SUPPLEMENTAL EXPERIMENTAL METHODS Plasmid Construction

ALIX Protein Expression Constructs. ALIX coding sequences were amplified and subcloned from an EST clone (von Schwedler et al., 2003), and ALIX proteins were expressed as either 6x-His or GST N-terminal fusion proteins. For ALIXBro1-V (residues 1-698) and ALIXV (residues 360-702), ALIX coding sequences were amplified with 5’NdeI and 3’BamHI restriction sites and inserted into the pET151/D-TOPO vector (Invitrogen) following the manufacturer’s instructions. ALIXBro1 (residues 1-359) was cloned between 5’NdeI and 3’BamHI restriction sites in a modified pET16b vector (Novagen) designed to contain a TEV protease cleavage site following the 6x-His tag. GST-ALIX expression constructs were generated by inserting ALIX coding sequences between 5’NdeI and 3’BamHI restriction sites in a pGEX2T vector (GE Healthcare) modified to contain a TEV protease cleavage site and 5’NdeI and 3’BamHI/BglII restriction sites following the GST gene (pGEX2T-TEV). HIV-1 p6Gag and EIAV p9Gag Expression Constructs. Genes encoding HIV-1NL4-3 p6Gag and EIAV p9Gag were amplified from the proviral R9 and pEV53B plasmids (see below), with 5’NdeI and 3’BamHI restriction sites, to allow expression as GST fusion protein