pc1 3  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: Cryo-electron tomography reveals coupled flavivirus replication, budding and maturation

doi: 10.1038/s41467-026-68483-4

Figure Lengend Snippet: A Ratio of cleaved M to uncleaved prM quantified by western blot in LGTV-infected cell lysates, treated as indicated, at 24 h p.i. Each dot, one biological replicate. B Percentage mature virions in tomograms of LGTV-infected cells that were untreated ( N = 29), supplemented with Furin Inhibitor I ( N = 23), or NH 4 Cl ( N = 14). Each dot, one tomogram. C Polyprotein of a chimeric LGTV, rLGTV T:prME , with prM and ecto-E from TBEV strain 93/783. Protease sites are shown in the structural protein, and the furin site sequences from TBEV strains 93/783 and Torö are shown highlighting the difference at position 86 (., identical sequence). D Percentage survival of Ips1 -/- mice infected intraperitoneally with rLGTV T:prME R86 ( N = 5) or Q86 ( N = 10). E As ( D ), but for mice infected intracranially with rLGTV T:prME R86 ( N = 9) or Q86 ( N = 10). F , G Schematic ( F ) and result ( G ) of enzymatic cleavage assay using furin or PC1/3 with peptides covering furin site sequences in ( C ) (“RTRR”), or peptides with impaired furin sites (“RTRA”). Four independent experiments performed in duplicates are shown. H prM and M protein levels in cell lysates and supernatant 48 h p.i. by immunoblotting using an anti-M antibody. Viral NS3 and cellular tubulin included as infection and loading control. Representative blots are shown. I Percent prM intensity of total prM+M quantified in supernatant western blots at 48 and 72 h p.i. Four independent experiments performed in duplicates. J Slice through tomogram of rLGTV T:prME Q86-infected cell. Orange arrows, immature virions. Scale bar 100 nm. K segmentation of the tomogram in ( J ). L Percentage mature virions in tomograms of cells infected with rLGTV T:prME R86 ( N = 7) and Q86 ( N = 7). Each dot, one tomogram. A , B , G , I , L Bars indicate average±standard deviation. ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. P values by unpaired two-tailed Student’s t test: A untreated vs inhibitor (20 µM; p = 0.0753, 40 µM; p = 0.0178, 60 µM; p = 0.0293), vs NH 4 Cl p = 0.00060), B untreated vs inhibitor p = 0.0037, vs NH 4 Cl p = 0.0028, G RTRR (R86 vs Q86) for furin p = 0.0184, for PC1/3 p = 2.91 × 10 −6 , I 48 h: p = 0.0085, 72 h: p = 0.2403, L p = 0.0290. In ( D , E ), Q86 vs R86 were assessed by log-rank (Mantel–Cox) test p = 0.2567.

Article Snippet: For furin, 3 U furin (Thermo Fisher, Waltham, MA, USA) was mixed with 100 μM of substrate in a total volume of 100 μl reaction buffer (100 mM HEPES pH7.5 + 1 mM CaCl2 + 0.5% Triton ×-100) at 30 °C for 3 h. For proprotein convertase 1/3 (PC1/3), 1 μg recombinant human PC1/3 (R&D Systems, Minneapolis, MN, USA) was mixed with 100 μM of substrate in a total volume of 100 μl reaction buffer (25 mM MES pH 6.0 + 5 mM CaCl2 + 1% (w/v) Brij-35) at 37 °C for 1 h. The emission at 490 nm measured every 3 min and the average rate calculated by linear regression.

Techniques: Western Blot, Infection, Sequencing, Cleavage Assay, Control, Standard Deviation, Two Tailed Test