Isre luc

Gene fragments of all 27 viral proteins (except PLpro of SARS-CoV-2) were gene synthesized (Sangon Biotech) with codon optimization. PLpro and orf6 of both SARS-CoV and SARS-CoV-2 were PCR amplified from cDNA reverse-transcribed from viral RNA extracted from viral supernatant. All gene fragments were cloned into pCAGEN expression vector with c-terminal FLAG-tag and confirmed by Sanger sequencing. Plasmids used have previously been detailed [33 (link)]. pEBG plasmid containing the GST-tagged RIG-I N-terminal card domains was used. Interferon-beta firefly luciferase reporter (IFNβ-luc), which comprises the promoter of IFNβ, is a kind gift of Professor Takashi Fujita, Kyoto University. Interferon-stimulated response element firefly luciferase reporter (ISRE-luc) which contains five repeats of ISRE enhancer element upstream of minimal TA promoter was purchased from Clontech. Renilla luciferase reporter (pRL-TK) that contains an HSV-thymidine kinase promoter was purchased from Promega.

To evaluate the promoter activity regulated by EcAtg5, luciferase reporter plasmids, including interferon-sensitive response element (ISRE)-Luc, IFN3-Luc, and nuclear factor (NF)-κB (Clontech, USA), were used for co-transfection. Briefly, GS cells were transiently transfected with the luciferase plasmids together with the indicated EcAtg5 expression vectors using Lipofectamine 2000 reagent. The pRL-SV40 Renilla luciferase vector was used as an internal control. Luciferase activity of total cell lysates was measured using a luciferase reporter assay system (Promega).