Fig. 5: NSP3 induces coagulation defects via fibrinogen.

a NSP3 with signal peptide, processed by Sec translocon and cleaved by Signal Peptidase I (SPI); SPI sequence in red, with cleavage site and non-signal regions indicated. b Glycosylation sites (score > 0.5) are mapped by amino acid (AA) position based on NetNGlyc 1.0 and NetOGlyc 4.0. c TMHMM predicts four transmembrane regions. d IB of whole cell extracts (WCEs) and supernatants from NSP3-V5 HepG2 cells using anti-V5 antibody for NSP3, with FGA and GAPDH (1:1,000) as controls. e WCEs and immunoprecipitates (IPs) from NSP3-V5 HepG2 and HEK293 cells were probed with FGA, NSP3, or GAPDH antibodies, normalized to input FGA. f FGA IPs from COVID-19 (n = 4) and control (n = 2) plasma were probed with NSP3 and FGA antibodies. g Representative micrographs show (n = 7-10) NSP3 co-localization with FGA in SARS-CoV-2-infected but not mock Huh-7 cells; nuclei in blue. Scale bar, 10 μm. FGA fluorescence in infected vs. mock cells (n = 100, P = 2.1e⁻⁷³, two-tailed paired t-test). h Coagulation was measured at 420 nm over time using varying NSP3 with fibrinogen, with thrombin-treated and untreated fibrinogen as controls. i Fibrinogen expression in indicated Huh-7 cells was measured by ELISA over time (n = 5 technical replicates; 3.2e⁻⁹  ≤ P  ≥ 9.7^e−2, two-sided paired t-test). j IB of saliva WCEs using FGA antibody; mean FGA levels (n = 10) from respective IB with band intensities normalized to GAPDH (1.1e⁻²² ≤ P ≥ 2.9 e⁻², one-tailed paired t-test). k FGA levels (P  =  2.5e⁻¹⁹, two-tailed paired t-test) in NSP3-V5 vs. empty vector HepG2 cells. Quantification (n = 10) based on WCEs from panel e using FGA antibody and GAPDH as LC. l IL-6 and CCL2 mRNA levels were measured in NSP3-transfected and untransfected M1 vs. M0 macrophages (n = 4 biological replicates; P = 7.6e⁻⁷ for IL-6, P = 5.4e⁻⁶ for CCL2; two-tailed paired t-test).  Data are mean ± SEM (g, i-l). IBs are representative (n = 2) with molecular weight markers (kDa). A clearer protein ladder from the same blot was overlaid (dashed line). Source data are provided as a Source Data file.