Journal of Molecular Biology
HIV Fusion Peptide Penetrates, Disorders, and Softens T-Cell Membrane Mimics
Introduction
Infection by enveloped viruses, such as human immunodeficiency virus type 1 (HIV-1), requires fusion of the virion membrane with the target cell membrane in order to transfer viral RNA into the target cell.1 Enveloped viruses use ectodomain glycoproteins to first dock with receptors on the T-cell membrane and then perturb the target membrane to form a pore.2 On the HIV-1 ectodomain, the glycoproteins gp160 are typically assembled as homotrimers3 and proteolytically cleaved to gp120 and gp41, which remain non-covalently associated. gp120 binds to the CD4 glycoprotein on the T cell4 and a chemokine coreceptor, primarily CCR5 and CXCR4.5 This binding causes a conformational change in gp41 that exposes the fusion peptide (FP23) that contains a highly hydrophobic sequence of 23 amino acids at the N-terminus of gp41.3, 6, 7 FP interacts in a nonspecific way with the target membrane and perhaps also with the virion membrane;8 this interaction provides the perturbation that allows a pore to form, thus allowing release of the HIV RNA into the target cell. The importance of FP23 is indicated by its ability to fuse and/or lyse liposomes and erythrocytes,9, 10 and mutations with a polar residue in this sequence drastically reduce fusogenic activity.11, 12 Although some investigations have studied longer13 and shorter14 N-terminal FP constructs from HIV (the 12-mer being the minimal FP15), FP23 is the most common choice.9, 10, 13 It may also be noted that a recent work presents evidence to show that the main pathway is endocytosis of the HIV virion, rather than fusion with the plasma membrane, and then the virion membrane fuses with the endosomal membrane near the nuclear membrane in lymphoid CEMss cells expressing CD4 and CXCR4.16 Even in this scenario, the mechanism of fusion between the virion and endosomal membranes would likely be similar to that at the plasma membrane, since the same HIV ectodomain is exposed at the virion membrane surface.17
Atomic-resolution structure is often achievable and valuable. The crystal structure of gp41 of simian immunodeficiency virus has been obtained18 as has the crystal structure of HIV-1 gp41 ectodomain.3 These structures share a similar coiled-coil core, which is thought to stabilize the interaction of the viral and T-cell membranes. In contrast, the crystal structure of the N-terminal FP has not been determined, since it is normally removed prior to crystallization due to its extreme hydrophobicity.3, 18 Even a 3D solution structure of ectodomain of simian immunodeficiency virus gp41 did not contain the 26 N-terminal amino acids.19 Furthermore, the membrane with which FP interacts is in a fluid state; hence, it should not be expected that physiological concentrations of FP will induce a crystalline state. Even if a method could be found to obtain a crystalline state of FP with lipids, it is unlikely that such a state would be biologically relevant because it would not be fully hydrated and the disorder due to the fluid nature of the membranes would not be present. Instead of atomic-level crystallography that utilizes sharp diffraction peaks, we use the diffuse x-ray scattering that is produced by fully hydrated, fluctuating bilayers to determine disordered structure at the nanoscale.20
Since gp41 acts as a trimer during HIV infection, a trimer construct of FP (FPtri) could be an important tool for studying its membrane interactions. A water-soluble monomer (FPwsm) had been synthesized, by attaching six lysines to the native FP, in order to ease synthesis steps.21 A similar FPwsm produced by genetic expression was found useful for solution NMR studies.22 FPtri enhanced lipid mixing in LM3 model fusion studies by 40 times compared to FPwsm.21 Solid-state NMR has shown that there is a strong correlation between membrane insertion depth and fusogenicity in a model membrane system of PC:PG (∼ 4:1) with 30 mol% cholesterol.23 In the present work, we study FP23 as well as the FPwsm and FPtri constructs in LM3. LM3 is a T-cell lipid membrane mimic consisting of a mixture of six lipids [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE):1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS):soy phosphatidylinositol (PI):egg sphingomyelin (ESM):cholesterol (Chol)], in a 10:5:2:1:2:10 mole ratio,24, 25 which our work shows is a liquid ordered bilayer that becomes more disordered when FP is added. As controls, we also compare results of adding FP23 to simpler lipid bilayers, starting with the favorite fully fluid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (diC18:1PC) bilayer, and we also add cholesterol. However, the DOPC bilayer is considerably thinner than LM3; thus, we have also studied 1,2-dierucoyl-sn-glycero-3-phosphocholine (diC22:1PC; with and without 30 mol% cholesterol), which is 7 Å thicker than DOPC.26 In addition to structure, our synchrotron low-angle x-ray scattering (LAXS) data determine the bending modulus, KC, which is a measure of membrane stiffness. Using our wide-angle x-ray scattering (WAXS) analysis,27 we also determine the orientational Sxray order parameters. In a third type of diffraction experiment, we show that FP23 increases the curvature of the hexagonal II phase forming lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), thus confirming that FP23 penetrates into the hydrocarbon interior. Finally, our volumetric data are fundamental for quantitative structural analysis.
Section snippets
Volumes
The volume shown for LM3 is the mole fraction weighted average of lipid (including cholesterol) in the LM3 mixture. Upon addition of FP23 mole fraction, xFP23 = [FP23]/([FP23] + [lipids]), of FP23 to any of the three bilayers in Table 1, the measured volume is VM = xFP23VFP + (1 − xFP23)VLipid. Using VFP23 = 2664 Å3 calculated from volumes of amino acids in crystals†, we show in the VL column in Table 1 that the volume of the average lipid molecule
Discussion
Although the most appropriate comparison was expected to be FPtri with the monomeric FPwsm from which FPtri was constructed, our LAXS (Fig. 1), KC (Table 3), WAXS (Fig. 6), and Sxray (Fig. 7) results show that monomeric FPwsm perturbs the LM3 bilayer far less than either monomeric FP23 or trimeric FPtri. We suggest that FPwsm did not partition into the LM3 membrane. This is at first surprising since both FPwsm and FPtri have the same proportion of additional lysines chemically attached to the
Lipids
LM3 is a mixture of six lipids (POPC:POPE:POPS:PI:ESM:Chol, 10:5:2:1:2:10) that mimics the T-cell lipid composition.24, 25 Lyophilized lipids were purchased from Avanti, Alabaster, AL: POPC (Lot 160–181PC-174), POPE (Lot 160–181PE-109), POPS (Lot 160–181PS-203), PI (Lot PPI-150), ESM (Lot ESM-101), and cholesterol (Lot CH-55). DOPC (Lot 181–228), diC22:1PC (Lot 221PC-33), and DOPE (Lots 181PE-304 and 243) were also purchased from Avanti Polar Lipids in the lyophilized form and used without
Acknowledgements
This research was supported by National Institutes of Health (NIH) Grant GM 44976 (S.T.-N. and J.F.N.), NIH Grant AI 47153 (W.Q. and D.P.W.), and the Howard Hughes Medical Institute (R.C.). X-ray scattering data were taken at CHESS, which is supported by the National Science Foundation and the NIH/National Institute of General Medical Sciences under National Science Foundation award DMR-0225180. We especially thank Dr. Arthur Woll for obtaining our beam and for general support during our data
References (74)
- et al.
Membrane fusion
Cell
(2003) - et al.
The amino-terminal peptide of HIV-1 glycoprotein 41 interacts with human erythrocyte membranes: peptide conformation, orientation and aggregation
Biochim. Biophys. Acta
(1992) - et al.
Membrane interactions of the synthetic N-terminal peptide of HIV-1 gp41 and its structural analogs
Biochim. Biophys. Acta
(1999) - et al.
Fusion peptides derived from the HIV type 1 glycoprotein 41 associate within phospholipid membranes and inhibit cell–cell fusion—structure–function study
J. Biol. Chem.
(1997) - et al.
The N-terminal 12 residue long peptide of HIV gp41 is the minimal peptide sufficient to induce significant T-cell-like membrane destabilization in vitro
J. Mol. Biol.
(2006) - et al.
HIV enters cells via endocytosis and dynamin-dependent fusion with endosomes
Cell
(2009) - et al.
The crystal structure of the SIV gp41 ectodomain at 1.47 Å resolution
J. Struct. Biol.
(1999) - et al.
Structure and plasticity of the human immunodeficiency virus gp41 fusion domain in lipid micelles and bilayers
Biophys. J.
(2007) - et al.
Order parameters and areas in fluid-phase oriented lipid membranes using wide angle x-ray scattering
Biophys. J.
(2008) - et al.
HIV-1 fusion peptide decreases bending energy and promotes curved fusion intermediates
Biophys. J.
(2007)