No abstract

The three-dimensional structure of the human immunodeficiency virus-type 1 (HIV-1) nucleocapsid protein (NC) bound to the SL3 stem-loop recognition element of the genomic Psi RNA packaging signal has been determined by heteronuclear magnetic resonance spectroscopy. Tight binding (dissociation constant, approximately 100 nM) is mediated by specific interactions between the amino- and carboxyl-terminal CCHC-type zinc knuckles of the NC protein and the G7 and G9 nucleotide bases, respectively, of the G6-G7-A8-G9 RNA tetraloop. A8 packs against the amino-terminal knuckle and forms a hydrogen bond with conserved Arg32, and residues Lys3 to Arg10 of NC form a 310 helix that binds to the major groove of the RNA stem and also packs against the amino-terminal zinc knuckle. The structure provides insights into the mechanism of viral genome recognition, explains extensive amino acid conservation within NC, and serves as a basis for the development of inhibitors designed to interfere with genome encapsidation.

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

The myristoylated matrix protein (myr-MA) of HIV functions as a regulator of intracellular localization, targeting the Gag precursor polyprotein to lipid rafts in the plasma membrane during virus assembly and dissociating from the membrane during infectivity for nuclear targeting of the preintegration complex. Membrane release is triggered by proteolytic cleavage of Gag, and it has, until now, been believed that proteolysis induces a conformational change in myr-MA that sequesters the myristyl group. NMR studies reported here reveal that myr-MA adopts myr-exposed [myr(e)] and -sequestered [myr(s)] states, as anticipated. Unexpectedly, the tertiary structures of the protein in both states are very similar, with the sequestered myristyl group occupying a cavity that requires only minor conformational adjustments for insertion. In addition, myristate exposure is coupled with trimerization, with the myristyl group sequestered in the monomer and exposed in the trimer (Kassoc ‫؍‬ 2.5 ؎ 0.6 ؋ 10 8 M ؊2 ). The equilibrium constant is shifted Ϸ20-fold toward the trimeric, myristate-exposed species in a Gag-like construct that includes the capsid domain, indicating that exposure is enhanced by Gag subdomains that promote self-association. Our findings indicate that the HIV-1 myristyl switch is regulated not by mechanically induced conformational changes, as observed for other myristyl switches, but instead by entropic modulation of a preexisting equilibrium.