PARTITIONING OF HIV-1 GAG AND GAG-RELATED PROTEINS TO MEMBRANES

The binding of HIV-1 Gag and Gag-related proteins to model membranes w as examined using three experimental systems: (i) large unilamellar ph ospholipid vesicles (LUVs) and recombinant Gag purified from Escherich ia coli; (ii) LUVs added to a mammalian cell extract in which Gag prot eins were expressed by a coupled transcription/translation system; and (iii) inside-out plasma membrane vesicles purified from human red blo od cells (RBC) and recombinant, purified Gag from E. coli. Several nov el aspects of HIV-1 Gag membrane interactions were observed: (i) Gag p roteins bound with high affinity to both model membranes with a negati vely charged surface and to RBC membranes. (ii) Binding of the Gag pre cursor and mature Gag proteins exhibited different sensitivities to io nic strength indicating that the precursor directed membrane binding t hrough interactions that were qualitatively and quantitatively distinc t from those of any of its individual domains. Studies using energy tr ansfer between tryptophan residues in the proteins and anthroyloxy-con taining probes inserted In the LUVs indicated that the orientation of the precursor and of the mature proteins on the membrane surface were distinct; (iii) Gag oligomers appear to have facilitated high-affinity binding under high salt conditions, suggesting that protein-protein i nteractions led to formation of stronger electrostatic or new hydropho bic membrane binding determinants. Since binding studies with model me mbranes permit quantitative analysis, these experimental approaches ma y permit identification of interactions that drive Gag assembly on the membrane.