FIG 4.

Translational shut off by NSP1 and TRIM25-independent mechanism by N blocks IFN induction. (A) HEK 293T cells were transfected with wither empty vector (pcDNA) and plasmids encoding wild-type NSP1 or mutant NSP1-KH164AA for 24 h. Cell lysates were then subjected to immunoblotting with antibodies against FLAG. (B) HEK 293T cells were transfected with empty vector (pcDNA) or plasmids encoding wild-type NSP1 or mutant NSP1-KH164AA, IFN-β-responsive firefly luciferase reporter, and control Renilla reporter. After 24 h, the cells were challenged with 100 HAU/ml of SeV for 16 h and then harvested for luciferase assays. The firefly reporter activities were normalized against Renilla reporter values, and the data are presented as fold activity relative to uninduced pcDNA empty vector control. (C and D) A549 cells were transfected with plasmid encoding wild-type NSP1, mutant (KH164AA) NSP1, or NS7B (negative control) for 24 h, after which cells were challenged with 100 HAU/ml of SeV for 8 h. Samples were then processed for indirect immunofluorescence microscopy using antibodies against FLAG and IRF3. The fluorescent intensities in the nucleus and cytoplasm were measured using Volocity software (n = 20). (E) HEK 293T cells were transfected with plasmids encoding SARS-CoV-2 N or NS3A proteins or empty vector (pcDNA). After 48 h, cell lysates were immunoprecipitated with anti-FLAG antibody and then subjected immunoblot analysis using antibodies against FLAG, TRIM25, and β-actin. (F) HEK 293T cells were transfected with a plasmid encoding FLAG-TRIM25 or empty vector (pcDNA) for 48 h, after which cell lysates were subjected to immunoblot analysis using antibodies against TRIM25 and β-actin. (G) HEK 293T-ACE2 cells were transfected with a plasmid encoding FLAG-tagged TRIM25 or empty vector (pcDNA) for 48 h. Cells were then infected with SARS-CoV-2 (MOI = 1) for 48 h, after which total RNA was harvested and subjected to qRT-PCR to quantify viral genomic RNA, which was normalized to ACTB mRNA level and expressed as fold values relative to pcDNA empty vector-transfected cells. (H) HEK 293T cells were transfected with plasmids encoding GST-tagged human RIG-I CARD domains (GST-h2CARD) or GST alone, together with HA-tagged ubiquitin (HA-Ub), V5-tagged human TRIM25 (hTRIM25-V5), and the indicated FLAG-tagged viral proteins. Clarified whole-cell lysates were subjected to GST pulldown (IP: GST), followed by immunoblot analysis with anti-GST, anti-HA, anti-V5, and anti-FLAG antibodies. Influenza A virus (IAV) NS1 served as a positive control for blocking TRIM25-mediated ubiquitination of the RIG-I CARD domains. Data are means ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.