Structural basis for targeting avian sarcoma virus Gag polyprotein to the plasma membrane for virus assembly

Abstract

For most retroviruses, including HIV-1, binding of the Gag polyprotein to the plasma membrane (PM) is mediated by interactions between Gag's N-terminal myristoylated matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) in the PM. The Gag protein of avian sarcoma virus (ASV) lacks the N-myristoylation signal but contains structural domains having functions similar to those of HIV-1 Gag. The molecular mechanism by which ASV Gag binds to the PM is incompletely understood. Here, we employed NMR techniques to elucidate the molecular determinants of the membrane-binding domain of ASV MA (MA₈₇) to lipids and liposomes. We report that MA₈₇ binds to the polar head of phosphoinositides such as PI(4,5)P₂ We found that MA₈₇ binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups, indicating that the MA₈₇-IP binding is governed by charge-charge interactions. Using a sensitive NMR-based liposome-binding assay, we show that binding of MA₈₇ to liposomes is enhanced by incorporation of PI(4,5)P₂ and phosphatidylserine. We also show that membrane binding is mediated by a basic surface formed by Lys-6, Lys-13, Lys-23, and Lys-24. Substitution of these residues to glutamate abolished binding of MA₈₇ to both IPs and liposomes. In an accompanying paper, we further report that mutation of these lysine residues diminishes Gag assembly on the PM and inhibits ASV particle release. These findings provide a molecular basis for ASV Gag binding to the inner leaflet of the PM and advance our understanding of the basic mechanisms of retroviral assembly.

Keywords: ASV; Gag protein; avian sarcoma virus; human immunodeficiency virus (HIV); inositol hexakisphosphate; isothermal titration calorimetry (ITC); liposome; myristoylated matrix; phosphatidylinositol 4,5-bisphosphate; phosphatidylserine; plasma membrane.

Figure 1.

Comparison of ASV MA₈₇ structures from this work and a previous study. Shown are two views of our NMR structure of MA₈₇ (red cartoon) overlaid with the previously determined structure (PDB entry 1A6S, light cyan). Structures were superimposed by minimizing the positional RMSD of N, C^α, and C′ atoms of residues 3–85.

Figure 2.

PI(4,5)P₂ binding to MA₈₇. A, overlay of two-dimensional ¹H-¹⁵N HSQC spectra upon titration of MA₈₇ with diC₄-PI(4,5)P₂ (100 μm, 35 °C; diC₄-PI(4,5)P₂/MA = 0:1 (black), 1:1 (magenta), 2:1 (orange), 4:1 (blue), 8:1 (green), and 16:1 (red)). Signals labeled in blue are shifted by 20 ppm due to aliasing. B, histogram of normalized ¹H-¹⁵N chemical shift changes versus residue number calculated from the HSQC spectra for MA₈₇ upon titration with diC₄-PI(4,5)P₂. C, cartoon representation and electrostatic map of the MA₈₇ structure highlighting basic residues (blue) that exhibited substantial chemical shift changes upon binding of diC₄-PI(4,5)P₂. Of note, Lys¹⁸ and Lys²⁴ (cyan) are in close proximity to the perturbed residues but do not appear to be perturbed by lipid binding.

Figure 3.

IP₆ binding to MA₈₇. Shown is an overlay of two-dimensional ¹H-¹⁵N HSQC spectra upon titration of MA₈₇ with IP₆ (100 μm, 35 °C; IP₆/MA = 0:1 (black), 0.25:1 (magenta), 0.5:1 (orange), 1:1 (blue), 2:1 (green), and 4:1 (red)). Signals labeled in blue are shifted by 20 ppm due to aliasing.

Figure 4.

ITC data for binding of IP₆ to MA₈₇. ITC data were obtained for titration of IP₆ (2.5 mm) into MA₈₇ (192 μm). DP is differential power.

Figure 5.

Structures of MA₈₇ bound to inositol phosphates. Shown are representative models of MA₈₇ bound to IP₆, I(1,4,5)P₃, and I(1,3,5)P₃ calculated in HADDOCK. For all complexes, the phosphate groups are poised to form salt bridges with the lysine amine groups.

Figure 6.

Interaction of MA₈₇ with LUVs. A, overlay of ¹H NMR spectra of MA₈₇ (50 μm) in the presence of constant amount of LUVs with (80 − x)% mol POPC, 20% mol POPS, and x = 0 (black), 1 (red), 2 (green), 3 (dark blue), 4 (light blue), 5 (yellow), and 7 (purple) mol % PI(4,5)P₂. The gray box marks the area of spectra integration. B, MA₈₇ binding to POPC LUVs as a function of increasing PI(4,5)P₂ or POPS concentrations. Molar percentages of POPS are indicated. C, isotherms of MA₈₇ binding to LUVs containing POPC, fixed 20% mol POPS, and varying amounts of PI(4,5)P₂ or PI(3,5)P₂. Solid lines are Hill equation fits to the experimental data represented by points with error bars. D, fitted parameters of Hill equation.

Figure 7.

Binding of MA₈₇ mutant proteins to LUVs. Shown is binding of ASV MA₈₇ mutants to LUVs containing POPC/POPS/PI(4,5)P₂ (65:30:5) as determined by ¹H NMR. Each sample contained 50 μm MA₈₇ and 231 μg of lipids. Error bars, S.D.

Figure 8.

Model of ASV MA₈₇–membrane interaction and comparison of HIV-1 and ASV MA structures. A, a model of MA₈₇ bound to a membrane bilayer constructed based on the structural data using a representative structure of MA₈₇. Favorable electrostatic interactions occur between the acidic polar head of PI(4,5)P₂ and a basic patch formed by Lys⁶, Lys¹³, and Lys²³ (blue). HADDOCK calculations suggest a potential membrane interaction with Lys²⁴ due to the close proximity of the side chain to the inositol ring. Membrane bilayer was generated in the VMD membrane builder plug-in (78). PI(4,5)P₂ was generated in Avogadro (75). B, cartoon representation of the ASV and HIV-1 MA structures (PDB entries 6CCJ and 2H3I, respectively), highlighting the similar structural motifs and basic residues involved in membrane binding (blue sticks). The myr group, residues 2–3, and residues 115–132 of HIV-1 MA are not shown for clarity.