Targeting CTP synthetase 1 to restore interferon induction and impede nucleotide synthesis in SARS-CoV-2 infection
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Abstract
Despite the global impact caused by the most recent SARS-CoV-2 pandemic, our knowledge of the molecular underpinnings of its highly infectious nature remains incomplete. We report here that SARS-CoV-2 exploits cellular CTP synthetase 1 (CTPS1) to promote CTP synthesis and suppress interferon (IFN) induction. In addition to catalyzing CTP synthesis, CTPS1 also deamidates interferon regulatory factor 3 (IRF3) to dampen interferon induction. Screening a SARS-CoV-2 expression library, we identified several viral proteins that interact with CTPS1. Functional analyses demonstrate that ORF8 and Nsp8 activate CTPS1 to deamidate IRF3 and negate IFN induction, whereas ORF7b and ORF8 activate CTPS1 to promote CTP synthesis. These results highlight CTPS1 as a signaling node that integrates cellular metabolism and innate immune response. Indeed, small-molecule inhibitors of CTPS1 deplete CTP and boost IFN induction in SARS-CoV-2-infected cells, thus effectively impeding SARS-CoV-2 replication and pathogenesis in mouse models. Our work uncovers an intricate mechanism by which a viral pathogen couples immune evasion to metabolic activation to fuel viral replication. Inhibition of the cellular CTPS1 offers an attractive means to develop antiviral therapy against highly mutagenic viruses.
IMPORTANCE
Our understanding of the underpinnings of highly infectious SARS-CoV-2 is rudimentary at best. We report here that SARS-CoV-2 activates CTPS1 to promote CTP synthesis and suppress IFN induction, thus coupling immune evasion to activated nucleotide synthesis. Inhibition of the key metabolic enzyme not only depletes the nucleotide pool but also boosts host antiviral defense, thereby impeding SARS-CoV-2 replication. Targeting cellular enzymes presents a strategy to counter the rapidly evolving SARS-CoV-2 variants.
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SciScore for 10.1101/2021.02.05.429959: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization Samples were randomized and analyzed on a Q-Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer coupled to Vanquish UHPLC system (Thermo Fisher). Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Single colonies were isolated and screened by immunoblotting with IRF3 antibody. IRF3suggested: NoneCelle were incubated with primary mouse monoclonal anti-Flag antibody and Alex Fluor 488 congugated goat secondary antibody, and analyzed with confocal microscope (Leica). anti-Flagsuggested: (Cell Signaling Technology …SciScore for 10.1101/2021.02.05.429959: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization Samples were randomized and analyzed on a Q-Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer coupled to Vanquish UHPLC system (Thermo Fisher). Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Single colonies were isolated and screened by immunoblotting with IRF3 antibody. IRF3suggested: NoneCelle were incubated with primary mouse monoclonal anti-Flag antibody and Alex Fluor 488 congugated goat secondary antibody, and analyzed with confocal microscope (Leica). anti-Flagsuggested: (Cell Signaling Technology Cat# 5407, RRID:AB_1950473)Experimental Models: Cell Lines Sentences Resources Mass Spectrometry Analysis for Deamidation Sites: To identify deamidation sites, HEK293T cells were transfected with plasmid containing IRF3-GST without or with that containing the enzyme-deficient CTPS1 mutant (Flag-CTPS1-ED) HEK293Tsuggested: NoneCTPS1 Enzymatic Activity Assay: Flag-CTPS1 expressed 293T stable cell line was transfected with plasmids containing SARS-CoV-2 ORF7b, ORF8, Nsp8 and empty vector for 40 h. 293Tsuggested: NoneDrug Treatment: For SARS-CoV-2 infection, Caco-2 or NHBE cells were pre-treated with compound 1 or its derivatives (5, 6, 8, 9, and 10) for 2 h. Caco-2suggested: NoneTo test the effect of compound 1 on IRF3 deamidation, Caco-2 cells expressing ORF8, or A549 and LoVo cells expressing Nsp8 were treated with 5 μM compound 1 for 4 h. A549suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)To analyze the effect of compound 1 on intracellular metabolites, ORF8 expressed Caco-2 or LoVo cells were treated with 5 μM compound 1 for 2 h. LoVosuggested: NCI-DTP Cat# LOVO, RRID:CVCL_0399)Experimental Models: Organisms/Strains Sentences Resources HEK293T, Irf3−/−Irf7−/− mouse embryonic fibroblasts (MEFs), Caco-2, LoVo and A549 cells were infected with the supernatant, supplied with polybrene (8 ug/ml), and centrifuged at 1800 rpm for 50 min at 30℃. Irf3−/−Irf7−/−suggested: NoneSoftware and Algorithms Sentences Resources Metabolites were detected and quantified as area under the curve based on retention time and accurate mass (5 ppm) using the TraceFinder 4.1 (Thermo Scientific) software. TraceFindersuggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from scite Reference Check: We found no unreliable references.
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