Viral fusion proteins: multiple regions contribute to membrane fusion

In recent years, the simple picture of a viral fusion protein interacting with the cell and/or viral membranes by means of only two localized segments (i.e. the fusion peptide and the transmembrane domain) has given way to a more complex picture in which multiple regions from the viral proteins interact with membranes. Indeed, possible roles in membrane binding and/or destabilization have been postulated for the N-terminal heptad repeats, pre-transmembrane segments, and other internal regions of fusion proteins from distant viruses (such as orthomyxo-, retro-, paramyxo-, or flaviviruses). This review focuses on the experimental evidence and functional models postulated so far about the role of these regions in the process of virus-induced membrane fusion.     

Peptide models for the membrane destabilizing actions of viral fusion proteins.

The fusion of enveloped viruses to target membranes is promoted by certain viral fusion proteins. However, many other proteins and peptides stabilize bilayer membranes and inhibit membrane fusion. We have evaluated some characteristics of the interaction of peptides that are models of segments of measles and influenza fusion proteins with membranes. Our results indicate that these models of the fusogenic domains of viral fusion proteins promote conversion of model membrane bilayers to nonbilayer phases. This is opposite to the effects of peptides and proteins that inhibit viral fusion. A peptide model for the fusion segment of the HA protein of influenza increased membrane leakage as well as promoted the formation of nonbilayer phases upon acidification from pH 7-5. We analyze the gross conformational features of the peptides, and speculate on how these conformational features relate to the structures of the intact proteins and to their role in promoting membrane fusion.     

Virus membrane fusion

Membrane fusion of enveloped viruses with cellular membranes is mediated by viral glycoproteins (GP). Interaction of GP with cellular receptors alone or coupled to exposure to the acidic environment of endosomes induces extensive conformational changes in the fusion protein which pull two membranes into close enough proximity to trigger bilayer fusion. The refolding process provides the energy for fusion and repositions both membrane anchors, the transmembrane and the fusion peptide regions, at the same end of an elongated hairpin structure in all fusion protein structures known to date. The fusion process follows several lipidic intermediate states, which are generated by the refolding process. Although the major principles of viral fusion are understood, the structures of fusion protein intermediates and their mode of lipid bilayer interaction, the structures and functions of the membrane anchors and the number of fusion proteins required for fusion, necessitate further investigations.     

Characterization of fusion determinants points to the involvement of three discrete regions of both E1 and E2 glycoproteins in the membrane fusion process of hepatitis C virus

Infection of eukaryotic cells by enveloped viruses requires the merging of viral and cellular membranes. Highly specific viral surface glycoproteins, named fusion proteins, catalyze this reaction by overcoming inherent energy barriers. Hepatitis C virus (HCV) is an enveloped virus that belongs to the genus Hepacivirus of the family Flaviviridae. Little is known about the molecular events that mediate cell entry and membrane fusion for HCV, although significant progress has been made due to recent developments in infection assays. Here, using infectious HCV pseudoparticles (HCVpp), we investigated the molecular basis of HCV membrane fusion. By searching for classical features of fusion peptides through the alignment of sequences from various HCV genotypes, we identified six regions of HCV E1 and E2 glycoproteins that present such characteristics. We introduced conserved and nonconserved amino acid substitutions in these regions and analyzed the phenotype of HCVpp generated with mutant E1E2 glycoproteins. This was achieved by (i) quantifying the infectivity of the pseudoparticles, (ii) studying the incorporation of E1E2 and their capacity to mediate receptor binding, and (iii) determining their fusion capacity in cell-cell and liposome/HCVpp fusion assays. We propose that at least three of these regions (i.e., at positions 270 to 284, 416 to 430, and 600 to 620) play a role in the membrane fusion process. These regions may contribute to the merging of viral and cellular membranes either by interacting directly with lipid membranes or by assisting the fusion process through their involvement in the conformational changes of the E1E2 complex at low pH.     

New insights into the mechanism of virus-induced membrane fusion

Infection by enveloped viruses requires fusion between the viral and cellular membranes, a process mediated by specific viral envelope glycoproteins. Information from studies with whole viruses, as well as protein dissection, has suggested that the fusion glycoprotein (F) from Paramyxoviridae, a family that includes major human pathogens, has two hydrophobic segments, termed fusion peptides. These peptides are directly responsible for the membrane fusion event. The recently determined three-dimensional structure of the pre-fusion conformation of the F protein supported these predictions and enabled the formulation of: (1) a detailed model for the initial interaction between F and the target membrane, (2) a new model for Paramyxovirus-induced membrane fusion that can be extended to other viral families, and (3) a novel strategy for developing better inhibitors of paramyxovirus infection.     

Fluorescent lipid probes in the study of viral membrane fusion

Fluorescent lipid probes are widely used in the observation of viral membrane fusion, providing a sensitive method to study fusion mechanism(s). Due to the wealth of data concerning liposome fusion, a variety of fusion assays has been designed including fluorescent probe redistribution, fluorescence dequenching, fluorescence resonance energy transfer and photosensitized labeling. These methods can be tailored for different virus fusion assays. For instance, virions can be loaded with membrane dye which dequenches at the moment of membrane merger. This allows for continuous observation of fusion and therefore kinetic information can be acquired. In the case of cells expressing viral envelope proteins, dye redistribution studies of lipidic and water-soluble fluorophores yield information about fusion intermediates. Lipid probes can be metabolically incorporated into cell membranes, allowing observation of membrane fusion in vitro with minimal chance of flip flop, non-specific transfer and formation of microcrystals. Fluorescent lipid probes have been incorporated into liposomes and/or reconstituted viral envelopes, which provide a well-defined membrane environment for fusion to occur. Interactions of the viral fusion machinery with the membrane can be observed through the photosensitized labeling of the interacting segments of envelope proteins with a hydrophobic probe. Thus, fluorescent lipid probes provide a broad repertoire of fusion assays and powerful tools to produce precise, quantitative data in real time required for the elucidation of the complex process of viral fusion.     

Lipids as modulators of membrane fusion mediated by viral fusion proteins

Enveloped viruses infect host cells by fusion of viral and target membranes. This fusion event is triggered by specific glycoproteins in the viral envelope. Fusion glycoproteins belong to either class I, class II or the newly described third class, depending upon their arrangement at the surface of the virion, their tri-dimensional structure and the location within the protein of a short stretch of hydrophobic amino acids called the fusion peptide, which is able to induce the initial lipid destabilization at the onset of fusion. Viral fusion occurs either with the plasma membrane for pH-independent viruses, or with the endosomal membranes for pH-dependent viruses. Although, viral fusion proteins are parted in three classes and the subcellular localization of fusion might vary, these proteins have to act, in common, on lipid assemblies. Lipids contribute to fusion through their physical, mechanical and/or chemical properties. Lipids can thus play a role as chemically defined entities, or through their preferential partitioning into membrane microdomains called "rafts", or by modulating the curvature of the membranes involved in the fusion process. The purpose of this review is to make a state of the art on recent findings on the contribution of cholesterol, sphingolipids and glycolipids in cell entry and membrane fusion of a number of viral families, whose members bear either class I or class II fusion proteins, or fusion proteins of the recently discovered third class.     

Inhibition of Nipah virus infection in vivo: targeting an early stage of paramyxovirus fusion activation during viral entry

In the paramyxovirus cell entry process, receptor binding triggers conformational changes in the fusion protein (F) leading to viral and cellular membrane fusion. Peptides derived from C-terminal heptad repeat (HRC) regions in F have been shown to inhibit fusion by preventing formation of the fusogenic six-helix bundle. We recently showed that the addition of a cholesterol group to HRC peptides active against Nipah virus targets these peptides to the membrane where fusion occurs, dramatically increasing their antiviral effect. In this work, we report that unlike the untagged HRC peptides, which bind to the postulated extended intermediate state bridging the viral and cell membranes, the cholesterol tagged HRC-derived peptides interact with F before the fusion peptide inserts into the target cell membrane, thus capturing an earlier stage in the F-activation process. Furthermore, we show that cholesterol tagging renders these peptides active in vivo: the cholesterol-tagged peptides cross the blood brain barrier, and effectively prevent and treat in an established animal model what would otherwise be fatal Nipah virus encephalitis. The in vivo efficacy of cholesterol-tagged peptides, and in particular their ability to penetrate the CNS, suggests that they are promising candidates for the prevention or therapy of infection by Nipah and other lethal paramyxoviruses.     

The membrane-active regions of the hepatitis C virus E1 and E2 envelope glycoproteins

We have identified the membrane-active regions of the full sequences of the HCV E1 and E2 envelope glycoproteins by performing an exhaustive study of membrane leakage, hemifusion, and fusion induced by 18-mer peptide libraries on model membranes having different phospholipid compositions. The data and their comparison have led us to identify different E1 and E2 membrane-active segments which might be implicated in viral membrane fusion, membrane interaction, and/or protein-protein binding. Moreover, it has permitted us to suggest that the fusion peptide might be located in the E1 glycoprotein and, more specifically, the segment comprised by amino acid residues 265-296. The identification of these membrane-active segments from the E1 and E2 envelope glycoproteins, as well as their membranotropic propensity, supports their direct role in HCV-mediated membrane fusion, sustains the notion that different segments provide the driving force for the merging of the viral and target cell membranes, and defines those segments as attractive targets for further development of new antiviral compounds.     

The membranotropic regions of the endo and ecto domains of HIV gp41 envelope glycoprotein

We have identified the membranotropic regions of the full sequence of the HIV gp41 envelope glycoprotein by performing an exhaustive study of membrane rupture, phospholipid-mixing and fusion induced by two 15-mer gp41-derived peptide libraries from HIV strains HIV_MN and HIV_consensus_B on model membranes having different phospholipid compositions. The data obtained for the two strains and its comparison have led us to identify different gp41 membranotropic segments in both ecto- and endodomains which might be implicated in viral membrane fusion and/or membrane interaction. The membranotropic segments corresponding to the gp41 ectodomain were the fusion domain, a stretch located on the N-heptad repeat region adjacent to the fusion domain, part of the immunodominant loop, the pre-transmembrane domain and the transmembrane domain. The membranotropic segments corresponding to the gp41 endodomain were mainly located at some specific parts of the previously described lentivirus lytic sequences. Significantly, the C-heptad repeat region and the Kennedy sequence located in the ectodomain and in the endodomain, respectively, presented no membranotropic activity in any model membrane assayed. The identification of these gp41 segments as well as their membranotropic propensity sustain the notion that different segments of gp41 provide the driving force for the merging of the viral and target cell membranes as well as they help us to define those segments as attractive targets for further development of new anti-viral compounds.     

The membranotropic regions of the endo and ecto domains of HIV gp41 envelope glycoprotein

We have identified the membranotropic regions of the full sequence of the HIV gp41 envelope glycoprotein by performing an exhaustive study of membrane rupture, phospholipid-mixing and fusion induced by two 15-mer gp41-derived peptide libraries from HIV strains HIV_MN and HIV_consensus_B on model membranes having different phospholipid compositions. The data obtained for the two strains and its comparison have led us to identify different gp41 membranotropic segments in both ecto- and endodomains which might be implicated in viral membrane fusion and/or membrane interaction. The membranotropic segments corresponding to the gp41 ectodomain were the fusion domain, a stretch located on the N-heptad repeat region adjacent to the fusion domain, part of the immunodominant loop, the pre-transmembrane domain and the transmembrane domain. The membranotropic segments corresponding to the gp41 endodomain were mainly located at some specific parts of the previously described lentivirus lytic sequences. Significantly, the C-heptad repeat region and the Kennedy sequence located in the ectodomain and in the endodomain, respectively, presented no membranotropic activity in any model membrane assayed. The identification of these gp41 segments as well as their membranotropic propensity sustain the notion that different segments of gp41 provide the driving force for the merging of the viral and target cell membranes as well as they help us to define those segments as attractive targets for further development of new anti-viral compounds.     

Role of membranotropic sequences from herpes simplex virus type I glycoproteins B and H in the fusion process

The entry of enveloped viruses involves attachment followed by close apposition of the viral and plasma membranes. Then, either on the cell surface or in an endocytotic vesicle, the two membranes fuse by an energetically unfavourable process requiring the destabilisation of membrane microenvironment in order to release the viral nucleocapsid into the cytoplasm. The core fusion machinery, conserved throughout the herpesvirus family, involves glycoprotein B (gB) and the non-covalently associated complex of glycoproteins H and L (gH/gL). Both gB and gH possess several hydrophobic domains necessary for efficient induction of fusion, and synthetic peptides corresponding to these regions are able to associate to membranes and induce fusion of artificial liposomes. Here, we describe the first application of surface plasmon resonance (SPR) to the study of the interaction of viral membranotropic peptides with model membranes in order to enhance our molecular understanding of the mechanism of membrane fusion. SPR spectroscopy data are supported by tryptophan fluorescence, circular dichroism and electron spin resonance spectroscopy (ESR). We selected peptides from gB and gH and also analysed the behaviour of HIV gp41 fusion peptide and the cationic antimicrobial peptide melittin. The combined results of SPR and ESR showed a marked difference between the mode of action of the HSV peptides and the HIV fusion peptide compared to melittin, suggesting that viral-derived membrane interacting peptides all act via a similar mechanism, which is substantially different from that of the non-cell selective lytic peptide melittin.     

The fusion peptide of Semliki Forest virus associates with sterol-rich membrane domains

Semliki Forest virus (SFV) is an enveloped alphavirus whose membrane fusion is triggered by low pH and promoted by cholesterol and sphingolipid in the target membrane. Fusion is mediated by E1, a viral membrane protein containing the putative fusion peptide. Virus mutant studies indicate that SFV's cholesterol dependence is controlled by regions of E1 outside of the fusion peptide. Both E1 and E1*, a soluble ectodomain form of E1, interact with membranes in a reaction dependent on low pH, cholesterol, and sphingolipid and form highly stable homotrimers. Here we have used detergent extraction and gradient floatation experiments to demonstrate that E1* associated selectively with detergent-resistant membrane domains (DRMs or rafts). In contrast, reconstituted full-length E1 protein or influenza virus fusion peptide was not associated with DRMs. Methyl beta-cyclodextrin quantitatively extracted both cholesterol and E1* from membranes in the absence of detergent, suggesting a strong association of E1* with sterol. Monoclonal antibody studies demonstrated that raft association was mediated by the proposed E1 fusion peptide. Thus, although other regions of E1 are implicated in the control of virus cholesterol dependence, once the SFV fusion peptide inserts in the target membrane it has a high affinity for membrane domains enriched in cholesterol and sphingolipid.     

Fusogenic domains in herpes simplex virus type 1 glycoprotein H

Infection of eukaryotic cells by enveloped viruses requires fusion between the viral envelope and the cellular plasma or endosomal membrane. The actual merging of the two membranes is mediated by viral envelope glycoproteins, which generally contain a highly hydrophobic region termed the fusion peptide. The entry of herpesviruses is mediated by three conserved proteins: glycoproteins B, H (gH), and L. However, how fusion is executed remains unknown. Herpes simplex virus type 1 gH exhibits features typical of viral fusion glycoproteins, and its ectodomain seems to contain a putative internal fusion peptide. Here, we have identified additional internal segments able to interact with membranes and to induce membrane fusion of large unilamellar vesicles. We have applied the hydrophobicity-at-interface scale proposed by Wimley and White (Wimley, W. C., and White, S. H. (1996) Nat. Struct. Biol. 3, 842-848) to identify six hydrophobic stretches within gH with a tendency to partition into the membrane interface, and four of them were able to induce membrane fusion. Experiments in which equimolar mixtures of gH peptides were used indicated that different fusogenic regions may act in a synergistic way. The functional and structural characterization of these segments suggests that herpes simplex virus type 1 gH possesses several fusogenic internal peptides that could participate in the actual fusion event.     

Membrane fusion mechanisms: the influenza hemagglutinin paradigm and its implications for intracellular fusion

The mechanism of membrane fusion induced by the influenza virus hemagglutinin (HA) has been extensively characterized. Fusion is triggered by low pH, which induces conformational changes in the protein, leading to insertion of a hydrophobic 'fusion peptide' into the viral membrane and the target membrane for fusion. Insertion perturbs the target membrane, and hour glass-shaped lipidic fusion intermediates, called stalks, fusing the outer monolayers of the two membranes, are formed. Stalk formation is followed by complete fusion of the two membranes. Structures similar to those formed by HA at the pH of fusion are found not only in many other viral fusion proteins, but are also formed by SNAREs, proteins involved in intracellular fusion. Substances that inhibit or promote HA-induced fusion because they affect stalk formation, also inhibit or promote intracellular fusion, cell–cell fusion and even intracellular fission similarly. Therefore, the mechanism of influenza HA-induced fusion may be a paradigm for many intracellular fusion events.     

Structural basis for membrane fusion by enveloped viruses.

Enveloped viruses such as HIV-1, influenza virus, and Ebola virus express a surface glycoprotein that mediates both cell attachment and fusion of viral and cellular membranes. The membrane fusion process leads to the release of viral proteins and the RNA genome into the host cell, initiating an infectious cycle. This review focuses on the HIV-1 gp41 membrane fusion protein and discusses the structural similarities of viral membrane fusion proteins from diverse families such as Retroviridae (HIV-1), Orthomyxoviridae (influenza virus), and Filoviridae (Ebola virus). Their structural organization suggests that they have all evolved to use a similar strategy to promote fusion of viral and cellular membranes. This observation led to the proposal of a general model for viral membrane fusion, which will be discussed in detail.     

Interaction of the most membranotropic region of the HCV E2 envelope glycoprotein with membranes. Biophysical characterization

The previously identified membrane-active regions of the hepatitis C virus (HCV) E1 and E2 envelope glycoproteins led us to identify different segments that might be implicated in viral membrane fusion, membrane interaction, and/or protein-protein binding. HCV E2 glycoprotein contains one of the most membranotropic segments, segment 603-634, which has been implicated in CD81 binding, E1/E2 and E2/E2 dimerization, and membrane interaction. Through a series of complementary experiments, we have carried out a study of the binding and interaction with the lipid bilayer of a peptide corresponding to segment 603-634, peptide E2_FP, as well as the structural changes induced by membrane binding that take place in both the peptide and the phospholipid molecules. Here, we demonstrate that peptide E2_FP binds to and interacts with phospholipid model membranes, modulates the polymorphic phase behavior of membrane phospholipids, is localized in a shallow position in the membrane, and is probably oligomerized in the presence of membranes. These data support the role of E2_FP in HCV-mediated membrane fusion, and sustain the notion that this segment of the E2 envelope glycoprotein, together with other segments of E2 and E1 glycoproteins, provides the driving force for the merging of the viral and target cell membranes.     

Viral fusion peptides and identification of membrane-interacting segments

Viral envelope glycoproteins promote infection by mediating fusion between viral and cellular membranes. Fusion occurs after dramatic conformational changes within fusion proteins, leading to the exposure of a short stretch of mostly apolar residues, termed the fusion peptide, which is presumed to insert into the membrane and initiate the fusion process. The typical global composition of fusion peptides, rich in hydrophobic but also in small amino acids such as alanine and glycine, was used here as bait to detect other peptidic segments that can insert into membranes. We so evidenced a similar composition in several cytotoxic peptides, which promote pore formation such as peptides involved in amyloidoses and hydrophobic alpha-hairpins of pore-forming toxins. It is suggested that the structural plasticity observed for several membrane active peptides can be conferred by this particular global amino acid composition, which could be thus used to predict such functional behavior from genome data.     

Membrane perturbation and fusion pore formation in influenza hemagglutinin-mediated membrane fusion. A new model for fusion

Low pH-induced fusion mediated by the hemagglutinin (HA) of influenza virus involves conformational changes in the protein that lead to the insertion of a "fusion peptide" domain of this protein into the target membrane and is thought to perturb the membrane, triggering fusion. By using whole virus, purified HA, or HA ectodomains, we found that shortly after insertion, pores of less than 26 A in diameter were formed in liposomal membranes. As measured by a novel assay, these pores stay open, or continue to close and open, for minutes to hours and persist after pH neutralization. With virus and purified HA, larger pores, allowing the leakage of dextrans, were seen at times well after insertion. For virus, dextran leakage was simultaneous with lipid mixing and the formation of "fusion pores," allowing the transfer of dextrans from the liposomal to the viral interior or vice versa. Pores did not form in the viral membrane in the absence of a target membrane. Based on these data, we propose a new model for fusion, in which HA initially forms a proteinaceous pore in the target, but not in the viral membrane, before a lipidic hemifusion intermediate is formed.     

Protein-induced fusion can be modulated by target membrane lipids through a structural switch at the level of the fusion peptide

Regulatory features of protein-induced membrane fusion are largely unclear, particularly at the level of the fusion peptide. Fusion peptides being part of larger protein complexes, such investigations are met with technical limitations. Here, we show that the fusion activity of influenza virus or Golgi membranes is strongly inhibited by minor amounts of (lyso)lipids when present in the target membrane but not when inserted into the viral or Golgi membrane itself. To investigate the underlying mechanism, we employ a membrane-anchored peptide system and show that fusion is similarly regulated by these lipids when inserted into the target but not when present in the peptide-containing membrane. Peptide-induced fusion is regulated by a reversible switch of secondary structure from a fusion-permissive alpha-helix to a nonfusogenic beta-sheet. The "on/off" activation of this switch is governed by minor amounts of (lyso)-phospholipids in targets, causing a drop in alpha-helix and a dramatic increase in beta-sheet contents. Concomitantly, fusion is inhibited, due to impaired peptide insertion into the target membrane. Our observations in biological fusion systems together with the model studies suggest that distinct lipids in target membranes provide a means for regulating membrane fusion by causing a reversible secondary structure switch of the fusion peptides.