Class I and class II viral fusion protein structures reveal similar principles in membrane fusion (Review)

Recent crystal structures of Flavivirus and Alphavirus fusion proteins (class II) confirm two major principles of protein machineries that mediate the merger of two opposing lipid bilayers. First, the fusion protein can bridge both membranes tethered by two membrane anchors. Second, refolding or domain rearrangement steps lead to the positioning of both anchors into close proximity at the same end of an elongated structure. Although these two steps are in principle sufficient to pull two opposing membranes together and initiate membrane fusion, accumulating evidence suggests that the process requires the concerted action of a number of fusion proteins at and outside the contact sites. This review will focus on the structures of viral class I and class II fusion proteins and their similarities in facilitating membrane fusion.     

Class I and class II viral fusion protein structures reveal similar principles in membrane fusion

Recent crystal structures of Flavivirus and Alphavirus fusion proteins (class II) confirm two major principles of protein machineries that mediate the merger of two opposing lipid bilayers. First, the fusion protein can bridge both membranes tethered by two membrane anchors. Second, refolding or domain rearrangement steps lead to the positioning of both anchors into close proximity at the same end of an elongated structure. Although these two steps are in principle sufficient to pull two opposing membranes together and initiate membrane fusion, accumulating evidence suggests that the process requires the concerted action of a number of fusion proteins at and outside the contact sites. This review will focus on the structures of viral class I and class II fusion proteins and their similarities in facilitating membrane fusion.     

Functional Analysis of the Transmembrane Domain in Paramyxovirus F Protein-Mediated Membrane Fusion

To enter cells, enveloped viruses use fusion-mediating glycoproteins to facilitate the merger of the viral and host cell membranes. These glycoproteins undergo large-scale irreversible refolding during membrane fusion. The paramyxovirus parainfluenza virus 5 mediates membrane merger through its fusion protein (F). The transmembrane (TM) domains of viral fusion proteins are typically required for fusion. The TM domain of F is particularly interesting in that it is potentially unusually long; multiple calculations suggest a TM helix length between 25 and 48 residues. Oxidative cross-linking of single-cysteine substitutions indicates the F TM trimer forms a helical bundle within the membrane. To assess the functional role of the paramyxovirus parainfluenza virus 5 F protein TM domain, alanine scanning mutagenesis was performed. Two residues located in the outer leaflet of the bilayer are critical for fusion. Multiple amino acid substitutions at these positions indicate the physical properties of the side chain play a critical role in supporting or blocking fusion. Analysis of intermediate steps in F protein refolding indicated that the mutants were not trapped at the open stalk intermediate or the prehairpin intermediate. Incorporation of a known F protein destabilizing mutation that causes a hyperfusogenic phenotype restored fusion activity to the mutants. Further, altering the curvature of the lipid bilayer by addition of oleic acid promoted fusion of the F protein mutants. In aggregate, these data indicate that the TM domain plays a functional role in fusion beyond merely anchoring the protein in the viral envelope and that it can affect the structures and steady-state concentrations of the various conformational intermediates en route to the final postfusion state. We suggest that the unusual length of this TM helix might allow it to serve as a template for formation of or specifically stabilize the lipid stalk intermediate in fusion.     

Influenza Hemifusion Phenotype Depends on Membrane Context: Differences in Cell–Cell and Virus–Cell Fusion

Influenza viral entry into the host cell cytoplasm is accomplished by a process of membrane fusion mediated by the viral hemagglutinin protein. Hemagglutinin acts in a pH-triggered fashion, inserting a short fusion peptide into the host membrane followed by refolding of a coiled-coil structure to draw the viral envelope and host membranes together. Mutations to this fusion peptide provide an important window into viral fusion mechanisms and protein–membrane interactions. Here, we show that a well-described fusion peptide mutant, G1S, has a phenotype that depends strongly on the viral membrane context. The G1S mutant is well known to cause a “hemifusion” phenotype based on experiments in transfected cells, where cells expressing G1S hemagglutinin can undergo lipid mixing in a pH-triggered fashion similar to virus but will not support fusion pores. We compare fusion by the G1S hemagglutinin mutant expressed either in cells or in influenza virions and show that this hemifusion phenotype occurs in transfected cells but that native virions are able to support full fusion, albeit at a slower rate and 10–100 × reduced infectious titer. We explain this with a quantitative model where the G1S mutant, instead of causing an absolute block of fusion, alters the protein stoichiometry required for fusion. This change slightly slows fusion at high hemagglutinin density, as on the viral surface, but at lower hemagglutinin density produces a hemifusion phenotype. The quantitative model thus reproduces the observed virus–cell and cell–cell fusion phenotypes, yielding a unified explanation where membrane context can control the observed viral fusion phenotype.     

Properties and structures of the influenza and HIV fusion peptides on lipid membranes: implications for a role in fusion

The fusion peptides of HIV and influenza virus are crucial for viral entry into a host cell. We report the membrane-perturbing and structural properties of fusion peptides from the HA fusion protein of influenza virus and the gp41 fusion protein of HIV. Our goals were to determine: 1), how fusion peptides alter structure within the bilayers of fusogenic and nonfusogenic lipid vesicles and 2), how fusion peptide structure is related to the ability to promote fusion. Fluorescent probes revealed that neither peptide had a significant effect on bilayer packing at the water-membrane interface, but both increased acyl chain order in both fusogenic and nonfusogenic vesicles. Both also reduced free volume within the bilayer as indicated by partitioning of a lipophilic fluorophore into membranes. These membrane ordering effects were smaller for the gp41 peptide than for the HA peptide at low peptide/lipid ratio, suggesting that the two peptides assume different structures on membranes. The influenza peptide was predominantly helical, and the gp41 peptide was predominantly antiparallel beta-sheet when membrane bound, however, the depths of penetration of Trps of both peptides into neutral membranes were similar and independent of membrane composition. We previously demonstrated: 1), the abilities of both peptides to promote fusion but not initial intermediate formation during PEG-mediated fusion and 2), the ability of hexadecane to compete with this effect of the fusion peptides. Taken together, our current and past results suggest a hypothesis for a common mechanism by which these two viral fusion peptides promote fusion.     

Role of Spike Protein Endodomains in Regulating Coronavirus Entry

Enveloped viruses enter cells by viral glycoprotein-mediated binding to host cells and subsequent fusion of virus and host cell membranes. For the coronaviruses, viral spike (S) proteins execute these cell entry functions. The S proteins are set apart from other viral and cellular membrane fusion proteins by their extensively palmitoylated membrane-associated tails. Palmitate adducts are generally required for protein-mediated fusions, but their precise roles in the process are unclear. To obtain additional insights into the S-mediated membrane fusion process, we focused on these acylated carboxyl-terminal intravirion tails. Substituting alanines for the cysteines that are subject to palmitoylation had effects on both S incorporation into virions and S-mediated membrane fusions. In specifically dissecting the effects of endodomain mutations on the fusion process, we used antiviral heptad repeat peptides that bind only to folding intermediates in the S-mediated fusion process and found that mutants lacking three palmitoylated cysteines remained in transitional folding states nearly 10 times longer than native S proteins. This slower refolding was also reflected in the paucity of postfusion six-helix bundle configurations among the mutant S proteins. Viruses with fewer palmitoylated S protein cysteines entered cells slowly and had reduced specific infectivities. These findings indicate that lipid adducts anchoring S proteins into virus membranes are necessary for the rapid, productive S protein refolding events that culminate in membrane fusions. These studies reveal a previously unappreciated role for covalently attached lipids on the endodomains of viral proteins eliciting membrane fusion reactions.     

Are fusion peptides a good model to study viral cell fusion?

Fusion peptides are hydrophobic and conserved sequences located within glycoprotein ectodomains that protrude from the virion surface. Direct participation of fusion peptides in the viral membrane fusion phenomenon has been inferred from genetic analyses showing that even a single residue substitution or a deletion within these sequences may completely block the process. However, the specific fusion peptide activities associated to the multi-step fusion mechanism are not well defined. Based on the assumption that fusion peptides are transferred into target membranes, biophysical methodologies have been applied to study integration into model membranes of synthetic fragments representing functional and non-functional sequences. From these studies, it is inferred that, following insertion, functional sequences generate target membrane perturbations and adopt specific structural arrangements within. Further characterization of these artificial systems may help in understanding the molecular processes that bring initial bilayer destabilizations to the eventual opening of a fusion pore.     

Interaction between dengue virus fusion peptide and lipid bilayers depends on peptide clustering

Dengue fever is one of the most widespread tropical diseases in the world. The disease is caused by a virus member of the Flaviviridae family, a group of enveloped positive sense single-stranded RNA viruses. Dengue virus infection is mediated by virus glycoprotein E, which binds to the cell surface. After uptake by endocytosis, this protein induces the fusion between viral envelope and endosomal membrane at the acidic environment of the endosomal compartment. In this work, we evaluated by steady-state and time-resolved fluorescence spectroscopy the interaction between the peptide believed to be the dengue virus fusion peptide and large unilamellar vesicles, studying the extent of partition, fusion capacity and depth of insertion in membranes. The roles of the bilayer composition (neutral and anionic phospholipids), ionic strength and pH of the medium were also studied. Our results indicate that dengue virus fusion peptide has a high affinity to vesicles composed of anionic lipids and that the interaction is mainly electrostatic. Both partition coefficient and fusion index are enhanced by negatively charged phospholipids. The location determined by differential fluorescence quenching using lipophilic probes demonstrated that the peptide is in an intermediate depth in the hemilayers, in-between the bilayer core and its surface. Ultimately, these data provide novel insights on the interaction between dengue virus fusion peptide and its target membranes, namely, the role of oligomerization and specific types of membranes.     

Combined NMR and EPR spectroscopy to determine structures of viral fusion domains in membranes

Methods are described to determine the structures of viral membrane fusion domains in detergent micelles by NMR and in lipid bilayers by site-directed spin labeling and EPR spectroscopy. Since in favorable cases, the lower-resolution spin label data obtained in lipid bilayers fully support the higher-resolution structures obtained by solution NMR, it is possible to graft the NMR structural coordinates into membranes using the EPR-derived distance restraints to the lipid bilayer. Electron paramagnetic dynamics and distance measurements in bilayers support conclusions drawn from NMR in detergent micelles. When these methods are applied to a structure determination of the influenza virus fusion domain and four point mutations with different functional phenotypes, it is evident that a fixed-angle boomerang structure with a glycine edge on the outside of the N-terminal arm is both necessary and sufficient to support membrane fusion. The human immunodeficiency virus fusion domain forms a straight helix with a flexible C-terminus. While EPR data for this fusion domain are not yet available, it is tentatively speculated that, because of its higher hydrophobicity, a critically tilted insertion may occur even in the absence of a kinked boomerang structure in this case.     

Fatty acids can substitute the HIV fusion peptide in lipid merging and fusion: an analogy between viral and palmitoylated eukaryotic fusion proteins

Various fusion proteins from eukaryotes and viruses share structural similarities such as a coiled coil motif. However, compared with eukaryotic proteins, a viral fusion protein contains a fusion peptide (FP), which is an N-terminal hydrophobic fragment that is primarily involved in directing fusion via anchoring the protein to the target cell membrane. In various eukaryotic fusion proteins the membrane targeting domain is cysteine-rich and must undergo palmitoylation prior to the fusion process. Here we examined whether fatty acids can replace the FP of human immunodeficiency virus type 1 (HIV-1), thereby discerning between the contributions of the sequence versus hydrophobicity of the FP in the lipid-merging process. For that purpose, we structurally and functionally characterized peptides derived from the N terminus of HIV fusion protein - gp41 in which the FP is lacking or replaced by fatty acids. We found that fatty acid conjugation dramatically enhanced the capability of the peptides to induce lipid mixing and aggregation of zwitterionic phospholipids composing the outer leaflet of eukaryotic cell membranes. The enhanced effect of the acylated peptides on membranes was further supported by real-time atomic force microscopy (AFM) showing nanoscale holes in zwitterionic membranes. Membrane-binding experiments revealed that fatty acid conjugation did not increase the affinity of the peptides to the membrane significantly. Furthermore, all free and acylated peptides exhibited similar α-helical structures in solution and in zwitterionic membranes. Interestingly, the fusogenic active conformation of N36 in negatively charged membranes composing the inner leaflet of eukaryotic cells is β-sheet. Apparently, N-terminal heptad repeat (NHR) can change its conformation as a response to a change in the charge of the membrane head group. Overall, the data suggest an analogy between the eukaryotic cysteine-rich domains and the viral fusion peptide, and mark the hydrophobic nature of FP as an important characteristic for its role in lipid merging.     

The unique transmembrane hairpin of flavivirus fusion protein E is essential for membrane fusion

The fusion of enveloped viruses with cellular membranes is mediated by proteins that are anchored in the lipid bilayer of the virus and capable of triggered conformational changes necessary for driving fusion. The flavivirus envelope protein E is the only known viral fusion protein with a double membrane anchor, consisting of two antiparallel transmembrane helices (TM1 and TM2). TM1 functions as a stop-transfer sequence and TM2 as an internal signal sequence for the first nonstructural protein during polyprotein processing. The possible role of this peculiar C-terminal helical hairpin in membrane fusion has not been investigated so far. We addressed this question by studying TM mutants of tick-borne encephalitis virus (TBEV) recombinant subviral particles (RSPs), an established model system for flavivirus membrane fusion. The engineered mutations included the deletion of TM2, the replacement of both TM domains (TMDs) by those of the related Japanese encephalitis virus (JEV), and the use of chimeric TBEV-JEV membrane anchors. Using these mutant RSPs, we provide evidence that TM2 is not just a remnant of polyprotein processing but, together with TM1, plays an active role in fusion. None of the TM mutations, including the deletion of TM2, affected early steps of the fusion process, but TM interactions apparently contribute to the stability of the postfusion E trimer and the completion of the merger of the membranes. Our data provide evidence for both intratrimer and intertrimer interactions mediated by the TMDs of E and thus extend the existing models of flavivirus membrane fusion.     

Probing the interaction between vesicular stomatitis virus and phosphatidylserine

The entry of enveloped animal viruses into their host cells always depends on membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion between the viral envelope and the endosomal membrane at the acidic environment of this compartment. In this work, we evaluated VSV interactions with membranes of different phospholipid compositions, at neutral and acidic pH, using atomic force microscopy (AFM) operating in the force spectroscopy mode, isothermal calorimetry (ITC) and molecular dynamics simulation. We found that the binding forces differed dramatically depending on the membrane phospholipid composition, revealing a high specificity of G protein binding to membranes containing phosphatidylserine (PS). In a previous work, we showed that the sequence corresponding amino acid 164 of VSV G protein was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Here, we used this sequence to explore VSV–PS interaction using ITC. We found that peptide binding to membranes was exothermic, suggesting the participation of electrostatic interactions. Peptide–membrane interaction at pH 7.5 was shown to be specific to PS and dependent on the presence of His residues in the fusion peptide. The application of the simplified continuum Gouy–Chapman theory to our system predicted a pH of 5.0 at membrane surface, suggesting that the His residues should be protonated when located close to the membrane. Molecular dynamics simulations suggested that the peptide interacts with the lipid bilayer through its N-terminal residues, especially Val¹⁴⁵ and His¹⁴⁸. Fabiana A.Carneiro and Pedro A. Lapido-Loureiro contributed equally to this work An erratum to this article can be found at     

Sec18p and Vam7p remodel trans-SNARE complexes to permit a lipid-anchored R-SNARE to support yeast vacuole fusion

Intracellular membrane fusion requires SNARE proteins in a trans-complex, anchored to apposed membranes. Proteoliposome studies have suggested that SNAREs drive fusion by stressing the lipid bilayer via their transmembrane domains (TMDs), and that SNARE complexes require a TMD in each docked membrane to promote fusion. Yeast vacuole fusion is believed to require three Q-SNAREs from one vacuole and the R-SNARE Nyv1p from its fusion partner. In accord with this model, we find that fusion is abolished when the TMD of Nyv1p is replaced by lipid anchors, even though lipid-anchored Nyv1p assembles into trans-SNARE complexes. However, normal fusion is restored by the addition of both Sec18p and the soluble SNARE Vam7p. In restoring fusion, Sec18p promotes the disassembly of trans-SNARE complexes, and Vam7p enhances their assembly. Thus, either the TMD of this R-SNARE is not essential for fusion, and TMD-mediated membrane stress is not the only mode of trans-SNARE complex action, or these SNAREs have more flexibility than heretofore appreciated to form alternate functional complexes that violate the 3Q:1R rule.     

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.     

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.     

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.     

HIV-1 Envelope Glycoprotein Biosynthesis, Trafficking, and Incorporation

The HIV-1 envelope (Env) glycoproteins play an essential role in the virus replication cycle by mediating the fusion between viral and cellular membranes during the entry process. The Env glycoproteins are synthesized as a polyprotein precursor (gp160) that is cleaved by cellular proteases to the mature surface glycoprotein gp120 and the transmembrane glycoprotein gp41. During virus assembly, the gp120/gp41 complex is incorporated as heterotrimeric spikes into the lipid bilayer of nascent virions. These gp120/gp41 complexes then initiate the infection process by binding receptor and coreceptor on the surface of target cells. Much is currently known about the HIV-1 Env glycoprotein trafficking pathway and the structure of gp120 and the extracellular domain of gp41. However, the mechanism by which the Env glycoprotein complex is incorporated into virus particles remains incompletely understood. Genetic data support a major role for the cytoplasmic tail of gp41 and the matrix domain of Gag in Env glycoprotein incorporation. Still to be defined are the identities of host cell factors that may promote Env incorporation and the role of specific membrane microdomains in this process. Here, we review our current understanding of HIV-1 Env glycoprotein trafficking and incorporation into virions.     

Designed protein mimics of the Ebola virus glycoprotein GP2 α-helical bundle: stability and pH effects

Ebola virus (EboV) belongs to the Filoviridae family of viruses that causes severe and fatal hemhorragic fever. Infection by EboV involves fusion between the virus and host cell membranes mediated by the envelope glycoprotein GP2 of the virus. Similar to the envelope glycoproteins of other viruses, the central feature of the GP2 ectodomain postfusion structure is a six-helix bundle formed by the protein's N- and C-heptad repeat regions (NHR and CHR, respectively). Folding of this six-helix bundle provides the energetic driving force for membrane fusion; in other viruses, designed agents that disrupt formation of the six-helix bundle act as potent fusion inhibitors. To interrogate determinants of EboV GP2-mediated membrane fusion, we designed model proteins that consist of the NHR and CHR segments linked by short protein linkers. Circular dichroism and gel filtration studies indicate that these proteins adopt stable α-helical folds consistent with design. Thermal denaturation indicated that the GP2 six-helix bundle is highly stable at pH 5.3 (melting temperature, T(m) , of 86.8 ± 2.0°C and van't Hoff enthalpy, ΔH(vH) , of -28.2 ± 1.0 kcal/mol) and comparable in stability to other viral membrane fusion six-helix bundles. We found that the stability of our designed α-helical bundle proteins was dependent on buffering conditions with increasing stability at lower pH. Small pH differences (5.3-6.1) had dramatic effects (ΔT(m) = 37°C) suggesting a mechanism for conformational control that is dependent on environmental pH. These results suggest a role for low pH in stabilizing six-helix bundle formation during the process of GP2-mediated viral membrane fusion.     

Initial size and dynamics of viral fusion pores are a function of the fusion protein mediating membrane fusion

Background information. Protein‐mediated merger of biological membranes, membrane fusion, is an important process. To investigate the role of fusogenic proteins in the initial size and dynamics of the fusion pore (a narrow aqueous pathway, which widens to finalize membrane fusion), two different fusion proteins expressed in the same cell line were investigated: the major glycoprotein of baculovirus Autographa californica (GP64) and the HA (haemagglutinin) of influenza X31. Results. The host Sf9 cells expressing these viral proteins, irrespective of protein species, fused to human RBCs (red blood cells) upon acidification of the medium. A high‐time‐resolution electrophysiological study of fusion pore conductance revealed fundamental differences in (i) the initial pore conductance; pores created by HA were smaller than those created by GP64; (ii) the ability of pores to flicker; only HA‐mediated pores flickered; and (iii) the time required for pore formation; HA‐mediated pores took much longer to form after acidification. Conclusion. HA and GP64 have divergent electrophysiological phenotypes even when they fuse identical membranes, and fusion proteins play a crucial role in determining initial fusion pore characteristics. The structure of the initial fusion pore detected by electrical conductance measurements is sensitive to the nature of the fusion protein.     

囊膜病毒膜融合分子机制研究进展

囊膜病毒通过病毒与宿主细胞膜融合的方式感染宿主,病毒囊膜蛋白介导了膜融合过程。根据这些囊膜蛋白在病毒囊膜表面的排列、蛋白结构及其在融合肽中的位置不同,可将囊膜病毒分为三类,其利用这些囊膜特殊的蛋白分子与受体相互作用完成膜融合。在分子水平上研究这一过程有助于认识病毒侵染的本质和发现关键环节,达到预防与治疗病毒病的目的。