Streptactin beads

Cells were lysed in 1% Digitonin lysis buffer (1% Digitonin (Calbiochem), 50 mM Tris–HCl, 5 mM MgCl₂, 150 mM NaCl; pH 7.5) containing 1 mM Pefabloc SC (Roche) and 10 μM Leupeptin (Roche). Lysates were incubated for 90 min at 4 °C. Cell fragments were pelleted 12,000g for 20 min at 4 °C. Post-nuclear supernatants were incubated overnight with StrepTactin beads (GE Healthcare), or FLAG-M2-coupled beads (Sigma). After four washes in 1% digitonin lysis buffer, proteins were eluted in elution buffer (For StrepTactin beads: 2.5 mM desthiobiotin, 150 mM NaCl, 100 mM Tris–HCl, 1 mM EDTA, pH 8; For FLAG-M2-coupled beads: 500 μg ml⁻¹ FLAG peptide, 150 mM NaCl, 100 mM Tris–HCl, pH 7.5) for 30 min on ice. Beads were pelleted, the supernatant was transferred to a new tube and subsequently denatured in Laemmli sample buffer containing DTT. Immunoblotting was performed as described below.

For the purification of CSPP1 constructs, HEK293T cells were transiently transfected with polyethyleneimine (Polysciences) with different StrepII-GFP-CSPP1 constructs. The cells were harvested 28 h after transfection. Cells from a 15-cm dish were lysed in 500 µl lysis buffer (50 mM HEPES, 300 mM NaCl, 1 mM MgCl₂, 1 mM DTT, 0.5% Triton X-100, pH 7.4) supplemented with protease inhibitors (Roche) on ice for 15 min. The lysate was cleared from debris by centrifugation and the supernatant was incubated with 20 µl StrepTactin beads (GE Healthcare) for 45 min. Beads were washed five times with a 300 mM salt wash buffer (50 mM HEPES, 300 mM NaCl, 1 mM MgCl₂, 1 mM EGTA, 1 mM DTT, and 0.05% Triton X-100; pH 7.4) and three times with a 150 mM salt wash buffer (similar to the 300 mM salt buffer but with 150 mM NaCl). The protein was eluted in elution buffer (similar to the 150 mM salt wash but supplemented with 2.5 mM d-Desthiobiotin [Sigma-Aldrich]) where the volume depended on the expression levels before harvesting. Purified proteins were snap-frozen and stored at −80°C.

HEK-293T (1–2×10⁷) cells were infected with WSN or P908/WSN recombinant viruses at a m.o.i. of 3. Six hours post-infection, cells were lysed in 0.5 ml of lysis buffer (20 mM MOPS-KOH pH 7.4, 120 mM of KCl, 0.5% Igepal), supplemented with Complete Protease Inhibitor Mixture (Roche). Cell lysates were processed and incubated wih StrepTactin beads (StrepTactin Sepharose High Performance, GE Healthcare) as described in [51] . After three washes with 1 ml of lysis buffer, protein complexes were eluted from StrepTactin beads with desthiobiotin (IBA). Purification samples were either diluted in Laemmli sample buffer and analyzed by western-blot, or diluted in Renilla lysis buffer (Promega) and submitted to Gaussia princeps luciferase enzymatic activity measurement, using the Renilla luciferase assay reagent (Promega) and a Berthold Centro XS luminometer.