融合蛋白与病毒入膜机制研究进展

包膜病毒感染细胞的第一步即病毒与靶细胞膜的融合,它由病毒包膜上的融合蛋白诱发,融合蛋白与受体分子相互作用后暴露出融合肽,它伸向靶膜使两膜紧密接近后,多肽周围的脂质分子进一步重排,通过中间态最后发生融合,本文将介绍近年来病毒融合蛋白及入膜机制研究进展。     

Hypothesis: spring-loaded boomerang mechanism of influenza hemagglutinin-mediated membrane fusion

Substantial progress has been made in recent years to augment the current understanding of structures and interactions that promote viral membrane fusion. This progress is reviewed with a particular emphasis on recently determined structures of viral fusion domains and their interactions with lipid membranes. The results from the different structural and thermodynamic experimental approaches are synthesized into a new proposed mechanism, termed the “spring-loaded boomerang” mechanism of membrane fusion, which is presented here as a hypothesis.     

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.     

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.     

Lipopolysaccharide (Endotoxin)-host defense antibacterial peptides interactions: Role in bacterial resistance and prevention of sepsis

Lipopolysaccharide (LPS) is the major molecular component of the outer membrane of Gram-negative bacteria and serves as a physical barrier providing the bacteria protection from its surroundings. LPS is also recognized by the immune system as a marker for the detection of bacterial pathogen invasion, responsible for the development of inflammatory response, and in extreme cases to endotoxic shock. Because of these functions, the interaction of LPS with LPS binding molecules attracts great attention. One example of such molecules are antimicrobial peptides (AMPs). These are large repertoire of gene-encoded peptides produced by living organisms of all types, which serve as part of the innate immunity protecting them from pathogen invasion. AMPs are known to interact with LPS with high affinities. The biophysical properties of AMPs and their mode of interaction with LPS determine their biological function, susceptibility of bacteria to them, as well as the ability of LPS to activate the immune system. This review will discuss recent studies on the molecular mechanisms underlying these interactions, their effects on the resistance of the bacteria to AMPs, as well as their potential to neutralize LPS-induced endotoxic shock.     

The interactions of the HIV gp41 fusion peptides with zwitterionic membrane mimics determined by NMR spectroscopy

The wild-type (wt) N-terminal 23-residue fusion peptide (FP) of the human immunodeficiency virus (HIV) fusion protein gp41 and its V2E mutant have been studied by nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles as membrane mimics. A number of NMR techniques have been used. Pulsed field-gradient diffusion measurements in DPC and in 4:1 DPC/sodium dodecylsulfate mixed micelles showed that there is no major difference between the partition coefficients of the fusogenic wt peptide and the V2E mutant in these micelles, indicating that there is no correlation between the activity of the fusion peptides and their membrane affinities. The nuclear Overhauser enhancement (NOE) patterns and the chemical shift index for these two peptides indicated that both FP are in an α helical conformation between the Ile⁴ to Leu¹² or to Ala¹⁵ region. Simulated annealing showed that the helical region extends from Ile⁴ to Met¹⁹. The two FPs share similar conformational characteristics, indicating that the conformation of the FP is not an important factor determining its activity. The spin-label studies, utilizing spin labels 5- and 16-doxystearic acids in the DPC micelles, provided clear indication that the wt FP inserts its N-terminus into the micelles while the V2E mutant does not insert into the micelles. The conclusion from the spin-label results is corroborated by deuterium amide proton exchange experiments. The correlation between the oblique insertion of the FP and its fusogenic activity is in excellent agreement with results from our molecular dynamics simulation and from other previous studies.     

Secondary structure, phospholipid membrane interactions, and fusion activity of two glutamate-rich analogs of influenza hemagglutinin fusion peptide

Two synthetic mutants of influenza HA2 fusion peptide (residues 1-25), containing Glu on the polar (residues 4,8-E5(4,8)) or the hydrophobic (residues 3,7-E5(3,7)) face of the amphipathic helix, were synthesized and labeled with NBD at the N-terminus. Introduction of Glu residues into the fusion peptide leads to increased sensitivity of various biochemical properties to pH compared to the wild type. The E5 peptides showed a decrease of alpha-helix content and increase of beta-sheet structure. Lipid binding was diminished, but not abolished even at high pH. The E5 analogs penetrate the lipid bilayer less deeply than the wild type, especially at high pH. The N-terminal half of the peptide showed significant variation of the depth of the penetration into the lipid bilayer. Both E5 peptides were fusion active. The properties of E5(3,7) were more affected by the Glu substitution and showed greater variation with pH than E5(4,8).     

Sphingolipids, cholesterol, and HIV-1: A paradigm in viral fusion

Our previous studies show that the depletion of cholesterol or sphingolipids (raft-associated lipids) from receptor-bearing adherent cell lines blocks HIV-1 entry and HIV-1 Env-mediated membrane fusion. Here we have evaluated the mechanism(s) by which these lipids contribute to the HIV-1 Env-mediated membrane fusion. We report the following: (1) GSL depletion from a suspension T lymphocyte cell line (Sup-T1) reduced subsequent fusion with HIV-1IIIB-expressing cells by 70%. (2) Cholesterol depletion from NIH3T3 cells bearing HIV-1 receptors (NIH3T3CD4R5/NIH3T3CD4X4) did not impair subsequent fusion with HeLa cells expressing the corresponding HIV-1 Envs. In contrast GSL depletion from these targets reduced fusion by 50% suggesting that GSL facilitate fusion in different ways. (3) GSL-deficient GM95 cells bearing high receptors fused with HIV-1 Env-expressing cells at 37°C with kinetics similar to that of GSL + NIH3T3 targets. Based on these observations, we propose that the plasma membrane cholesterol is required to maintain the integrity of receptor pools whereas GSLs are involved in stabilizing the coupling of inter-receptor pools.     

Determinants of membrane association in the SH4 domain of Fyn: Roles of N-terminus myristoylation and side-chain thioacylation

The SH4 domain of Fyn, a member of the Src family of tyrosine kinases, though rich in polar amino acid residues, anchors to the cytosolic face of membranes upon fatty acylation. In order to probe the requirement of specific fatty acylation at the N-terminus and at the side-chain of this domain for membrane-association, we have studied the interaction of peptides corresponding to the polar segment of the SH4 domain of Fyn and its mono- and dually fatty acylated analogs with model membranes. While the polar segment without covalently linked fatty acids (KDKEATKLTEW-amide) does not interact with lipid vesicles, peptides with one covalently linked fatty acid at the N-terminus or in the side-chain, associate with zwitterionic and anionic lipids to varying degrees. The interaction of dually acylated peptides (Myr-GK(ε-myr)KDKEATKLTEW-amide and Myr-GC(S-pal)KDKEATKLTEW-amide) with lipids depends on the linkage between fatty acyl side-chain and peptide backbone. The peptide chain associates with membranes only when the side-chain acylation is via an amide bond and not via a thioester bond. Our investigations indicate that acylation is essential for membrane targeting and unacylated polar stretch of the SH4 domain does not have a role in membrane-anchoring. Side-chain acylation via a thioester bond not only provides membrane anchorage but also directs the peptide chain away from the bilayer which might be important to enable the full length protein to interact with other signaling partners.     

Investigations of antimicrobial peptides in planar film systems

Planar systems - monolayers and films - constitute a useful platform for studying membrane-active peptides. Here, we summarize varied approaches for studying peptide organization and peptide-lipid interactions at the air/water interface, and focus on three representative antimicrobial membrane-associated peptides—alamethicin, gramicidin, and valinomycin. Experimental data, specifically surface pressure/area isotherms and Brewster angle microscopy images, provided information on peptide association and the effects of the lipid monolayers on peptide surface organization. In general, film analysis emphasized the effects of lipid layers in promoting peptide association and aggregation at the air/water interface. Importantly, the data demonstrated that in many cases peptide domains are phase-separated within the phospholipid monolayers, suggesting that this behavior contributes to the biological actions of membrane-active antimicrobial peptides.     

What studies of fusion peptides tell us about viral envelope glycoprotein-mediated membrane fusion (Review)

This review describes the numerous and innovative methods used to study the structure and function of viral fusion peptides. The systems studied include both intact fusion proteins and synthetic peptides interacting with model membranes. The strategies and methods include dissecting the fusion process into intermediate stages, comparing the effects of sequence mutations, electrophysiological patch clamp methods, hydrophobic photolabelling, video microscopy of the redistribution of both aqueous and lipophilic fluorescent probes between cells, standard optical spectroscopy of peptides in solution (circular dichroism and fluorescence) and attenuated total reflection-Fourier transform infrared spectroscopy of peptides bound to planar bilayers. Although the goal of a detailed picture of the fusion pore has not been achieved for any of the intermediate stages, important properties useful for constraining the development of models are emerging. For example, the presence of x-helical structure in at least part of the fusion peptide is strongly correlated with activity; whereas, β-structure tends to be less prevalent, associated with non-native experimental conditions, and more related to vesicle aggregation than fusion. The specific angle of insertion of the peptides into the membrane plane is also found to be an important characteristic for the fusion process. A shallow penetration, extending only to the central aliphatic core region, is likely responsible for the destabilization of the lipids required for coalescence of the apposing membranes and fusion. The functional role of the fusion peptides (which tend to be either nonpolar or aliphatic) is then to bind to and dehydrate the outer bilayers at a localized site; and thus reduce the energy barrier for the formation of highly curved, lipidic 'stalk’ intermediates. In addition, the importance of the formation of specific, ‘higher-order’ fusion peptide complexes has also been shown. Recent crystallographic structures of core domains of two more fusion proteins (in addition to influenza haemagglutinin) has greatly facilitated the development of prototypic models of the fusion site. This latter effort will undoubtedly benefit from the insights and constraints gained from the studies of fusion peptides.     

Molecular dynamics simulations of the influenza hemagglutinin fusion peptide in micelles and bilayers: conformational analysis of peptide and lipids

Molecular dynamics simulations of the influenza hemagglutinin fusion peptide in two differently sized dodecylphosphocholine micelles and a palmitoyl oleoyl phosphatidylcholine bilayer were generated to analyze the influence of the environment. Four independent trajectories (5 ns each for the bilayer, and 2 ns each for the micelles) were generated for each system. The peptide lies at the surface of the micelles, while its N-terminal region inserts deeply in the bilayer. This leads to a substantial increase of the solvation and rigidity of the peptide in micelles as compared to the bilayer. The average structures, nevertheless, are similar in all three systems and agree reasonably with micelle-based NMR structures. When in the bilayer, the peptide increases the chain gauche population and area of adjacent lipids in the same binding leaflet, while it has the opposite effect for the nearby lipids of the other leaflet. These changes, which occur spontaneously to fill voids and defects, cause a decrease in the thickness of the membrane in the neighborhood of the peptide. They would be expected to promote positive curvature, as consistent with the formation of the convex bulge, or "nipple", in the initial stage of membrane fusion. An extension of the classical surfactant theory of Israelachvili based on shapes is proposed to introduce the concept of a "dynamically induced shape" of the membrane lipids by the peptide.     

Interaction of Influenza Virus Fusion Peptide with Lipid Membranes: Effect of Lysolipid

The effect of lysophosphatidylcholine (LPC) on lipid vesicle fusion and leakage induced by influenza virus fusion peptides and the peptide interaction with lipid membranes were studied by using fluorescence spectroscopy and monolayer surface tension measurements. It was confirmed that the wild-type fusion peptide-induced vesicle fusion rate increased several-fold between pH 7 and 5, unlike a mutated peptide, in which valine residues were substituted for glutamic acid residues at positions 11 and 15. This mutated peptide exhibited a much greater ability to induce lipid vesicle fusion and leakage but in a less pH-dependent manner compared to the wild-type fusion peptide. The peptide-induced vesicle fusion and leakage were well correlated with the degree of interaction of these peptides with lipid membranes, as deduced from the rotational correlation time obtained for the peptide tryptophan fluorescence. Both vesicle fusion and leakage induced by the peptides were suppressed by LPC incorporated into lipid vesicle membranes in a concentration-dependent manner. The rotational correlation time associated with the peptide’s tryptophan residue, which interacts with lipid membranes containing up to 25 mole % LPC, was virtually the same compared to lipid membranes without LPC, indicating that LPC-incorporated membrane did not affect the peptide interaction with the membrane. The adsorption of peptide onto a lipid monolayer also showed that the presence of LPC did not affect peptide adsorption.     

流行性出血热患者尿液中融合细胞的检测及其意义

我们观察到流行性出血热患者尿液中的“多核巨细胞”并非是上皮细胞脱落后的偶然堆积,而是细胞之间发生了融合。经免疫组化染色发现所有融合细胞内均有特异性EHF病毒抗原颗粒。提示流行性出血热病毒的直接作用与尿液中融合细胞的形成有关。     

Bilayer stabilizing peptides and the inhibition of viral infection: antimeasles activity of carbobenzoxy-Ser-Leu-amide

A number of carbobenzoxy-dipeptide-amides raise the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine (stabilizes the bilayer). The potency of the peptides in stabilizing the bilayer phase is Z-Tyr-Leu-NH₂= Z-Gly-Phe-NH₂>Z-Ser-Leu-NH₂>Z-Gly-Leu-NH₂>Z-Gly-Gly-NH₂. A linear correlation was found between the respective HPLC retention time parameterk for the peptide and the slope of the bilayer stabilization curve determined with model membranes by differential scanning calorimetry. One dipeptide, Z-Ser-Leu-NH₂, reduces measles virus cytopathic effect (CPE) in Vero cells. The mechanism by which this peptide reduces the CPE is not known, although some peptides which raise the bilayer to hexagonal phase transition temperature of phospholipids inhibit membrane fusion.Abbreviations Z carbobenzoxy - DEPE dielaidoylphosphatidylethanolamine - DSC differential scanning calorimetry - HPLC high pressure liquid chromatography - CPE cytopathic effect To whom correspondence should be addressed.     

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.     

血纤蛋白粘附肽与低分子量单链尿激酶融合基因在大肠杆菌中的表达

人工合成了血纤蛋白粘附肽基因,构建了粘附肽与低分子量单链尿激酶cDNA的融合基因,在大肠杆菌中表达了融合基因。融合基因表达产物的抗原性和天然尿激酶相同,并具有尿激酶的溶纤活性和粘附肽的抗纤维蛋白单体聚合的功能。     

Both heptad repeats of human respiratory syncytial virus fusion protein are potent inhibitors of viral fusion

Heptad repeat regions (HR1 and HR2) are highly conserved peptides located in F(1) of paramyxovirus envelope proteins. They are important in the process of virus fusion and form six-helix bundle structure (trimer of HR1 and HR2 heterodimer) post-fusion, similar to those found in the fusion proteins of other enveloped viruses, such as retrovirus HIV. Both HR1 and HR2 show potent inhibition for virus fusion in some members of paramyxovirus. However, in other members, only HR2 gives strong inhibition whereas HR1 does not. Human respiratory syncytial virus (hRSV) is a member of paramyxovirus and its crystal structure of HR1 and HR2 six-helix bundle was solved lately. Although hRSV HR2 inhibition was reported, nevertheless the effect of HR1 on virus fusion is not known. In this study, hRSV HR1 and HR2 were expressed as fusion protein separately in Escherichia coli system and their complex assembly and virus fusion inhibition effect have been analysed. It shows that both HR1 and HR2 (in the fusion form with 50-amino-acid fusion partner) of hRSV F protein give strong inhibition on virus fusion (IC(50) values are 1.68 and 2.93 microM, respectively) and they form stable six-helix bundle in vitro with both in the fusion protein form.     

Determination of the minimal fusion peptide of bovine leukemia virus gp30

In this study, we determined the minimal N-terminal fusion peptide of the gp30 of the bovine leukemia virus on the basis of the tilted peptide theory. We first used molecular modelling to predict that the gp30 minimal fusion peptide corresponds to the 15 first residues. Liposome lipid-mixing and leakage assays confirmed that the 15-residue long peptide induces fusion in vitro and that it is the shortest peptide inducing optimal fusion since longer peptides destabilize liposomes to the same extent but not shorter ones. The 15-residue long peptide can thus be considered as the minimal fusion peptide. The effect of mutations reported in the literature was also investigated. Interestingly, mutations related to glycoproteins unable to induce syncytia in cell-cell fusion assays correspond to peptides predicted as non-tilted. The relationship between obliquity and fusogenicity was also confirmed in vitro for one tilted and one non-tilted mutant peptide.