In vitro binding assay

8xHis-ZZ fused BICD2 (full-length and truncated versions) and BICDR1 purified by His-Trap column were mixed separately with SV40 intact viruses (CsCl purified) in at least 72 times excess in molar ratio. Incubation/binding buffer included 50 mM Hepes, pH 7.5, 150 mM NaCl, 0.4 mM DTT, and 0.4 mM EGTA with BSA 0.1% and Triton-X100 0.02%. IgG Sepharose resin preequilibration was done per the manufacturer’s protocol in the presence of 0.1–0.2% BSA. 100 µl packed resin was treated with 200 µl of each protein sample mixture at 4°C for 2 h on tube roller. Unbound proteins were washed out with 5 × 3 ml binding buffer, 2 × 3 ml binding buffer + high salt (400 mM NaCl), and 4 × 3 ml TEV buffer (10 mM Tris, pH 8.0, 150 mM KCl, 2 mM MgCl₂, 0.5 mM EGTA, and 10% glycerol). The proteins were eluted from the IgG Sepharose in 200 µl TEV buffer containing 60 µg/ml TEV protease. Following SDS-PAGE with immunoblotting, eluted SV40 was detected by anti-VP1 antibody, and eluted BICD2s and BICDR1 were visualized by Coomassie blue staining. For Fig. S2 D, native SV40 was treated with or without DTT and EGTA and incubated with BICD2 in PBS with 1 mM MgCl_2. Samples were then rotated with 1 µg BICD2 antibody overnight at 4°C. The next day, Protein A/G agarose beads (Pierce) were washed three times with PBS and rotated with the lysate for 20 min at 4°C. The tubes were then centrifuged at 7,000 rpm for 1 min and the beads washed three times with PBS. SDS sample buffer was added to the beads and boiled for 10 min at 95°C. Samples were then run on an SDS-PAGE gel and assessed by Western blot. Three replicates of each single assay were used to quantify the results using ImageJ.