when expression of the ORF3a mRNA is regulated quite differently in the context of SARS-CoV-2 infected cells than in transfected cells. Even in the context of transfected cells this claim would need experimental evidence, such as qRT-PCR for transcriptional analysis, post-translational modification studies, or protein folding studies.) A: The qRT-PCR experiment was conducted, and the results were shown in the revised Figure 1D. We found no significant differences in mRNA levels between ORF3a-WT and -Q57H in all transfection conditions. In response to the reviewer’s suggestion, we removed the statement "Differences in protein expression levels between WT and mutant ORF3a are more likely to occur via post-translational processes rather than transcriptional regulation” from the result section. (2. The results negative related to ORF3a effects on inflammasome activation, ER stress, and autophagy in this paper differ from other reported studies. The authors should discuss potential reasons for these discrepancies. One obvious difference regarding inflammasome activation is the use of H9c2 cells, a cardiomyoblast cell line, in the current work. It is likely that these cells are not competent for inflammasome activation since Figure 3A shows that pharmacologic activation of inflammasomes by Nigericin fails to activate inflammasomes (IL-beta or caspase-1 cleavage) in these cells.) A: We used the established protocol (Using the higher concentration of nigericin (NG) with longer stimulation, see PMID: 34432650) and confirmed that NG can indeed activate inflammatory signaling in H9c2 cells as reported previously (see revised Figure 3A). Nevertheless, following the reviewer’s comment, we added the following sentence to the discussion section: “H9c2 cells may be less competent for inflammasome activation”. (-The manuscript lacks detailed mechanistic studies on how the Q57H mutation could affect extrinsic apoptosis pathway activation. For example, the authors should have examined the effect of individual death receptor knockdown on ORF3a induction of the extrinsic pathway for apoptosis to both identify the receptor activated and confirm their results. Additionally, the authors should investigate and discuss the potential alterations in ORF3a interaction with death receptors, and any differences in downstream signaling activation between WT and Q57H. Comparative studies between WT and Q57H regarding their interactions and signaling pathways would significantly enhance the manuscript's contribution to the field.) A: Following the reviewer’s suggestions, we further investigated the potential molecular mechanism underlying the ORF3a-mediated apoptosis and its alteration by the Q57H mutation. Although we did not screen the ORF3a interaction with all eight types of human death receptors due to the lack of reliable antibodies for co-immunoprecipitations, we found that ORF3a (but not Q57H) increases the protein expression of FADD, a crucial adaptor protein that acts as a bridge between death receptors and caspases to trigger extrinsic apoptotic pathways. The previous report showed that ORF3a expression at the plasma membrane (PM) is required for activating the apoptotic signaling (PMID: 32555321), ORF3a expression at the PM may enhance the stability of FADD protein within the death-inducing signaling complex (DISC) located just beneath the PM. Moreover, we found that the protein expression level of Q57H at the PM is lower thsan that of WT, which may contribute to the reduced activation of the extrinsic apoptotic pathways. We have included this new data in Figure 4D and E and revised the result section, figure legends, and discussion section accordingly. We appreciate the constructive comments from the reviewers and editor. We have addressed all of them in our revisions. (It would have strengthened the paper to have directly measured the relative amounts of ORF3a-p2A protein with an anti-ORF3a antibody.) A: Following the reviewer’s suggestion, we used an anti-ORF3a antibody instead of an anti-GFP antibody to re-evaluate the expression levels of WT-ORF3a and ORF3a-Q57H mutant, and replaced the previous data obtained using the GFP antibody with this new data (see Revised Figure 1A-C). When the high amounts of plasmid (≥1.0 µg/well ) were transfected, ORF3a-WT and -Q57H constructs produced similar levels of ORF3a protein, as assessed with ORF3a-specific antibody (see revised Figure 1B and C). However, under lower amounts of plasmid transfection (0.5 µg/well), we found a significantly higher protein expression of Q57H compared to WT-ORF3a (see revised Figure 1B and C). Based on this new data, we have revised the abstract, result section, and discussion section. (It is not clear what the relevance of transcriptional regulation in transfected cells is when expression of the ORF3a mRNA is regulated quite differently in the context of SARS-CoV-2 infected cells than in transfected cells. Even in the context of transfected cells this claim would need experimental evidence, such as qRT-PCR for transcriptional analysis, post-translational modification studies, or protein folding studies.) A: The qRT-PCR experiment was conducted, and the results were shown in the revised Figure 1D. We found no significant differences in mRNA levels between ORF3a-WT and -Q57H in all transfection conditions. In response to the reviewer’s suggestion, we removed the statement "Differences in protein expression levels between WT and mutant ORF3a are more likely to occur via post-translational processes rather than transcriptional regulation” from the result section. (2. The results negative related to ORF3a effects on inflammasome activation, ER stress, and autophagy in this paper differ from other reported studies. The authors should discuss potential reasons for these discrepancies. One obvious difference regarding inflammasome activation is the use of H9c2 cells, a cardiomyoblast cell line, in the current work. It is likely that these cells are not competent for inflammasome activation since Figure 3A shows that pharmacologic activation of inflammasomes by Nigericin fails to activate inflammasomes (IL-beta or caspase-1 cleavage) in these cells.) A: We used the established protocol (Using the higher concentration of nigericin (NG) with longer stimulation, see PMID: 34432650) and confirmed that NG can indeed activate inflammatory signaling in H9c2 cells as reported previously (see revised Figure 3A). Nevertheless, following the reviewer’s comment, we added the following sentence to the discussion section: “H9c2 cells may be less competent for inflammasome activation”. (-The manuscript lacks detailed mechanistic studies on how the Q57H mutation could affect extrinsic apoptosis pathway activation. For example, the authors should have examined the effect of individual death receptor knockdown on ORF3a induction of the extrinsic pathway for apoptosis to both identify the receptor activated and confirm their results. Additionally, the authors should investigate and discuss the potential alterations in ORF3a interaction with death receptors, and any differences in downstream signaling activation between WT and Q57H. Comparative studies between WT and Q57H regarding their interactions and signaling pathways would significantly enhance the manuscript's contribution to the field.) A: Following the reviewer’s suggestions, we further investigated the potential molecular mechanism underlying the ORF3a-mediated apoptosis and its alteration by the Q57H mutation. Although we did not screen the ORF3a interaction with all eight types of human death receptors due to the lack of reliable antibodies for co-immunoprecipitations, we found that ORF3a (but not Q57H) increases the protein expression of FADD, a crucial adaptor protein that acts as a bridge between death receptors and caspases to trigger extrinsic apoptotic pathways. The previous report showed that ORF3a expression at the plasma membrane (PM) is required for activating the apoptotic signaling (PMID: 32555321), ORF3a expression at the PM may enhance the stability of FADD protein within the death-inducing signaling complex (DISC) located just beneath the PM. Moreover, we found that the protein expression level of Q57H at the PM is lower thsan that of WT, which may contribute to the reduced activation of the extrinsic apoptotic pathways. We have included this new data in Figure 4D and E and revised the result section, figure legends, and discussion section accordingly. Competing Interests: None. Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 23 Apr 2024 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 Version 2 (revision) 22 Nov 25 read read Version 1 23 Apr 24 read read Julian Leibowitz , Texas A&M School of Medicine, Bryan, USA Sarisa Na Pombejra , Texas A&M University School of Medicine, Bryan, USA Wei Liu , Henan Agricultural University, Zhengzhou, China Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 Liu W. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 30 Dec 2025 | for Version 2 Wei Liu , Henan Agricultural University, Zhengzhou, China 0 Views copyright © 2026 Liu W. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions I have carefully examined the revised manuscript and the authors' response to my earlier review comments. The authors have provided comprehensive and satisfactory responses to all the questions and concerns I raised previously. Corresponding revisions have been appropriately implemented throughout the manuscript, effectively addressing the issues identified in the initial review. Upon a detailed re-evaluation of the revised version, I have not found any new scientific flaws, logical inconsistencies, or technical inadequacies. I therefore recommend the manuscript for publication without further revisions. Competing Interests No competing interests were disclosed. Reviewer Expertise Virus infection and immunity; Interaction between viruses and host cells I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Liu W. Peer Review Report For: SARS-CoV-2-ORF3a variant Q57H reduces its pro-apoptotic activity in host cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :331 ( https://doi.org/10.5256/f1000research.190658.r435123) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-331/v2#referee-response-435123 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Leibowitz J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 25 Nov 2025 | for Version 2 Julian Leibowitz , Microbial Pathogenesis and Immunology, Texas A&M School of Medicine, Bryan, Texas, USA 0 Views copyright © 2025 Leibowitz J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions This revised manuscript is significantly improved from the prior submission and is now approved. I have no further comments. Competing Interests No competing interests were disclosed. Reviewer Expertise Replication and pathogenesis of coronaviruses I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Leibowitz J. Peer Review Report For: SARS-CoV-2-ORF3a variant Q57H reduces its pro-apoptotic activity in host cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :331 ( https://doi.org/10.5256/f1000research.190658.r435124) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-331/v2#referee-response-435124 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2024 Liu W. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 27 Jun 2024 | for Version 1 Wei Liu , Henan Agricultural University, Zhengzhou, China 0 Views copyright © 2024 Liu W. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions In their manuscript, Landherr et al. showed that SARS-CoV-2-ORF3a variant Q57H reduces its pro-apoptotic activity in host cells. As a consequence, SARS-CoV-2-ORF3a-Q57H variant does not exhibit lower protein expression, but rather exhibits a relatively higher expression compared to the WT. Finally, the authors demonstrated that SARS-CoV-2-ORF3a-Q57H variant expression causes less activation of the extrinsic apoptotic pathway in the host cells. This study adds may support the potential molecular linkage between this major mutation and a mild phenotype. Despite the interesting phenotypic observations, several major issues must be addressed before further consideration. 1) The writing lacks clarity and logical coherence, and these issues must be thoroughly resolved to enhance the manuscript's overall quality and scientific rigor. For instance, the sentence “we hypothesized that the SARS-CoV-2-ORF3a-Q57H variant produces less protein expression in host cells than” in the final part of the Introduction, is more appropriate to move this hypothetical sentence in the Discussion. 2)- In Figure 1B, there are two GFP indicator arrow? In addition, no trend of increasing concentration was observed. The WB-blot need to be repeated. 3)- In Figure 1D, the band intensity should be was normalized to the value from Tubulin. 4)- In Figure 2A, the molecular weight of the caspase-3 should be displayed. 5)- In Figure 3, the molecular weight of the proteins should be displayed. Figure 3A, the “cleaved” IL-1β should be shown in the panel. Figure 3G, although statistical analysis shows no significant difference in LC3-II to LC3-I, the he expression of LC3-II suggests that there is the activation in t ORF3a-WT and -Q57H. Maybe more representative WB-blot need to be displayed. 6)- In Figure 4, the tBid or Bid need to be indicated clearly in panel. 7)- “caspase-8” “Caspase 8” are inconsistent in manuscript. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Virus infection and immunity; Interaction between viruses and host cells I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 08 Nov 2025 Jin O-Uchi, Medicine, University of Minnesota Twin Cities, Minneapolis, 55455, USA We appreciate the constructive comments from the reviewers and editor. We have addressed all of them in our revisions. 1) The writing lacks clarity and logical coherence, and these issues must be thoroughly resolved to enhance the manuscript's overall quality and scientific rigor. For instance, the sentence “we hypothesized that the SARS-CoV-2-ORF3a-Q57H variant produces less protein expression in host cells than” in the final part of the Introduction, is more appropriate to move this hypothetical sentence in the Discussion. A: Following the reviewer’s suggestion, we moved this sentence to the revised discussion section. 2) In Figure 1B, there are two GFP indicator arrow? In addition, no trend of increasing concentration was observed. The WB-blot need to be repeated. A: The two arrows indicate the cleaved form (corresponding to the size of GFP) and non-cleaved (the size of ORF3a + P2A + GFP) form of ORF3a-P2A-GFP. The reviewer is correct that we could not observe a concentration-dependent increase in GFP expression from the transfection of either ORF3a-P2A-GFP or ORF3a-Q57H-P2A-GFP. Following another reviewer’s suggestion, we changed our assessment method to use an anti-ORF3a antibody. We have now observed a concentration-dependent increase in ORF3a protein levels. We included ths new data set as a revised Figure 1. The original Figure 1 using anti-GFP antibody has been moved to the online Supplementary Materials as a Supplementary Figure (see https://doi.org/10.6084/m9.figshare.30473513.v1). 3) In Figure 1D, the band intensity should be normalized to the value from Tubulin . A: The band intensity was normalized to the value from tubulin The 0.5 µg GFP/tubulin was set as 1 and showed as relative values in each panel (See online Supplementary Figure 1D). We also clarified all the normalization procedures in each panel in the main figure set 4) In Figure 2A, the molecular weight of the caspase-3 should be displayed . A: Added. See also the supplementary materials https://doi.org/10.6084/m9.figshare.30473513.v1 5-1) In Figure 3, the molecular weight of the proteins should be displayed. A: Added. See also the supplementary materials https://doi.org/10.6084/m9.figshare.30473513.v1 5-2) Figure 3A, the “cleaved” IL-1β should be shown in the panel. A: Added “cleaved”. 5-3) Figure 3G, although statistical analysis shows no significant difference in LC3-II to LC3-I, the expression of LC3-II suggests that there is the activation in the ORF3a-WT and -Q57H. Maybe more representative WB-blot need to be displayed. A: We performed three additional independent experiments and analyzed a total of 8 experiments. We confirmed that there is still no significant difference. New representative images were shown. See also the supplementary materials https://doi.org/10.6084/m9.figshare.30473513.v1 6) In Figure 4, the tBid or Bid need to be indicated clearly in panel. A: Added the label for tBid to Figure 4 C. 7) “caspase-8” “Caspase 8” are inconsistent in manuscript. A: Revised and made consistent with caspase-8 in the revised manuscript. View more View less Competing Interests none reply Respond Report a concern Liu W. Peer Review Report For: SARS-CoV-2-ORF3a variant Q57H reduces its pro-apoptotic activity in host cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :331 ( https://doi.org/10.5256/f1000research.160166.r290352) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-331/v1#referee-response-290352 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2024 Leibowitz J et al. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 24 Jun 2024 | for Version 1 Julian Leibowitz , Microbial Pathogenesis and Immunology, Texas A&M School of Medicine, Bryan, Texas, USA Sarisa Na Pombejra , Microbial Pathogenesis and Immunology, Texas A&M University School of Medicine, Bryan, Texas, USA 0 Views copyright © 2024 Leibowitz J et al. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions In this manuscript Landherr et al compare the effect of the common SARS-CoV-2 ORF3a Q57H mutation to the parental sequence on host cells using ectopic over expression strategies. The manuscript presents the interesting finding that over expression of the ORF3a Q57H protein decreases apoptosis relative to that observed by the parental protein, and that this difference is due to greater activation of the extrinsic apoptosis pathway by the parental protein. These data are the strength of the manuscript. However, there are several critical issues not adequately explained or addressed. Below are specific comments and suggestions to improve the manuscript. Specific Comments 1. Differences in ORF3a Protein Expression: -The expression strategy employed is somewhat unusual, in creating an ORF3a-p2A-GFP fusion construct in which the primary translation product undergoes efficient self-cleavage mediated by its picornavirus p2A protease domain, liberating GFP and ORF3a-p2A polypeptides. This allows tracking transfected cells by GFP fluorescence and the comparison of ORF3a-p2A expression between variant and parental proteins indirectly by performing immunoblots for GFP and quantitating the amount of the fusion proteins and liberated GFP. They report that, contrary to a computational prediction by Wang et al that the Q57H variant will be less stable than the parental protein, that it is somewhat more stable. A weakness of this approach is it ignores the possibility that the p2A-GFP fusion to ORF3a has a stabilizing effect on the fusion protein. Furthermore, by utilizing immunoblots to GFP it makes the assumption that liberated ORF3a-p2A peptides from parental and Q57H variants degrade at equivalent rates and that GFP immunoblots reflect the amount of ORF3a-p2A protein. It would have strengthened the paper to have directly measured the relative amounts of ORF3a-p2A protein with an anti-ORF3a antibody. -The statement "Differences in protein expression levels between WT and mutant ORF3a are more likely to occur via post translational processes rather than transcriptional regulation" (Results, Paragraph 1). It is not clear what the relevance of transcriptional regulation in transfected cells is when expression of the ORF3a mRNA is regulated quite differently in the context of SARS-CoV-2 infected cells than in transfected cells. Even in the context of transfected cells this claim would need experimental evidence, such as qRT-PCR for transcriptional analysis, post-translational modification studies, or protein folding studies. 2. Differing Results From Previous Studies: -The results negative related to ORF3a effects on inflammasome activation, ER stress, and autophagy in this paper differ from other reported studies. The authors should discuss potential reasons for these discrepancies. One obvious difference regarding inflammasome activation is the use of H9c2 cells, a cardiomyoblast cell line, in the current work. It is likely that these cells are not competent for inflammasome activation since Figure 3A shows that pharmacologic activation of inflammasomes by Nigericin fails to activate inflammasomes (IL-beta or caspase-1 cleavage) in these cells. 3. Mechanistic Studies on Q57H Mutation: -The manuscript lacks detailed mechanistic studies on how the Q57H mutation could affect extrinsic apoptosis pathway activation. For example, the authors should have examined the effect of individual death receptor knockdown on ORF3a induction of the extrinsic pathway for apoptosis to both identify the receptor activated and confirm their results. Additionally, the authors should investigate and discuss the potential alterations in ORF3a interaction with death receptors, and any differences in downstream signaling activation between WT and Q57H. Comparative studies between WT and Q57H regarding their interactions and signaling pathways would significantly enhance the manuscript's contribution to the field. Conclusion: The manuscript presents interesting findings but requires substantial improvements in experimental support and detailed mechanistic studies. Addressing the points mentioned above will strengthen the manuscript and provide a more comprehensive understanding of the ORF3a protein's role and its mutations. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Replication and pathogenesis of coronaviruses We confirm that we have read this submission and believe that we have an appropriate level of expertise to state that we do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (1) Author Response 08 Nov 2025 Jin O-Uchi, Medicine, University of Minnesota Twin Cities, Minneapolis, 55455, USA We appreciate the constructive comments from the reviewers and editor. We have addressed all of them in our revisions. (It would have strengthened the paper to have directly measured the relative amounts of ORF3a-p2A protein with an anti-ORF3a antibody.) A: Following the reviewer’s suggestion, we used an anti-ORF3a antibody instead of an anti-GFP antibody to re-evaluate the expression levels of WT-ORF3a and ORF3a-Q57H mutant, and replaced the previous data obtained using the GFP antibody with this new data (see Revised Figure 1A-C). When the high amounts of plasmid (≥1.0 µg/well ) were transfected, ORF3a-WT and -Q57H constructs produced similar levels of ORF3a protein, as assessed with ORF3a-specific antibody (see revised Figure 1B and C). However, under lower amounts of plasmid transfection (0.5 µg/well), we found a significantly higher protein expression of Q57H compared to WT-ORF3a (see revised Figure 1B and C). Based on this new data, we have revised the abstract, result section, and discussion section. (It is not clear what the relevance of transcriptional regulation in transfected cells is when expression of the ORF3a mRNA is regulated quite differently in the context of SARS-CoV-2 infected cells than in transfected cells. Even in the context of transfected cells this claim would need experimental evidence, such as qRT-PCR for transcriptional analysis, post-translational modification studies, or protein folding studies.) A: The qRT-PCR experiment was conducted, and the results were shown in the revised Figure 1D. We found no significant differences in mRNA levels between ORF3a-WT and -Q57H in all transfection conditions. In response to the reviewer’s suggestion, we removed the statement "Differences in protein expression levels between WT and mutant ORF3a are more likely to occur via post-translational processes rather than transcriptional regulation” from the result section. (2. The results negative related to ORF3a effects on inflammasome activation, ER stress, and autophagy in this paper differ from other reported studies. The authors should discuss potential reasons for these discrepancies. One obvious difference regarding inflammasome activation is the use of H9c2 cells, a cardiomyoblast cell line, in the current work. It is likely that these cells are not competent for inflammasome activation since Figure 3A shows that pharmacologic activation of inflammasomes by Nigericin fails to activate inflammasomes (IL-beta or caspase-1 cleavage) in these cells.) A: We used the established protocol (Using the higher concentration of nigericin (NG) with longer stimulation, see PMID: 34432650) and confirmed that NG can indeed activate inflammatory signaling in H9c2 cells as reported previously (see revised Figure 3A). Nevertheless, following the reviewer’s comment, we added the following sentence to the discussion section: “H9c2 cells may be less competent for inflammasome activation”. (-The manuscript lacks detailed mechanistic studies on how the Q57H mutation could affect extrinsic apoptosis pathway activation. For example, the authors should have examined the effect of individual death receptor knockdown on ORF3a induction of the extrinsic pathway for apoptosis to both identify the receptor activated and confirm their results. Additionally, the authors should investigate and discuss the potential alterations in ORF3a interaction with death receptors, and any differences in downstream signaling activation between WT and Q57H. Comparative studies between WT and Q57H regarding their interactions and signaling pathways would significantly enhance the manuscript's contribution to the field.) A: Following the reviewer’s suggestions, we further investigated the potential molecular mechanism underlying the ORF3a-mediated apoptosis and its alteration by the Q57H mutation. Although we did not screen the ORF3a interaction with all eight types of human death receptors due to the lack of reliable antibodies for co-immunoprecipitations, we found that ORF3a (but not Q57H) increases the protein expression of FADD, a crucial adaptor protein that acts as a bridge between death receptors and caspases to trigger extrinsic apoptotic pathways. The previous report showed that ORF3a expression at the plasma membrane (PM) is required for activating the apoptotic signaling (PMID: 32555321), ORF3a expression at the PM may enhance the stability of FADD protein within the death-inducing signaling complex (DISC) located just beneath the PM. Moreover, we found that the protein expression level of Q57H at the PM is lower thsan that of WT, which may contribute to the reduced activation of the extrinsic apoptotic pathways. We have included this new data in Figure 4D and E and revised the result section, figure legends, and discussion section accordingly. View more View less Competing Interests None. reply Respond Report a concern Leibowitz J and Na Pombejra S. Peer Review Report For: SARS-CoV-2-ORF3a variant Q57H reduces its pro-apoptotic activity in host cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :331 ( https://doi.org/10.5256/f1000research.160166.r290354) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-331/v1#referee-response-290354 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. 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