While the inhibitory antiviral effect of Poly6 in Calu-3 cells was not observed in Vero-E6 in either RT-qPCR or plaque assays, RDV and HCQ showed significant antiviral activity. inhibitors, or blocking antibodies that Poly6 can exert an anti-SARS-CoV-2 effect in an IFN-I-dependent manner. We also found that Poly6 inhibits IL-6 production enhanced by SARS-CoV-2 in infected Calu-3 cells at both the transcription and the translation levels, mediated via IL-10 induction in an IFN-I-dependent manner. These results indicate the feasibility of Poly6 as an IFN-I-inducing COVID-19 drug with potent antiviral and anti-inflammatory activities. 0.01, and *** indicates 0.001. The results are offered as mean values SD. Abbreviations: RDV, remdesivir, HCQ, hydroxychloroquine sulfate. 2. Materials and Methods 2.1. Viruses and Cell Cultures The human lung epithelial cell lines Calu-3 cells and Vero-E6 were managed at 37 C with 5% CO2 in Dulbeccos altered Eagles medium (WELGENE, Gyeongsan, Korea), 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), and penicillin and streptomycin (100 U/mL; Gibco, Carlsbad, CA, USA). The SARS-CoV-2 computer virus (CoV/KOR/KCDC03/2020) was provided by the National Culture Collection for Pathogens (NCCP), Korea Centers for Disease Control and Prevention (KCDC). All work with the infectious computer virus was performed in a biosafety level 3 laboratory and approved by the Seoul National University (SNU) and the Korean Disease Control and Prevention Agency (KDCA). 2.2. Compounds and Reagents The Poly6 (GRLVFQ) peptide from your HBV polymerase region overlapping with the preS1 deletion region, mainly discovered from occult HBV contamination or severe liver disease progressive patients, was synthesized by the (9-fluorenylmethoxycarbonyl Fmoc)-based solid-phase method and characterized by Peptron Inc. (Cheongju, Korea) (Physique 1A and Physique S1). Remdesivir (GS-5734, RDV), hydroxychloroquine sulfate (S4430, HCQ), and ruxolitinib (INCB018424) were purchased from Selleck Chemicals (Selleck, Houston, TX, USA). Unless otherwise indicated, all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.3. Infections Calu-3 cells and Vero E6 cells were plated in 24-well plates (200,000 cells/well). The next day, N-Desmethylclozapine Rabbit Polyclonal to Cytochrome P450 46A1 the cells were infected with SARS-CoV-2 (Calu-3, MOI = 0.1; Vero, MOI = 0.01) for 1 h. After media change, the drugs, including Poly6 and two positive controls, i.e., RDV and HCQ, were added to the culture media. 2.4. Quantification of SARS-CoV-2 Viral RNA Genome Copy Number by RT-qPCR Cell supernatants were harvested using TRIzol LS reagent (Invitrogen, Carlsbad, CA, USA), and RNA was purified using the chloroform manual method. cDNA was N-Desmethylclozapine synthesized (Intron), and viral RNA was quantified using the SensiFAST Probe Lo-ROX N-Desmethylclozapine kit (Bioline, Merdian Biosciences Inc., Memphis, TN, USA). 2.5. Cell Cytotoxicity Assay Calu-3 and Vero E6 cells were seeded (1 104 cells) in 96-well microplates and incubated with increasing concentrations of Poly6 for 24 h. According to the manufacturers protocol, cell viability was decided using the MTS assay kit (Promega, Fitchburg, WI, USA). 2.6. Immunofluorescence and DoseCResponse Curve (DRC) Analysis Calu-3 cells were seeded and cultivated in N-Desmethylclozapine two-chamber glass slides (Nunc, Roskilde, Denmark) for 12 h before the experiment. Ten points were assigned for each drug, with concentrations ranging from 0.1 to 50 M for Poly6 and RDV and from 0.49 N-Desmethylclozapine to 250 M for HCQ. The cells were infected with SARS-CoV-2 at a multiplicity of contamination (MOI) of 0.1 in the BSL-3 facility. After 24 h, the cells were fixed with 4% PFA and permeabilized with 0.1% Triton-X 100 for 10 min. The cells were stained with main (1:100, overnight at 4 C) and secondary (1:1000, 2 h at room heat) antibodies in 1% BSA in PBS and mounted in a mounting medium made up of DAPI (Vectashield, Vetor Laboratories, Inc., Burlingame, CA, USA). Images were captured and analyzed using software to quantify the infection ratio (Leica Software analysis, LAS X and Image J program, version 1.52a), and antiviral activity was measured. DRCs were calculated using Prism7 software (GraphPad, San Diego, CA, USA), with doseCresponse-inhibition nonlinear analysis. EC50 and CC50 values were calculated. 2.7. IFN-I Neutralization Assay.