Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly

Abstract

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

Incorporation of Glycosylphosphatidylinositol-Anchored Granulocyte- MacrophageColony-Stimulating Factor or CD40 Ligand Enhances Immunogenicity of Chimeric Simian Immunodeficiency Virus-Like Particles

“…As an alternative, we generated membrane-bound GM-CSF by tethering it with GPI anchors from CD59 or LFA3. Interestingly, the GPIanchored GM-CSF was found to be incorporated at higher levels into the VLPs than GM-CSF TM-gp160 , presumably because GPI-anchored proteins associate with lipid rafts, which are used as sites for HIV assembly (34,41). In addition, the enhanced activity of SIV VLPs with GPI-anchored GM-CSF in vivo could possibly be attributed to greater flexibility and less steric constraints of the GPI anchor.…”

Point Mutations in the HIV-1 Matrix Protein Turn Off the Myristyl Switch

“…Chemical shift changes of "group 2" were observed for WT myrMA upon addition of di-C 4 -PI(4,5)P 2 and NMR-based structural studies confirmed that they reflect di-C 4 -PI(4,5)P 2 -dependent exposure of the myristyl group. 31 In addition, intermolecular NOEs between protein residues and myristate group are clearly observed in the 3D 13 C-edited/ 12 C-double-half-filtered NOE experiment obtained for the V7R:di-C 4 -PI(4,5)P 2 complex (Figure 7), indicating that di-C 4 -PI(4,5)P 2 binding does not trigger myristyl exposure.…”

“…In addition, the relative NOE intensities and cross-peak patterns matched the data obtained for the WT myr(s)MA protein. 27,31 The main differences between the structures are localized within the disordered loop formed by residues Gly-2 -Ser-9. For V7R, the side chain of Arg-7 is poised to make a salt bridge with the side chain of Glu-52, and the mythelene groups of Arg-7 contribute to the hydrophobic interactions with the methylene groups of the myristate ( Figure 6).…”

Monoclonal Antibodies to Phosphatidylinositol Phosphate Neutralize Human Immunodeficiency Virus Type 1: Role of Phosphate-Binding Subsites

“…However, one simple hypothesis to explain the poor neutralization by anti-PIP in the pseudovirus/ TZM-bl system compared to that in the PBMC assay is that the anti-PIP antibodies might neutralize HIV-1 by binding to viral lipids when the lipid bilayer of the virus directly interacts with the lipid bilayer of the target cell. A further possibility is that anti-PIP reacts with phosphatidylinositol-4,5-bisphosphate during viral assembly and budding (27). In either case, such binding of anti-PIP to the viral lipids might be sterically hindered by excessively high levels of adjacent CCR5 protein on the TZM-bl target cell when the viral and plasma membrane lipid bilayers are closely apposed.…”