Fig 2: TRIM4 is required for MDA5 K63-linked polyubiquitination and Nsp8 inhibition in antiviral responses.A-B. HEK293T cells were co-transfected with an empty vector or nsp8-expressing plasmid, plus scramble shRNA or shTRIM4 (A), or empty vector or TRIM4-expressing plasmid (B) for 24 h, and then left uninfected or infected with EMCV (MOI = 0.25) for 18 h. The total RNA was extracted, reverse-transcribed and detected with real-time PCR analysis for IFNA, ISG15, TNF and EMCV 3D gene expression. The results are shown as the mean ± SD (n = 3), *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, by Sidak’s multiple comparisons test. C. Flag-nsp8, GFP-MDA5 and/or HA-TRIM4-expressing plasmids were co-transfected into HEK293T cells as indicated for 36 h. Then cell lysates were subjected to coimmunoprecipitation with anti-HA beads, followed by immunoblotting with the indicated antibodies. D-E. GST or His-tagged fusion proteins were immobilized on GST-beads or Ni-NTA resins as indicated, and then incubated with Flag-nsp8 protein purified from lysates of HEK293T cells for 2 hours(D). For sequential double pull-down assays, the glutathione agarose beads were eluted with GSH solution(E). After five washing, the beads were analyzed by immunoblotting analysis. F-G. HEK293T cells were transiently transfected with GFP-nsp8 expressing plasmids (F) or Spike-deleted SARS-CoV-2 replicon BAC DNA (G) for 36h. The cell lysates were subjected to coimmunoprecipitation using anti-GFP beads (F) or anti-nsp8 antibody plus protein A/G beads (G), and analyzed by western blotting as indicated to detect endogenous MDA5 and TRIM4. H. GST-MDA5 and Flag-TRIM4-expressing plasmids were co-transfected into HEK293T cells with HA-K63-Ub expressing plasmid, followed with uninfected or infected with EMCV (MOI = 0.25) for 18 h. The cells were collected, lysed and subjected to ubiquitination assay with immunoprecipitation with anti-GST beads and western blotting with antibodies as indicated. I. Flag-nsp8 and GST-MDA5 expressing plasmids were co-transfected into HEK293T cells with scramble shRNA or shTRIM4 as indicated. The cell extracts were subjected to in vivo ubiquitination assay as indicated. J.GST-MDA5, His-TRIM4 and/or Flag-nsp8 plasmids were co-transfected with HA-K63-Ub expressing plasmid into uninfected HEK293T cells or cells harboring SARS-CoV-2 replicon for 36h. The K63-polyubiquitination of MDA5 was analyzed as described above. K-L. Purified GST-tagged MDA5 CARDs protein was bound to GST-beads and then incubated for 1 h at 37°C with TRIM4 protein (K) and/or nsp8 protein (L) in in vitro ubiquitination reaction. The beads were washed three times with PBS containing 0.1% SDS and 0.2% Triton X-100 and then immunoblotted with antibodies to Ub.