Figure 1.

Yeast two hybrid interaction between the HIV-1 p6 late domain and ALIX. (A) Alanine-scanning mutagenesis of the ALIX-binding region of p6. The HIV-1 p6 (pNL4-3, NIH AIDS Research and Reference Reagent Program) derived-DNA fragment was generated by PCR and inserted in frame with the Gal4-DBD of pGBKT7 (Clonetech). ALIX was PCR-generated from plasmid pGAD AIP-1/ALIX [23] and fused in frame with the Gal4 AD of pACT2 (Clontech). Yeast strain AH109 (MATa, trp-901, leu2-3, 112, ura3-52, his3-200, gal4Δ, gal80Δ, LYS2::GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2; URA3::MEL1UAS-MEL1TATA-lacZ, MEL1) was cotransformed with pGBKT7 and pACT2 derivatives. The relative strength of the protein interaction between bait and prey was determined in yeast transformants grown at 30°C in SD/-Leu/-Trp selection medium by measuring β-galactosidase activity according to the protocol described in the Yeast β-Galactosidase Assay Kit from Pierce. Values are referred to 100% β-galactosidase activity measured in yeast cells cotransformed with wild-type p6 and ALIX proteins. Liquid culture assays were performed in triplicate. In the histogram, the lack of activity is indicated by triangles. (B). ALIX and fragments thereof were tested for interaction with Gal4 DBD-HIV-1 p6 and/or GAL4 DBD-p9 EIAV_UK [33]. Bro1: Bro1-rhophilin-like domain; PRR: proline-rich region. Deletions and point mutations were generated using a splice-overlap extension method [34]. A reference value of 1 was set to β-galactosidase activity resulting from the interaction of ALIX with either p9 or p6 wild-type proteins. The lack of activity is indicated by triangles.