Human TRIM5α binds CA from restricted MLV virions. (A) HeLa cells were infected with VSV G-pseudotyped, N- and B-tropic MLV-GFP vectors after normalization for RT activity and infectivity on non-restrictive Mus dunni tail fibroblasts. The percentage of infected (GFP-positive) cells was determined by flow cytometry. (B) 293T cells were transfected with plasmids encoding glutathione S-transferase (GST) fusions with full-length TRIM5α or with TRIM5 lacking the SPRY domain. Cells were lysed (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.1% SDS) and mixed for 2 hrs at 4°C with virions (N-MLV or B-MLV) that had been concentrated by acceleration through 25% sucrose. GST fusions and associated proteins were enriched on glutathione-sepharose beads and immunoblotted with goat anti-MLV CA antibody (CA pull-out), or anti-GST antibody (bottom panel). Unbound CA remaining in the binding reaction was probed with anti-MLV CA antibody (CA input). TRIM5 protein domains fused to GST are indicated schematically on the bottom left: RF, ring finger; BB, B box; CC, coiled-coil.