Fig 1.

Mapping of Alix Bro1 residues involved in the interaction with HIV-1 and EIAV NC domains. (A and B) GST, GST-NCp6, and GST-NCp9 fusion proteins were expressed in E. coli, captured on glutathione-conjugated beads, and subsequently incubated with lysates from 293T cells expressing WT HA-tagged Bro1 domain or the indicated Bro1 mutants (Q8K, K2R, and Q8/K2R). Captured proteins and cell lysates were analyzed by SDS-PAGE and Western blotting. GST fusion proteins were visualized by Coomassie blue staining. (C) Mutations that compromise the NC binding interface in Bro1 do not affect Alix V domain interaction with HIV-1 p6. Pulldown assays were performed as described above, with the only difference being that full-length HA-tagged Alix WT and the indicated mutants were used instead of the isolated Bro1 domain.