During the late phase of HIV type 1 (HIV-1) replication, newly synthesized retroviral Gag proteins are targeted to the plasma membrane of most hematopoietic cell types, where they colocalize at lipid rafts and assemble into immature virions. Membrane binding is mediated by the matrix (MA) domain of Gag, a 132-residue polypeptide containing an N-terminal myristyl group that can adopt sequestered and exposed conformations. Although exposure is known to promote membrane binding, the mechanism by which Gag is targeted to specific membranes has yet to be established . Here we show that PI(4,5)P 2 binds directly to HIV-1 MA, inducing a conformational change that triggers myristate exposure. Related phosphatidylinositides PI, PI(3)P, PI(4)P, PI(5)P, and PI(3,5)P 2 do not bind MA with significant affinity or trigger myristate exposure. Structural studies reveal that PI(4,5)P 2 adopts an ''extended lipid'' conformation, in which the inositol head group and 2-fatty acid chain bind to a hydrophobic cleft, and the 1-fatty acid and exposed myristyl group bracket a conserved basic surface patch previously implicated in membrane binding. Our findings indicate that PI(4,5)P 2 acts as both a trigger of the myristyl switch and a membrane anchor and suggest a potential mechanism for targeting Gag to membrane rafts. matrix protein ͉ membrane targeting ͉ NMR ͉ lipid rafts

During the late phase of HIV-1 replication, newly synthesized retroviral Gag proteins are targeted to lipid raft regions of specific cellular membranes, where they assemble and bud to form new virus particles. Gag binds preferentially to the plasma membrane (PM) of most hematopoietic cell types, a process mediated by interactions between the cellular PM marker phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P 2 ) and Gag's N-terminally-myristoylated matrix (MA) domain. We recently demonstrated that PI(4,5)P 2 binds to a conserved cleft on MA and promotes myristate exposure, suggesting a role as both a direct membrane anchor and myristyl switch trigger. Here we show that PI(4,5)P 2 is also capable of binding to MA proteins containing point mutations that inhibit membrane binding in vitro, and in vivo, including V7R, L8A and L8I. However, these mutants do not exhibit PI(4,5)P 2 -or concentration-dependent myristate exposure. NMR studies of V7R and L8A MA reveal minor structural changes that appear to be responsible for stabilizing the myristate-sequestered (myr (s)) species and inhibiting exposure. Unexpectedly, the myristyl group of a revertant mutant with normal PM targeting properties (V7R,L21K) is also tightly sequestered and insensitive to PI(4,5) P 2 binding. This mutant binds PI(4,5)P 2 with two-fold higher affinity compared with the native protein, suggesting a potential compensatory mechanism for membrane binding. KeywordsHuman immunodeficiency virus type-1 (HIV-1); Gag; myristyl (myr); matrix (MA); phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ); analytical ultracentrifugation (AU); nuclear magnetic resonance (NMR)

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