![]() Consistent with our mRNA results, M pro strongly suppressed IFN-α-induced ISRE promoter activity in a dose-dependent manner in HEK-293T cells, Vero E6 cells, and A549 cells ( Fig. ![]() Considering the existence of IFN-stimulated response elements (ISREs) within the ISG promoter regions, we assessed the ISRE-dependent transcription by dual-luciferase reporter assay in HEK-293T cells, Vero E6 cells, and A549 cells. In contrast, IFN stimulation failed to induce the expression of ISGs in cells overexpressing M pro. 1A, gene expression substantially increased in response to IFN stimulation in the control cells. To further investigate the effect of M pro on the transcription of other ISGs, we selected an effective dose of 1000 U/mL and an intermediate time of 8 h for subsequent analysis. As expected, the expression of IFIT3 increased over time ( Fig. Similarly, we treated cells with 1000 U/mL IFN-α for 0, 4, 8, or 12 h to measure IFIT3 transcription. S1A, IFIT3 was significantly induced in an IFN dose-dependent manner and reached a saturation point at approximately 1000 U/mL. ).The cells were first treated for eight hours with 0, 250, 500, 1000, or 2000 U/mL of recombinant human IFN-α, and then collected for measuring mRNA levels of IFIT3 using quantitative PCR. KeywordsĪbbreviations: SARS-CoV-2 ( severe acute respiratory syndrome coronavirus 2), COVID-19 ( coronavirus disease 2019), ISG ( interferon-stimulated gene), Mpro ( main protease), HDAC ( Histone deacetylases), DCP1A ( decapping mRNA 1A), ssRNA ( single strandRNA), ARDS ( acute respiratory distress syndrome), IFN ( Interferon), STAT ( signal transduction and transcriptional activation proteins), IRF9 ( IFN regulator factor 9), ISGF3 ( IFN-stimulating gene factor 3), ISRE ( IFN-stimulated response element), TSA ( trichostatin A), PDCoV ( porcine deltacoronavirus), NEMO ( nuclear factor-κB essential modulator), DAC ( deacetylation domain), NLS ( nuclear location sequence), Cp ( cleavage product), PLpro ( papain-like protease), OAS ( 2′,5′-oligoadenylates), REC8 ( REC8 meiotic recombination protein), MAP3K14 ( mitogen-activated protein kinase kinase kinase 14), CP ( cleaved peptide), IFIT ( tetratricopeptide repeats), MIX ( mix paired-like homeobox), Usp18 ( ubiquitin specific peptidase 18), GBP1 ( guanylate binding protein 1) In conclusion, our findings clearly demonstrate that SARS-CoV-2 M pro constitutes a critical anti-immune effector that modulates the IFN/ISG system at multiple levels, thus providing a novel molecular explanation for viral immune evasion, and allowing for new therapeutic approaches against COVID-19 infection. In addition, M pro from different genera of coronaviruses has the protease activity to cleave both HDAC2 and DCP1A, even though the alphacoronaviruse M pro exhibits weaker catalytic activity in cleaving HDAC2. Interestingly, M pro also abolishes the activity of ISG effector decapping mRNA 1A (DCP1A) by cleaving it at residue Q343. Our analysis shows that to inhibit the ISG production, M pro cleaves histone deacetylases (HDACs) rather than directly targeting IFN signal transducers. Here, we show that the SARS-CoV-2 main protease (M pro) significantly suppresses the expression and transcription of downstream ISGs driven by IFN-stimulated response elements (ISREs) in a dose-dependent manner, and similar negative regulations were observed in two mammalian epithelial cell lines (simian Vero E6 and human A549). ![]() However, whether SARS-CoV-2 evades IFN antiviral immunity by manipulating ISG activation remains to be elucidated. All coronaviruses studied thus far have to first overcome the inhibitory effects of the IFN/ISG system before establishing efficient viral replication. A critical role in protecting the host against invading pathogens is carried out by interferon-stimulated genes (ISGs), the primary effectors of the type I interferon (IFN) response. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), constitutes an emerging human pathogen of zoonotic origin. Glycobiology and Extracellular Matrices. ![]()
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