Fig 2: 3CLpro cleaves RPAP1 and PTBP1, altering PTBP1 localization(A) Locations of 3CLpro cleavage sites in RPAP1 and PTBP1 identified by TAILS neo-N-terminal peptides (red) and Edman sequencing (green). Representative MS/MS spectra of cleaved neo-N-terminal peptides.(B) SDS-PAGE and Edman sequencing of human recombinant RPAP1 and PTBP1 incubated with 3CLpro+/− inhibitor GC376, or 3CLpro-C145A (1:5 mol/mol, E:S). Δ-substrate, no sequence obtained.(C) MALDI-TOF-MS kinetic analyses of 3CLpro cleavage of synthetic P4–P4’ peptides.(D) RPAP1 and PTBP1 immunoblots of primary HAECs lysates from 5 donors incubated with 3CLpro or 3CLpro-C145A (1:200 w/w, E:S) for 18 h, 37°C.(E and F) (E) Immunoblots of 3CLpro, N-protein and (F) PTBP1 of Vero E6 cells infected with SARS-CoV-2 at a MOI 0.1 for 24 (n = 4) and 48 (n = 4) hpi, or mock (n = 3) ∗, unspecific bands.(G) Subcellular localization of PTBP1 by confocal imaging. SARS-CoV-2-infected Vero E6 cells (N = 5, scale bar 50 μm). Cyan boxes, Spike-negative (S–) uninfected cells. Green boxes, Spike-positive (S+) infected cells. Enlarged detail of mock-infected, S+ and S– cells in the same field, scale bar 10 μm. (H) Nuclear to cytoplasmic ratio of PTBP1 was quantified for mock and Spike-positive SARS-CoV-2 infected cells. Statistical significance was assessed by Student’s t test (mean ± SD, N = 5, n = 51 cells, ∗p ≤ 0.05). β-actin and β-tubulin loading controls.See also Figures S2–S5.