Construction and Characterization of vIRF1 Mutants

Based on the pHAGE-vIRF1-Flag expressing plasmid described in the previous studies [45 (link)], the vIRF1 domain-mutant plasmids (vIRF1-MD1-Flag, vIRF1-MD2-Flag), and the vIRF1 single lysine-mutant plasmids were constructed to replace the indicated lysine site(s) with arginine by Tsingke Biotechnology Co., Ltd. (Beijing, China), as well as the Lys406 and Lys442 co-mutant vIRF1 plasmid (vIRF1-K406R+K442R-Flag). The sirtuins expressing plasmids (SIRTs-1~7-Myc), USP10-Flag, IRF3-Myc and STING-Myc were generated based on the pCDH vector (Tsingke Biotechnology Co., Ltd., China). pcDNA3.1-p300-HA was kindly provided by Dr. Xiaoming Wang (Nanjing Medical University), and pcDNA3.1-CBP-HA was constructed based on a generous gift from Dr. Xiao Han (Nanjing Medical University). A 300 bp fragment of IFN-β promoter (-280 to +20) was amplified and subcloned into pGL3-basic vector containing the firefly luciferase reporter gene (Promega, USA). The mpCDH plasmid was used as the short hairpin RNA (shRNA) expressing lentiviral vector, and the shRNA sequences to USP10 were listed in S1 Table. All plasmids were prepared using Vazyme FastPure Plasmid Mini Kit and confirmed by DNA sequencing. HEK293T cells were transfected with Lipofectamine 2000 Reagent (Invitrogen, USA), and EA.hy926 cells was transfected with Effectence transfection reagent (Qiagen, China).