Membrane fusion promoters and inhibitors have contrasting effects on lipid bilayer structure and undulations.
(9/3607)
It has been established that the fusion of both biological membranes and phospholipid bilayers can be modulated by altering their lipid composition (Chernomordik et al., 1995 .J. Membr. Biol. 146:3). In particular, when added exogenously between apposing membranes, monomyristoylphosphatidylcholine (MMPC) inhibits membrane fusion, whereas glycerol monoleate (GMO), oleic acid (OA), and arachidonic acid (AA) promote fusion. This present study uses x-ray diffraction to investigate the effects of MMPC, GMO, OA, and AA on the bending and stability of lipid bilayers when bilayers are forced together with applied osmotic pressure. The addition of 10 and 30 mol% MMPC to egg phosphatidylcholine (EPC) bilayers maintains the bilayer structure, even when the interbilayer fluid spacing is reduced to approximately 3 A, and increases the repulsive pressure between bilayers so that the fluid spacing in excess water increases by 5 and 15 A, respectively. Thus MMPC increases the undulation pressure, implying that the addition of MMPC promotes out-of-plane bending and decreases the adhesion energy between bilayers. In contrast, the addition of GMO has minor effects on the undulation pressure; 10 and 50 mol% GMO increase the fluid spacing of EPC in excess water by 0 and 2 A, respectively. However, x-ray diffraction indicates that, at small interbilayer separations, GMO, OA, or AA converts the bilayer to a structure containing hexagonally packed scattering units approximately 50 A in diameter. Thus GMO, OA, or AA destabilizes bilayer structure as apposing bilayers are brought into contact, which could contribute to their role in promoting membrane fusion. (+info)
Evidence for the extended phospholipid conformation in membrane fusion and hemifusion.
(10/3607)
Molecular-level mechanisms of fusion and hemifusion of large unilamellar dioleoyl phosphatidic acid/phosphocholine (DOPA/DOPC, 1:1 molar ratio) vesicles induced by millimolar Ca2+ and Mg2+, respectively, were investigated using fluorescence spectroscopy. In keeping with reduction of membrane free volume Vf, both divalent cations increased the emission polarization for 1,6-diphenyl-1,3, 5-hexatriene (DPH). An important finding was a decrease in excimer/monomer emission intensity ratio (Ie/Im) for the intramolecular excimer-forming probe 1, 2-bis[(pyren-1-)yl]decanoyl-sn-glycero-3-phosphocholine (bis-PDPC) in the course of fusion and hemifusion. Comparison with another intramolecular excimer-forming probe, namely, 1-[(pyren-1)-yl]decanoyl-2-[(pyren-1)-yl]tetradecanoyl-sn-gl ycero-3-p hosphocholine (PDPTPC), allowed us to exclude changes in acyl chain alignment to be causing the decrement in Ie/Im. As a decrease in Vf should increase Ie/Im for bis-PDPC and because contact site between adhering liposomes was required we conclude the most feasible explanation to be the adoption of the extended conformation (P.K.J., Chem. Phys. Lipids 63:251-258) by bis-PDPC. In this conformation the two acyl chains are splaying so as to become embedded in the opposing leaflets of the two adhered bilayers, with the headgroup remaining between the adjacent surfaces. Our data provide evidence for a novel mechanism of fusion of the lipid bilayers. (+info)
Differential inhibition of human immunodeficiency virus type 1 fusion, gp120 binding, and CC-chemokine activity by monoclonal antibodies to CCR5.
(11/3607)
The CC-chemokine receptor CCR5 mediates fusion and entry of the most commonly transmitted human immunodeficiency virus type 1 (HIV-1) strains. We have isolated six new anti-CCR5 murine monoclonal antibodies (MAbs), designated PA8, PA9, PA10, PA11, PA12, and PA14. A panel of CCR5 alanine point mutants was used to map the epitopes of these MAbs and the previously described MAb 2D7 to specific amino acid residues in the N terminus and/or second extracellular loop regions of CCR5. This structural information was correlated with the MAbs' abilities to inhibit (i) HIV-1 entry, (ii) HIV-1 envelope glycoprotein-mediated membrane fusion, (iii) gp120 binding to CCR5, and (iv) CC-chemokine activity. Surprisingly, there was no correlation between the ability of a MAb to inhibit HIV-1 fusion-entry and its ability to inhibit either the binding of a gp120-soluble CD4 complex to CCR5 or CC-chemokine activity. MAbs PA9 to PA12, whose epitopes include residues in the CCR5 N terminus, strongly inhibited gp120 binding but only moderately inhibited HIV-1 fusion and entry and had no effect on RANTES-induced calcium mobilization. MAbs PA14 and 2D7, the most potent inhibitors of HIV-1 entry and fusion, were less effective at inhibiting gp120 binding and were variably potent at inhibiting RANTES-induced signaling. With respect to inhibiting HIV-1 entry and fusion, PA12 but not PA14 was potently synergistic when used in combination with 2D7, RANTES, and CD4-immunoglobulin G2, which inhibits HIV-1 attachment. The data support a model wherein HIV-1 entry occurs in three stages: receptor (CD4) binding, coreceptor (CCR5) binding, and coreceptor-mediated membrane fusion. The antibodies described will be useful for further dissecting these events. (+info)
The cholesterol requirement for sindbis virus entry and exit and characterization of a spike protein region involved in cholesterol dependence.
(12/3607)
Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped alphaviruses that enter cells via low-pH-triggered fusion in the endocytic pathway and exit by budding from the plasma membrane. Previous studies with cholesterol-depleted insect cells have shown that SFV requires cholesterol in the cell membrane for both virus fusion and efficient exit of progeny virus. An SFV mutant, srf-3, shows efficient fusion and exit in the absence of cholesterol due to a single point mutation in the E1 spike subunit, proline 226 to serine. We have here characterized the role of cholesterol in the entry and exit of SIN, an alphavirus quite distantly related to SFV. Growth, primary infection, fusion, and exit of SIN were all dramatically inhibited in cholesterol-depleted cells compared to control cells. Based on sequence differences within the E1 226 region between SFV, srf-3, and SIN, we constructed six SIN mutants with alterations within this region and characterized their cholesterol dependence. A SIN mutant, SGM, that had the srf-3 amino acid sequence from E1 position 224 to 235 showed increases of approximately 100-fold in infection and approximately 250-fold in fusion with cholesterol-depleted cells compared with infection and fusion of wild-type SIN. Pulse-chase analysis demonstrated that SGM exit from cholesterol-depleted cells was markedly more efficient than that of wild-type SIN. Thus, similar to SFV, SIN was cholesterol dependent for both virus entry and exit, and the cholesterol dependence of both steps could be modulated by sequences within the E1 226 region. (+info)
Cyanovirin-N binds to gp120 to interfere with CD4-dependent human immunodeficiency virus type 1 virion binding, fusion, and infectivity but does not affect the CD4 binding site on gp120 or soluble CD4-induced conformational changes in gp120.
(13/3607)
Cyanovirin-N (CV-N), an 11-kDa protein isolated from the cyanobacterium Nostoc ellipsosporum, potently inactivates diverse strains of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus. While it has been well established that the viral surface envelope glycoprotein gp120 is a molecular target of CV-N, the detailed mechanism of action is of further interest. We compared matched native and CV-N-treated virus preparations in a panel of assays that measure viral replication, assessing successive stages of the viral life cycle. CV-N-treated virions failed to infect cells as detected by p24 production and quantitative PCR for HIV-1 reverse transcription products, whereas treatment of the target cells did not block infection, confirming that CV-N acts at the level of the virus, not the target cell, to abort the initial infection process. Compared to native HIV-1 preparations, CV-N-treated HIV-1 virions showed impaired CD4-dependent binding to CD4(+) T cells and did not mediate "fusion from without" of CD4(+) target cells. CV-N also blocked HIV envelope glycoprotein Env-induced, CD4-dependent cell-cell fusion. Mapping studies with monoclonal antibodies (MAbs) to defined epitopes on the HIV-1 envelope glycoprotein indicated that CV-N binds to gp120 in a manner that does not occlude or alter the CD4 binding site or V3 loop or other domains on gp120 recognized by defined MAbs and does not interfere with soluble CD4-induced conformational changes in gp120. Binding of CV-N to soluble gp120 or virions inhibited subsequent binding of the unique neutralizing MAb 2G12, which recognizes a glycosylation-dependent epitope. However, prior binding of 2G12 MAb to gp120 did not block subsequent binding by CV-N. These results help clarify the mechanism of action of CV-N and suggest that the compound may act in part by preventing essential interactions between the envelope glycoprotein and target cell receptors. This proposed mechanism is consistent with the extensive activity profile of CV-N against numerous isolates of HIV-1 and other lentiviruses and supports the potential broad utility of this protein as a microbicide to prevent the sexual transmission of HIV. (+info)
Subdomain folding and biological activity of the core structure from human immunodeficiency virus type 1 gp41: implications for viral membrane fusion.
(14/3607)
The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) consists of two subunits, gp120 and gp41. The extraviral portion (ectodomain) of gp41 contains an alpha-helical domain that likely represents the core of the fusion-active conformation of the molecule. Here we report the identification and characterization of a minimal, autonomous folding subdomain that retains key determinants in specifying the overall fold of the gp41 ectodomain core. This subdomain, designated N34(L6)C28, is formed by covalent attachment of peptides N-34 and C-28 by a short flexible linker in place of the normal disulfide-bonded loop sequence. N34(L6)C28 forms a highly thermostable, alpha-helical trimer. Point mutations within the envelope protein complex that abolish membrane fusion and HIV-1 infectivity also impede the formation of the N34(L6)C28 core. Moreover, N34(L6)C28 is capable of inhibiting HIV-1 envelope-mediated membrane fusion. Taken together, these results indicate that the N34(L6)C28 core plays a direct role in the membrane fusion step of HIV-1 infection and thus provides a molecular target for the development of antiviral pharmaceutical agents. (+info)
Cloning and sequencing of a protein involved in phagosomal membrane fusion in Paramecium.
(15/3607)
An mAb was raised to the C5 phagosomal antigen in Paramecium multimicronucleatum. To determine its function, the cDNA and genomic DNA encoding C5 were cloned. This antigen consisted of 315 amino acid residues with a predicted molecular weight of 36,594, a value similar to that determined by SDS-PAGE. Sequence comparisons uncovered a low but significant homology with a Schizosaccharomyces pombe protein and the C-terminal half of the beta-fructofuranosidase protein of Zymomonas mobilis. Lacking an obvious transmembrane domain or a possible signal sequence at the N terminus, C5 was predicted to be a soluble protein, whereas immunofluorescence data showed that it was present on the membranes of vesicles and digestive vacuoles (DVs). In cells that were minimally permeabilized but with intact DVs, C5 was found to be located on the cytosolic surface of the DV membranes. Immunoblotting of proteins from the purified and KCl-washed DVs showed that C5 was tightly bound to the DV membranes. Cryoelectron microscopy also confirmed that C5 was on the cytosolic surface of the discoidal vesicles, acidosomes, and lysosomes, organelles known to fuse with the membranes of the cytopharynx, the DVs of stages I (DV-I) and II (DV-II), respectively. Although C5 was concentrated more on the mature than on the young DV membranes, the striking observation was that the cytopharyngeal membrane that is derived from the discoidal vesicles was almost devoid of C5. Approximately 80% of the C5 was lost from the discoidal vesicle-derived membrane after this membrane fused with the cytopharyngeal membrane. Microinjection of the mAb to C5 greatly inhibited the fusion of the discoidal vesicles with the cytopharyngeal membrane and thus the incorporation of the discoidal vesicle membranes into the DV membranes. Taken together, these results suggest that C5 is a membrane protein that is involved in binding and/or fusion of the discoidal vesicles with the cytopharyngeal membrane that leads to DV formation. (+info)
Structural basis for paramyxovirus-mediated membrane fusion.
(16/3607)
Paramyxoviruses are responsible for significant human mortality and disease worldwide, but the molecular mechanisms underlying their entry into host cells remain poorly understood. We have solved the crystal structure of a fragment of the simian parainfluenza virus 5 fusion protein (SV5 F), revealing a 96 A long coiled coil surrounded by three antiparallel helices. This structure places the fusion and transmembrane anchor of SV5 F in close proximity with a large intervening domain at the opposite end of the coiled coil. Six amino acids, potentially part of the fusion peptide, form a segment of the central coiled coil, suggesting that this structure extends into the membrane. Deletion mutants of SV5 F indicate that putative flexible tethers between the coiled coil and the viral membrane are dispensable for fusion. The lack of flexible tethers may couple a final conformational change in the F protein directly to the fusion of two bilayers. (+info)