Figure 4.

ALIX and TSG101 bind to MA. The relative affinity for MA compared with p6 is higher for ALIX.A, Gag-GFP-VLP release from HEK293 cells recapitulating published data for p6 dependence of HIV-1 release. 2 days after transfection with CMV promoter expression vectors for the indicated Gag versions, VLPs were harvested from the culture medium, and cell lysates were prepared. Gag was detected by immunoblotting with anti-GFP antibodies. A short (a) and a long (b) exposure are shown. B, Gag-GFP schematic. C–E, coimmunoprecipitations (IP) of epitope-tagged ALIX or TSG101 with GFP-tagged Gag versions or MA expressed from CMV promoter vectors in HEK293 cells, indicating that ALIX binding to Gag is reduced but not prevented by p6 deletion and that ALIX binds to MA. TSG101 binding to Gag more strongly depends on p6. GFP-tagged proteins were immunoprecipitated with anti-GFP antibodies in the presence of 150 mm NaCl (C and D) or as indicated (E), and coimmunopreciptated ALIX or TSG101 was detected with antibodies recognizing the epitope tag. F–I, pulldown experiments showing that ALIX and TSG101 bind to GST-MA expressed in E. coli. The relative affinity for MA compared with p6 is higher for ALIX. GST-tagged Gag fragments (MA (aa 1–132), CA (aa 133–363), and p6 (aa 448–500)) were bound to GSH-Sepharose and incubated with extract of HEK293 cells expressing epitope-tagged ALIX or TSG101 from a CMV promoter vector. Bead-bound proteins were analyzed by immunoblotting with the indicated antibodies. α-Actin served as control, showing specific ESCRT protein binding.