Fig 2: Electroporated-antibody-dependent neutralization assay (EDNA) for coronavirus AA titration of anti-MHV polyclonal serum was added either directly to media (extracellular) or electroporated into L929 cells (intracellular), and then, cells were infected with MHV-A59. The kinetics of cell growth was monitored by measuring total cell area.BPhase-contrast images of cells at 48 h post-infection. Scale bar = 200 µm.CAt 48 h post-infection, the viability of cells infected with MHV-A59 in the presence of intracellular or extracellular antiserum was determined by ATP luminescence assay. Increasing doses of antiserum results in increased cell survival.DKinetics of cell growth following MHV-A59 infection in the presence of a titration of electroporated anti-N antibody.EPhase-contrast images of cells at 48 h post-infection. Scale bar = 200 µm.FQuantification of cell viability by ATP luminescence assay at 48 h post-infection in the presence of electroporated anti-N or anti-GFP antibodies.G, HWT or TRIM21 KO L929 cells were infected with MHV-A59 in the presence of electroporated polyclonal antiserum (G) or anti-N antibody (H) and quantified by cell area 48 h later. Co-electroporation of protein A/G with anti-N antibody into WT cells mimics the TRIM21 KO phenotype.IL929 cells were infected with MHV-A59 for 4 h, and then, cyclohexamide (CHX) was added to block further viral protein synthesis. After 5 h, cells were electroporated with anti-N or anti-GFP antibodies and left for a further 3 h before being western blotted for cellular N protein levels (* denotes a non-specific band). Data information: Data were analysed using a Student's t-test. Error bars depict the mean +/- SEM. All data represent at least two independent replicates. Source data are available online for this figure.

Fig 3: TRIM21 silencing protects cells from DEN-induced oxidative damage.TRIM21–/– MEFs reconstituted with vector, HA-TRIM21 WT, HA-TRIM21 LD, and HA-TRIM21 W381/383A mutant. (A) Cells were lysed in RIPA buffer with 1% SDS and subjected to WB. (B–E) Cells were treated with DEN (20 mM) for 6 hours. (B) Cells were harvested and probed for indicated proteins by WB. (C) Cells were stained with H2DCFDA and analyzed by flow cytometry. Quantification of relative H2DCFDA intensity (geometry mean of 3 repeats) is shown on the right. ***P < .001. (D) Cells were subjected to IF with Keap1 and p62 antibodies and observed under deconvolution microscope. Cells with p62/Keap1 aggregates were counted blindly. Data shown are the averages plus SD of at least 3 countings with over 200 cells. ***P < .001. (E) Cells were separated into Triton X-100 (1%) soluble and insoluble fractions, and 30-µg soluble proteins and corresponding volume of insoluble proteins were used for WB. The numbers below Western blot panels indicate the ratios of absolute levels of insoluble and soluble proteins, respectively.

Fig 4: TRIM21–/–livers are protected from DEN-induced liver damage. (A) Eight-week-old TRIM21+/+ and TRIM21–/– mice were treated with 200-mg/kg DEN via intraperitoneal injection. Livers were collected 48 hours later and processed for IHC analysis. Mean integrated optical density was determined for p62, Keap1, and cleaved caspase-3 (C-C3) because they are predominantly localized in the cytosol. Cells with positive nuclear staining of 8-oxo-dG and γH2A.X staining were counted and indicated as the percentage to total cells. Representative images are shown. Data are presented as mean ± SEM of 3 replicates. (B) Liver tissues were lysed in RIPA buffer and subjected to WB for indicated proteins. ∗∗P < .01. ∗∗∗P < .001.

Fig 5: N antibodies inhibit SARS-CoV-2 replication intracellularly A, BVero cells OE ACE2 and TMPRSS2 were infected with SARS-CoV-2 in the presence of IgG or anti-N antibodies added directly into media or electroporated into cells. Viral replication was then determined by RT–qPCR (A) or plaque assay (B). Electroporation of anti-N antibodies significantly inhibits SARS-CoV-2 replication (***P < 0.0002).C, DAs with A&B, except with a titration of electroporated anti-N antibodies.EWestern blot of 293T cells and 293T ACE2 OE/TRIM21 KOs alone or reconstituted with empty vector (EV) or an endogenous promoter-driven TRIM21 vector (T21) (Zeng et al, 2019).F, G293T ACE2 OE/TRIM21 KO cells reconstituted with EV or TRIM21, electroporated with IgG or anti-N antibodies, and infected with SARS-CoV-2. Viral replication was then determined by RT–qPCR (F) or plaque assay (G). Electroporation of anti-N antibodies significantly inhibits SARS-CoV-2 replication only in TRIM21-reconstituted cells (*P < 0.05). Data information: All data represent at least three independent replicates. Error bars depict the mean ± SEM. Statistical comparisons were performed using a one-way (A, B) or two-way (F, G) ANOVA. Source data are available online for this figure.