Entry mechanisms of enveloped viruses. Implications for fusion of intracellular membranes

Enveloped viruses infect cells by a mechanism involving membrane fusion. This process is mediated and triggered by specific viral membrane glycoproteins. Evidence is accumulating that fusion of intracellular membranes, as occurs during endocytosis and transport between intracellular organelles, also requires the presence of specific proteins. The relevance of elucidating the mechanisms of virus fusion for a better understanding of fusion of intracellular membranes is discussed.     

Membrane fusion: a structural perspective on the interplay of lipids and proteins

The fusion of biological membranes is governed by the carefully orchestrated interplay of membrane proteins and lipids. Recently determined structures of fusion proteins, individual domains of fusion proteins and their complexes with regulatory proteins and membrane lipids have yielded much suggestive insight into how viral and intracellular membrane fusion might proceed. These structures may be combined with new knowledge on the fusion of pure lipid bilayer membranes in an attempt to begin to piece together the complex puzzle of how biological membrane fusion machines operate on membranes.     

Assembly and composition of intracellular particles formed by Moloney murine leukemia virus.

Assembly of type C retroviruses such as Moloney murine leukemia virus (M-MuLV) ordinarily occurs at the plasma membranes of infected cells and absolutely requires the particle core precursor protein, Pr65gag. Previously we have shown that Pr65gag is membrane associated and that at least a portion of intracellular Pr65gag protein appears to be routed to the plasma membrane by a vesicular transport pathway. Here we show that intracellular particle formation can occur in M-MuLV-infected cells. M-MuLV immature particles were observed by electron microscopy budding into and within rough endoplasmic reticulum, Golgi, and vacuolar compartments. Biochemical fractionation studies indicated that intracellular Pr65gag was present in nonionic detergent-resistant complexes of greater than 150S. Additionally, viral RNA and polymerase functions appeared to be associated with intracellular particles, as were Gag-beta-galactosidase fusion proteins which have the capacity to be incorporated into virions. Immature intracellular particles in postnuclear lysates could be proteolytically processed in vitro to mature forms, while extracellular immature M-MuLV particles remained immature as long as 10 h during incubations. The occurrence of M-MuLV-derived intracellular particles demonstrates that Pr65gag can associate with intracellular membranes and indicates that if a plasma membrane Pr65gag receptor exists, it also can be found in other membrane compartments. These results support the hypothesis that intracellular particles may serve as a virus reservoir during in vivo infections.     

Fusion of Newcastle disease virus with liposomes: role of the lipid composition of liposomes

We demonstrate here that fusion occurs between the membrane of the Newcastle disease virus (NDV) and liposomes. Fluorescence dequenching studies (using Rhodamine-bearing viral envelopes) revealed the mixing of the lipids constituting the viral and liposomal membrane. The digestion of internal viral proteins by trypsin-containing liposomes indicated the mixing of the internal aqueous compartments. This last assay is independent of exchange of lipids between liposomal and viral membrane in the absence of fusion. Investigation of the effects of liposomal composition indicated that the presence of phosphatidylethanolamine and gangliosides are essential to optimize fusion. The fact that the Newcastle disease virus membrane can fuse with liposome also confirms that fusion must be determined by the viral proteins and could be mostly independent of the nature or presence of the host proteins.     

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.     

Probing the dynamics of intact cells and nuclear envelope precursor membrane vesicles by deuterium solid state NMR spectroscopy

Membrane dynamics is an essential part of many cellular mechanisms such as intracellular trafficking, membrane fusion/fission and mitotic organelle reconstitution. The dynamics of membranes is dependent primarily on their phospholipid and cholesterol composition and how these molecules are ordered in relation to one another. To determine the physical status of membranes in whole cells or purified membranes of subcellular compartments we have developed a novel application exploiting solid-state (2)H-NMR spectroscopy. We utilise this method to probe the dynamics of intact sperm and nuclear envelope precursor membranes. We show, using mass spectrometry, that either multilamellar or small unilamellar vesicles of deuterium-labelled palmitoyl-oleoylphosphatidylcholine can be used to probe the dynamics of sperm cells or nuclear envelope precursor membrane vesicles, respectively. Using (2)H-NMR we determine the order parameters of sperm cells and nuclear envelope precursor membrane vesicles. We demonstrate that whole sperm membranes are more dynamic than nuclear envelope precursor membranes due to the higher cholesterol levels of the latter. Our new application can be exploited as a generic method for monitoring membrane dynamics in whole cells, various subcellular membrane compartments and membrane domains in subcellular compartments.     

Role of lipids in virus replication

Viruses intricately interact with and modulate cellular membranes at several stages of their replication, but much less is known about the role of viral lipids compared to proteins and nucleic acids. All animal viruses have to cross membranes for cell entry and exit, which occurs by membrane fusion (in enveloped viruses), by transient local disruption of membrane integrity, or by cell lysis. Furthermore, many viruses interact with cellular membrane compartments during their replication and often induce cytoplasmic membrane structures, in which genome replication and assembly occurs. Recent studies revealed details of membrane interaction, membrane bending, fission, and fusion for a number of viruses and unraveled the lipid composition of raft-dependent and -independent viruses. Alterations of membrane lipid composition can block viral release and entry, and certain lipids act as fusion inhibitors, suggesting a potential as antiviral drugs. Here, we review viral interactions with cellular membranes important for virus entry, cytoplasmic genome replication, and virus egress.     

Probing the dynamics of intact cells and nuclear envelope precursor membrane vesicles by deuterium solid state NMR spectroscopy

Membrane dynamics is an essential part of many cellular mechanisms such as intracellular trafficking, membrane fusion/fission and mitotic organelle reconstitution. The dynamics of membranes is dependent primarily on their phospholipid and cholesterol composition and how these molecules are ordered in relation to one another. To determine the physical status of membranes in whole cells or purified membranes of subcellular compartments we have developed a novel application exploiting solid-state ²H-NMR spectroscopy. We utilise this method to probe the dynamics of intact sperm and nuclear envelope precursor membranes. We show, using mass spectrometry, that either multilamellar or small unilamellar vesicles of deuterium-labelled palmitoyl-oleoylphosphatidylcholine can be used to probe the dynamics of sperm cells or nuclear envelope precursor membrane vesicles, respectively. Using ²H-NMR we determine the order parameters of sperm cells and nuclear envelope precursor membrane vesicles. We demonstrate that whole sperm membranes are more dynamic than nuclear envelope precursor membranes due to the higher cholesterol levels of the latter. Our new application can be exploited as a generic method for monitoring membrane dynamics in whole cells, various subcellular membrane compartments and membrane domains in subcellular compartments.     

Membrane Fusion

The fusion of biological membranes results in two bilayer-based membranes merging into a single membrane. In this process the lipids have to undergo considerable rearrangement. The nature of the intermediates that are formed during this rearrangement has been investigated. Certain fusion proteins facilitate this process. In many cases short segments of these fusion proteins have a particularly important role in accelerating the fusion process. Studies of the interaction of model peptides with membranes have allowed for increased understanding at the molecular level of the mechanism of the promotion of membrane fusion by fusion proteins. There is an increased appreciation of the roles of several independent segments of fusion proteins in promoting the fusion process.Many of the studies of the fusion of biological membranes have been done with the fusion of enveloped viruses with other membranes. One reason for this is that the number of proteins involved in viral fusion is relatively simple, often requiring only a single protein. For many enveloped viruses, the structure of their fusion proteins has certain common elements, suggesting that they all promote fusion by an analogous mechanism. Some aspects of this mechanism also appears to be common to intracellular fusion, although several proteins are involved in that process which is more complex and regulated than is fusion.     

Syntaxin 6: the promiscuous behaviour of a SNARE protein

Vesicle flow within the cell is responsible for the dynamic maintenance of and communication between intracellular compartments. In addition, vesicular transport is crucial for communication between the cell and its surrounding environment. The ability of a vesicle to recognise and fuse with an appropriate compartment or vesicle is determined by its protein and lipid composition as well as by proteins in the cytosol. SNARE proteins present on both vesicle as well as target organelle membranes provide one component necessary for the process of membrane fusion. While in mammalian cells the main focus of interest about SNARE function has centred on those involved in exocytosis, recent data on SNAREs involved in intracellular membrane-trafficking steps have provided a deeper insight into the properties of these proteins. We take, as an example, the promiscuous SNARE syntaxin 6, a SNARE involved in multiple membrane fusion events. The properties of syntaxin 6 reveal similarities but also differences in the behaviour of intracellular SNAREs and the highly specialised exocytotic SNARE molecules.     

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.     

Mixed and non-cognate SNARE complexes. Characterization of assembly and biophysical properties.

Assembly of soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) proteins between two opposing membranes is thought to be the key event that initiates membrane fusion. Many new SNARE proteins have recently been localized to distinct intracellular compartments, supporting the view that sets of specific SNAREs are specialized for distinct trafficking steps. We have now investigated whether other SNAREs can form complexes with components of the synaptic SNARE complex including synaptobrevin/VAMP 2, SNAP-25, and syntaxin 1. When the Q-SNAREs syntaxin 2, 3, and 4, and the R-SNARE endobrevin/VAMP 8 were used in various combinations, heat-resistant complexes were formed. Limited proteolysis revealed that these complexes contained a protease-resistant core similar to that of the synaptic complex. All complexes were disassembled by the ATPase N-ethylmaleimide-sensitive fusion protein and its cofactor alpha-SNAP. Circular dichroism spectroscopy showed that major conformational changes occur during assembly, which are associated with induction of structure from unstructured monomers. Furthermore, no preference for synaptobrevin was observed during the assembly of the synaptic complex when endobrevin/VAMP 8 was present in equal concentrations. We conclude that cognate and non-cognate SNARE complexes are very similar with respect to biophysical properties, assembly, and disassembly, suggesting that specificity of membrane fusion in intracellular membrane traffic is not due to intrinsic specificity of SNARE pairing.     

Capturing a fusion intermediate of influenza hemagglutinin with a cholesterol-conjugated peptide, a new antiviral strategy for influenza virus

We previously described fusion-inhibitory peptides that are targeted to the cell membrane by cholesterol conjugation and potently inhibit enveloped viruses that fuse at the cell surface, including HIV, parainfluenza, and henipaviruses. However, for viruses that fuse inside of intracellular compartments, fusion-inhibitory peptides have exhibited very low antiviral activity. We propose that for these viruses, too, membrane targeting via cholesterol conjugation may yield potent compounds. Here we compare the activity of fusion-inhibitory peptides derived from the influenza hemagglutinin (HA) and show that although the unconjugated peptides are inactive, the cholesterol-conjugated compounds are effective inhibitors of infectivity and membrane fusion. We hypothesize that the cholesterol moiety, by localizing the peptides to the target cell membrane, allows the peptides to follow the virus to the intracellular site of fusion. The cholesterol-conjugated peptides trap HA in a transient intermediate state after fusion is triggered but before completion of the refolding steps that drive the merging of the viral and cellular membranes. These results provide proof of concept for an antiviral strategy that is applicable to intracellularly fusing viruses, including known and emerging viral pathogens.     

Peptides and Membrane Fusion: Towards an Understanding of the Molecular Mechanism of Protein-Induced Fusion

Processes such as endo- or exocytosis, membrane recycling, fertilization and enveloped viruses infection require one or more critical membrane fusion reactions. A key feature in viral and cellular fusion phenomena is the involvement of specific fusion proteins. Among the few well-characterized fusion proteins are viral spike glycoproteins responsible for penetration of enveloped viruses into their host cells, and sperm proteins involved in sperm-egg fusion. In their sequences, these proteins possess a ``fusion peptide,' a short segment (up to 20 amino acids) of relatively hydrophobic residues, commonly found in a membrane-anchored polypeptide chain. To simulate protein-mediated fusion, many studies on peptide-induced membrane fusion have been conducted on model membranes such as liposomes and have employed synthetic peptides corresponding to the putative fusion sequences of viral proteins, or de novo synthesized peptides. Here, the application of peptides as a model system to understand the molecular details of membrane fusion will be discussed in detail. Data obtained from these studies will be correlated to biological studies, in particular those that involve viral and sperm-egg systems. Structure-function relationships will be revealed, particularly in the context of protein-induced membrane perturbations and bilayer-to-nonbilayer transition underlying the mechanism of fusion. We will also focus on the involvement of lipid composition of membranes as a potential regulating factor of the topological fusion site in biological systems. Received: 3 August 1998/Revised: 15 October 1998     

A simple guide to biochemical approaches for analyzing protein-lipid interactions

Eukaryotic cells contain many different membrane compartments with characteristic shapes, lipid compositions, and dynamics. A large fraction of cytoplasmic proteins associate with these membrane compartments. Such protein-lipid interactions, which regulate the subcellular localizations and activities of peripheral membrane proteins, are fundamentally important for a variety of cell biological processes ranging from cytoskeletal dynamics and membrane trafficking to intracellular signaling. Reciprocally, many membrane-associated proteins can modulate the shape, lipid composition, and dynamics of cellular membranes. Determining the exact mechanisms by which these proteins interact with membranes will be essential to understanding their biological functions. In this Technical Perspective, we provide a brief introduction to selected biochemical methods that can be applied to study protein-lipid interactions. We also discuss how important it is to choose proper lipid composition, type of model membrane, and biochemical assay to obtain reliable and informative data from the lipid-interaction mechanism of a protein of interest.     

Poly(ethylene glycol) (PEG)-mediated fusion between pure lipid bilayers: a mechanism in common with viral fusion and secretory vesicle release?

Membrane fusion is fundamental to the life of eukaryotic cells. Cellular trafficking and compartmentalization, import of food stuffs and export of waste, inter-cellular communication, sexual reproduction, and cell division are all dependent on this basic process. Yet, little is known about the molecular mechanism(s) by which fusion occurs. It is known that fusing membranes must somehow be docked and brought into close contact. Specific proteins, many of which have been identified within the past decade, accomplish this. An electrical connection or 'fusion pore' is established between compartments surrounded by the fusing membranes. Three primary views of the mechanism of pore formation during secretory and viral fusion have been proposed within the past decade. In one view, a protein ring forms an initial transient connection that expands slowly by recruiting lipid so as to form a lipidic junction. In another view, the initial fusion pore consists of a protein-lipid complex that transforms slowly until the fusion proteins dissociate from the complex to form an irreversible lipidic pore. In a third view, the initial pore is a transient lipid pore that fluctuates between open and closed states before either expanding irreversibly or closing. Recent work has helped define the mechanism by which poly(ethylene glycol) (PEG) mediates fusion of highly curved model membranes composed only of synthetic phospholipids. PEG is a highly hydrated polymer that can bring vesicle membranes to near molecular contact by making water between them thermodynamically unfavourable. Disrupted packing in the contacting monolayers of these vesicle membranes is necessary to induce fusion. The time course and sequence of molecular events of the ensuing fusion process have also been defined. This sequence of events involves the formation of an initial, transient intermediate in which outer leaflet lipids have mixed and small transient pores join fusing compartments ('stalk'). The transient intermediate transforms in 1-3 min to a fusion-committed, second intermediate ('septum') that then 'pops' to form the fusion pore. Inner leaflet mixing, which is shown to be distinct from outer leaflet mixing, accompanies contents mixing that marks formation of the fusion pore. Both the sequence of events and the activation energies of these events correspond well to those observed in viral membrane fusion and secretory granule fusion. These results strongly support the contention that both viral and secretory fusion events occur by lipid molecule rearrangements that can be studied and defined through the use of PEG-mediated vesicle fusion as a model system. A possible mechanism by which fusion proteins might mediate this lipidic process is described.     

Peripherin-2: an intracellular analogy to viral fusion proteins

The C-terminus of the intracellular retinal rod outer segment disk protein peripherin-2 binds to membranes, adopts a helical conformation, and promotes membrane fusion, which suggests an analogy to the structure and function of viral envelope fusion proteins. Nuclear magnetic resonance (NMR) data and fluorescence data show that a 63-residue polypeptide comprising the C-terminus of bovine peripherin-2 (R284-G346) binds to the membrane mimetic, dodecylphosphocholine micelles. High-resolution NMR studies reveal that although this C-terminal fragment is unstructured in solution, the same fragment adopts helical structure when bound to the micelles. The C-terminus may be a member of the class of intrinsically unstructured protein domains. Using methods developed for the G-protein coupled receptor rhodopsin, a model for the structure of the transmembrane domain of peripherin-2 was constructed. Previously published data showed that both peripherin-2 and viral fusion proteins are transmembrane proteins that promote membrane fusion and have a fusion peptide sequence within the protein that independently promotes membrane fusion. Furthermore, the fusion-active sequence of peripherin-2 exhibits a sequence motif that matches the viral fusion peptide of influenza hemagglutinin (HA). These observations collectively suggest that the mechanism of intracellular membrane fusion induced by peripherin-2 and the mechanism of enveloped viral fusion may have features in common.     

Inhibition of murine leukemia virus envelope protein (env) processing by intracellular expression of the env N-terminal heptad repeat region

A conserved structural motif in the envelope proteins of several viruses consists of an N-terminal, alpha-helical, trimerization domain and a C-terminal region that refolds during fusion to bind the N-helix trimer. Interaction between the N and C regions is believed to pull viral and target membranes together in a crucial step during membrane fusion. For several viruses with type I fusion proteins, C regions pack as alpha-helices in the grooves between N-helix monomers, and exogenously added N- and C-region peptides block fusion by inhibiting the formation of the six-helix bundle. For other viruses, including influenza virus and murine leukemia virus (MLV), there is no evidence for comparably extended C-region alpha-helices, although a short, non-alpha-helical interaction structure has been reported for influenza virus. We tested candidate N-helix and C-region peptides from MLV for their ability to inhibit cell fusion but found no inhibitory activity. In contrast, intracellular expression of the MLV N-helix inhibited fusion by efficiently blocking proteolytic processing and intracellular transport of the envelope protein. The results highlight another mechanism by which the N-helix peptides can inhibit fusion.     

Structures and Mechanisms of Viral Membrane Fusion Proteins: Multiple Variations on a Common Theme

Recent work has identified three distinct classes of viral membrane fusion proteins based on structural criteria. In addition, there are at least four distinct mechanisms by which viral fusion proteins can be triggered to undergo fusion-inducing conformational changes. Viral fusion proteins also contain different types of fusion peptides and vary in their reliance on accessory proteins. These differing features combine to yield a rich diversity of fusion proteins. Yet despite this staggering diversity, all characterized viral fusion proteins convert from a fusion-competent state (dimers or trimers, depending on the class) to a membrane-embedded homotrimeric prehairpin, and then to a trimer-of-hairpins that brings the fusion peptide, attached to the target membrane, and the transmembrane domain, attached to the viral membrane, into close proximity thereby facilitating the union of viral and target membranes. During these conformational conversions, the fusion proteins induce membranes to progress through stages of close apposition, hemifusion, and then the formation of small, and finally large, fusion pores. Clearly, highly divergent proteins have converged on the same overall strategy to mediate fusion, an essential step in the life cycle of every enveloped virus.     

Evidence against an essential role of COPII-mediated cargo transport to the endoplasmic reticulum-Golgi intermediate compartment in the formation of the primary membrane of vaccinia virus

Vaccinia virus assembles two distinct lipoprotein membranes. The primary membrane contains nonglycosylated proteins, appears as crescents in the cytoplasm, and delimits immature and mature intracellular virions. The secondary or wrapping membrane contains glycoproteins, is derived from virus-modified trans-Golgi or endosomal cisternae, forms a loose coat around some intracellular mature virions, and becomes the envelope of extracellular virions. Although the mode of formation of the wrapping membrane is partially understood, we know less about the primary membrane. Recent reports posit that the primary membrane originates from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). According to this model, viral primary membrane proteins are cotranslationally inserted into the ER and accumulate in the ERGIC. To test the ERGIC model, we employed Sar1(H79G), a dominant negative form of the Sar1 protein, which is an essential component of coatomer protein II (COPII)-mediated cargo transport from the ER to the ERGIC and other post-ER compartments. Overexpression of Sar1(H79G) by transfection or by a novel recombinant vaccinia virus with an inducible Sar1(H79G) gene resulted in retention of ERGIC 53 in the ER but did not interfere with localization of viral primary membrane proteins in factory regions or with formation of viral crescent membranes and infectious intracellular mature virions. Wrapping of intracellular mature virions and formation of extracellular virions did not occur, however, because some proteins that are essential for the secondary membrane were retained in the ER as a consequence of Sar1(H79G) overexpression. Our data argue against an essential role of COPII-mediated cargo transport and the ERGIC in the formation of the viral primary membrane. Instead, viral membranes may be derived directly from the ER or by a novel mechanism.