Figure 2
From: Refined study of the interaction between HIV-1 p6 late domain and ALIX
Characterization of the HIV-1 p6 ΔSQKQ -ALIX interaction. (A) HIV-1 p6 (1–52) structure according to [20]. (B) HIV-1 p6 (1–52), p6 ΔSQKQ and EIAV p9 interaction with either full length ALIX, or ALIXΔPRR as determined in yeast two-hybrid assay and revealed by α-galactosidase expression quantified by densitometry. Data are expressed as percentage of the maximal activity observed after the cotransformation with mutant p6ΔSQK and ALIX proteins. (C) Interaction determined by GST-pull-down. GST fusion proteins were obtained by subcloning of HIV-1 p6, p6ΔSQKQ and EIAV p9 domains into pGEX-KT (GE Healthcare). Purified GST-proteins bound to glutathione-beads were mixed with cell lysates containing either ALIX-HA or ALIXΔPRR-HA proteins. Co-precipitated proteins were detected by western blotting using an anti-HA monoclonal antibody (Clone HA.11) and quantified by densitometry. Results were expressed in percentage of the band intensity measured in the presence of the GST-p9 construct. Equivalent loads of the GST fusion proteins were verified by Coomassie blue staining of the glutathione-bound fraction. The lanes marked Input contain 10% of the cell extract used for binding experiments. (D) L-domain function as determined using a complementation assay [6, 20, 21, 35]. 293T cells were cotransfected with 300 ng of HIV proviral plasmid (Nlδp6) that lacks the p6 L domain, 200 ng of plasmid expressing a truncated HIV Gag protein (Gagδp6) fused to the p6 domain of Gag mutated on the PTAP L domain (PTAP/LIRL) or to the p6 PTAP/LIRLΔSQKQ and 200 ng of plasmid expressing myc tagged ALIX (1–868) or an empty vector. Virion samples pelleted through 20% sucrose cushions, Gag expression and Myc-ALIX were analyzed [21] by western blotting with a mouse antibody anti-HIV CAp24 serum (Biodesign International) and with a monoclonal antibody anti-Myc (Santa Cruz Biotechnology). Virion was also measured 48 h later using an infection assay with MAGIC-5B (HeLa-CD4/CCR5 LTR-lacZ) indicator cells for HIV-1. Error bars in infectivity assays represented standard deviations of three separate experiments.