Ifn beta pgl3

The following pLVX- SARS-CoV-2-Strep plasmids obtained from Addgene were used in this study: NSP1 (No 141367), NSP2 (No 141368), NSP4 (No 141369), NSP5 (No 141371), NSP7 (No 141373), NSP8 (No 141374), NSP9 (No 141375), NSP10 (No 141376), NSP11 (No 141377), NSP12 (No 141378), NSP13 (No 141379), NSP14 (No 141380), and NSP15 (No 141381) [28 ]. Additional plasmids include: pLVX-EGFP-2xStrep (Addgene No 141395), pHAGE-N-FLAG-HA TBK1 (Addgene No 131791) IFN-Beta_pGL3 (Addgene No 102597), pNL1.1.TK [Nluc/TK] (Promega), p.GL4.45 ISRE-Luc (Promega), and p.GL4.32 NFκB-Luc (Promega). pLVX-nsp1-2xStrep containing either the K164A/H165A (NSP1^KH) or the R125A/K126A (NSP1^RK) mutations were generated using QuickChange site directed mutagenesis (Agilent) with the primers 5’-CTCGCGAGTAACGCCACTGCTAGCTGCCGTATTCCAGTTTTCCTGGAAA-3’ and 5’-TTTCCAGGAAAACTGGAATACGGCAGCTAGCAGTGGCGTTACTCGCGAG-3’ (KH) and 5’-CCTGCGCCCTTGTTCCCATTTGCTGCCAACAGCACCTTCCGATATGC-3’ and 5’-GCATATCGGAAGGTGCTGTTGGCAGCAAATGGGAACAAGGGCGCAGG-3’ (RK).
The sequences of all plasmids were verified by DNA sequence (Genewiz) and are available upon request. Transfections were performed using Polyjet transfection reagent (SignaGen Laboratories) according to manufacturer’s instructions.

The RIOK3 open reading frame was PCR amplified from Addgene plasmid #20618 (pWZL Neo Myr FLAG), which was a gift from William Hahn and Jean Zhao (Dana-Farber Cancer Institute, Boston, MA, USA) [21 (link)]. The RIOK3 ORF was cloned into phRL-CMV (Promega) between the XbaI and NheI restriction sites. The X2 variant was PCR amplified from cDNA obtained from infected HEK293 cells and was also cloned into phRL-CMV between the XbaI and NheI restriction sites. The pGL3-IFNB plasmid was obtained from Addgene, plasmid #102597 (IFN-Beta_pGL3), which was a gift from Nicolas Manel (Institut Curie, Paris, France) [22 (link)].

HEK293 cells were seeded into 24-well plates in an amount of 1 × 10⁵ cells/well. The next day, cells were transfected with 0.5 µg of plasmid IFN-Beta_pGL3 (Addgene) encoding luciferase under the control of IFN-β promoter and 0.5 µg of either plasmids encoding HCV NS5B, or HIV-1 RT, or inactivated HIV-1 RT, or empty vector pVax1 using 1 µL Lipofectamine LTX and 0.5 µL Plus Reagent. Twenty hours post transfection, cells were harvested, lysed using RLB buffer (Promega, Madison, WI, USA), centrifuged, and supernatant was assessed for luciferase activity using the Luciferase Assay System (Promega) as recommended by the manufacturer. Luminescence was measured on a luminometer (Promega).