Retroviruses use short peptide motifs (late domains) located within the viral Gag protein to recruit cellular factors that facilitate virus budding. The YPXnL late domains of HIV and other lentiviruses recruit ALIX/AIP1, which also functions in vesicle formation at the multi-vesicular body and in the abscission stage of cytokinesis. In this study from the Hill, Sundquist, and Myszka labs, we determined the crystal structures of ALIX in complex with the YPXnL late domains from HIV-1 and EIAV. The two distinct late domains bind at the same site on the ALIX V domain but adopt different conformations that allow them to make equivalent contacts. Binding studies and functional assays were performed to verify the importance of key interface residues and these studies also showed that binding affinities are tuned by context-dependent effects. Overall, the study revealed how YPXnL late domains recruit ALIX to facilitate virus budding and how ALIX can bind both YPXnL sequences with n=1 and n=3.

Overlaid structures of the YPXnL late domains (sticks) of HIV-1 p6Gag (green) and EIAV p9Gag (turquoise) binding to arm 2 of the ALIX V domain (surface with binding site residues highlighted in yellow and critical binding residues underlined). Note that the intervening 38LTS40 residues of the HIV-1 YPXnL (n=3) motif form a helical turn, which positions Leu41 in an equivalent position to Leu26 of the EIAV p9Gag YPXnL late domain (n=1).