Insights into a Structure-Based Mechanism of Viral Membrane Fusion

A number of different viral spike proteins, responsible for membrane fusion, show striking similarities in their core structures. The prospect of developing a general structure-based mechanism seems plausible in light of these newly determined structures. Influenza hemagglutinin (HA) is the best-studied fusion machine, whose action has previously been described by a hypothetical spring-loaded model. This model has recently been extended to explain the mechanism of other systems, such as HIV gp120–gp41. However, evidence supporting this idea is insufficient, requiring re-examination of the mechanism of HA-induced membrane fusion. Recent experiments with a shortened construct of HA, which is able to induce lipid mixing, have provided evidence for an alternative scenario for HA-induced membrane fusion and perhaps that of other viral systems.     

Synchronized Retrovirus Fusion in Cells Expressing Alternative Receptor Isoforms Releases the Viral Core into Distinct Sub-cellular Compartments

Disparate enveloped viruses initiate infection by fusing with endosomes. However, the highly diverse and dynamic nature of endosomes impairs mechanistic studies of fusion and identification of sub-cellular sites supporting the nucleocapsid release. We took advantage of the extreme stability of avian retrovirus-receptor complexes at neutral pH and of acid-dependence of virus-endosome fusion to isolate the latter step from preceding asynchronous internalization/trafficking steps. Viruses were trapped within endosomes in the presence of NH₄Cl. Removal of NH₄Cl resulted in a quick and uniform acidification of all subcellular compartments, thereby initiating synchronous viral fusion. Single virus imaging demonstrated that fusion was initiated within seconds after acidification and often culminated in the release of the viral core from an endosome. Comparative studies of cells expressing either the transmembrane or GPI-anchored receptor isoform revealed that the transmembrane receptor delivered the virus to more fusion-permissive compartments. Thus the identity of endosomal compartments, in addition to their acidity, appears to modulate viral fusion. A more striking manifestation of the virus delivery to distinct compartments in the presence of NH₄Cl was the viral core release into the cytosol of cells expressing the transmembrane receptor and into endosomes of cells expressing the GPI-anchored isoform. In the latter cells, the newly released cores exhibited restricted mobility and were exposed to a more acidic environment than the cytoplasm. These cores appear to enter into the cytosol after an additional slow temperature-dependent step. We conclude that the NH₄Cl block traps the virus within intralumenal vesicles of late endosomes in cells expressing the GPI-anchored receptor. Viruses surrounded by more than one endosomal membrane release their core into the cytoplasm in two steps – fusion with an intralumenal vesicle followed by a yet unknown temperature-dependent step that liberates the core from late endosomes.     

A Quantitative and Kinetic Fusion Protein-Triggering Assay Can Discern Distinct Steps in the Nipah Virus Membrane Fusion Cascade

The deadly paramyxovirus Nipah virus (NiV) contains a fusion glycoprotein (F) with canonical structural and functional features common to its class. Receptor binding to the NiV attachment glycoprotein (G) triggers F to undergo a two-phase conformational cascade: the first phase progresses from a metastable prefusion state to a prehairpin intermediate (PHI), while the second phase is marked by transition from the PHI to the six-helix-bundle hairpin. The PHI can be captured with peptides that mimic F''s heptad repeat regions, and here we utilized a NiV heptad repeat peptide to quantify PHI formation and the half-lives (t_1/2) of the first and second fusion cascade phases. We found that ephrinB2 receptor binding to G triggered ∼2-fold more F than that triggered by ephrinB3, consistent with the increased rate and extent of fusion observed with ephrinB2- versus ephrinB3-expressing cells. In addition, for a series of hyper- and hypofusogenic F mutants, we quantified F-triggering capacities and measured the kinetics of their fusion cascade phases. Hyper- and hypofusogenicity can each be manifested through distinct stages of the fusion cascade, giving rise to vastly different half-lives for the first (t_1/2, 1.9 to 7.5 min) or second (t_1/2, 1.5 to 15.6 min) phase. While three mutants had a shorter first phase and a longer second phase than the wild-type protein, one mutant had the opposite phenotype. Thus, our results reveal multiple critical parameters that govern the paramyxovirus fusion cascade, and our assays should help efforts to elucidate other class I membrane fusion processes.Nipah (NiV) and Hendra (HeV) viruses are emerging members of the new Paramyxoviridae genus Henipavirus (12, 19). The Paramyxoviridae family comprises important viral pathogens, such as measles, mumps, human parainfluenza, respiratory syncytial, and Newcastle disease viruses and the henipaviruses (HNV), and NiV is its deadliest known member (4, 5). NiV has a broad host range and causes respiratory and neurological symptoms that often lead to encephalitis and a mortality rate of up to 75% in humans (21, 47). It can also spread efficiently and cause morbidity in economically important livestock (21). NiV is a biosafety level 4 (BSL4) pathogen and is considered a select agent with bio- and agro-terrorism potential. Both animal-to-human and human-to-human transmissions have been documented (4, 5), underscoring the need for research and treatment development. Since microvascular endothelial cell-cell fusion (syncytium formation) is a pathognomonic hallmark of NiV infection (50), understanding virus-cell and cell-cell membrane fusion should assist in the development of therapeutics to target this aspect of NiV pathobiology.Paramyxovirus membrane fusion requires the coordinated action of the attachment (G, HN, or H) and fusion (F) glycoproteins, and numerous canonical structural and functional features of G/HN/H and F proteins are conserved among paramyxoviruses (20, 23, 46, 48). G/HN/H proteins have a receptor-binding globular domain formed by a six-bladed beta-propeller connected to its transmembrane anchor via a flexible stalk domain (10, 51). For NiV and HeV, both ephrinB2 (B2) and ephrinB3 (B3) can be used as cell receptors (8, 33, 34), although B2 appears to be the higher-affinity receptor (34). B2 or B3 receptors bind to and activate G, which in turn triggers a conformation cascade in F that leads to membrane fusion (1). HNV F proteins are trimeric class I fusion proteins with structural/functional features common to their class (23, 52). HNV F proteins are synthesized as precursors that are cleaved and hence activated into a metastable conformation, poised for enabling membrane fusion. Cleavage generates a new N terminus that contains a hydrophobic fusion peptide (48). For NiV and HeV, the precursor (F₀) reaches the plasma membrane uncleaved, but endocytosis exposes F₀ to cathepsin L in the endosomes, cleaving F₀ to generate mature disulfide-linked F₁ and F₂ subunits that are trafficked back to the cell surface (14, 31). The structures of the retroviral Moloney murine leukemia virus p15E, lentiviral human immunodeficiency virus type 1 (HIV-1) gp41, Ebola virus GP2, influenza virus HA, and paramyxovirus SV5 and NiV-F fusion proteins all share similar trimeric coiled-coil core structures (6, 11, 17, 27, 53) and, in general, similar membrane fusion mechanisms (22, 23, 48).Receptor binding to paramyxoviral G/HN/H triggers a conformational cascade in F, leading to membrane fusion (Fig. ​(Fig.1).1). Although the determinants for F triggering on G/HN/H have not been defined clearly, evidence suggests that the stalk domain (7, 13, 24, 28, 29) and, at least for NiV, a region at the base of the globular domain of G (1) are involved in F triggering. Additionally, recent evidence indicates an interaction between the stalk region of the measles virus H protein and the globular domain of the cognate F protein (35). Once triggered, F progresses through a prehairpin intermediate (PHI) (Fig. 1A and B). In the PHI conformation, the fusion peptide is harpooned into the host cell membrane, and the N- and C-terminal heptad repeat domains (HR1 and HR2, respectively) are exposed. The HR domains then coalesce into the postfusion six-helix-bundle (6HB) hairpin conformation. In the 6HB, the transmembrane and fusion peptide domains are juxtaposed, bringing viral and target cell membranes together and driving membrane fusion (Fig. ​(Fig.1C)1C) (30, 48). Much evidence suggests that 6HB formation is coincident with membrane merger and that synthetic HR1 and HR2 peptides only bind to and inhibit fusion intermediates (e.g., PHI) prior to 6HB formation (9, 30, 37, 43, 48). Additionally, HR1 peptides can inhibit an earlier fusion intermediate than that inhibited by HR2 peptides (43), and HR2 peptides are invariably more potent inhibitors of fusion than HR1 peptides. HR2 peptides trap the PHI by binding to the radial interstices formed by the trimeric HR1 core, inhibiting 6HB formation and membrane fusion (22, 23, 48). Altogether, there is much evidence to support the fusion cascade shown in Fig. ​Fig.11 and the use of HR2 peptides to physically capture fusion intermediates (9, 30, 43, 48).Open in a separate windowFIG. 1.Nipah virus fusion cascade. The schematic shows the NiV fusion cascade broken down into three major stages. (A) EphrinB2 or ephrinB3 binding to NiV-G triggers the metastable NiV-F protein through allosteric mechanisms that are still being elucidated. (B) After F is triggered, it forms the PHI, in which a fusion peptide is harpooned into the host cell membrane. The PHI can be captured by peptides that mimic the NiV-F HR1 (orange-striped cylinder) or HR2 (green-striped cylinder) region and bind the F HR2 or HR1 region, respectively. (C) The HR1 and HR2 regions in the PHI coalesce to form the 6HB conformation, bringing the viral and cell membranes together and facilitating virus-host membrane fusion and viral entry. The viral membrane can be replaced by a cell membrane expressing the F and G glycoproteins in cell-cell fusion, resulting in syncytium formation. We term the transitions from A to B and from B to C phases I and II, respectively, of the fusion cascade. (D) Schematic representation of the F-triggering assay, showing its four main steps: (1) receptor binding at 4°C, (2) biotinylated HR2 peptide addition and induction of F triggering at 37°C, (3) fixation at 4°C with paraformaldehyde, and (4) signal amplification at 4°C. In the “time-of-addition” and “time-of-stopping” experiments, step 2 was modified as indicated in the text. The HR2 peptide (green hatched column) is shown with its N-terminal biotin modification (red star). Blue stars, streptavidin-APC; black, three-pronged symbols, activator; blue symbols with red octagons, enhancer.We previously developed a fluorescence-activated cell sorting (FACS)-based NiV-F-triggering assay by measuring the amount of HR2 peptide binding to F/G-expressing cells triggered by cell surface ephrinB2 (1). In this study, we further optimized our assay for robust quantification of HR2 peptide binding and used this assay to monitor the differential degree of F triggering induced by B2 or B3. In addition, through “time-of-addition” and “time-of-stopping” experiments (described below), we show that this HR2 binding assay can measure the half-lives of various fusion intermediates, i.e., the transition times from the prefusion (PF) state to PHI and from PHI to 6HB. Using a panel of hyper- and hypofusogenic mutants, we show that hyper- and hypofusogenicity can each be manifested through distinct effects on the half-lives of these fusion intermediates and/or the absolute amounts of F triggering. Thus, we elucidated the impacts of different mutations on individual steps of the fusion cascade. Since HR2 peptides can generally capture the PHI of class I fusion proteins, our assays should help efforts to understand fusion processes mediated by other class I fusion proteins.     

Proteins of Newcastle Disease Virus and of the Viral Nucleocapsid

Newcastle disease virus was found to contain three major proteins. The structure unit of the viral nucleocapsid appears to be monomeric and to consist of a single large protein of an approximate molecular weight of 62,000.     

Dynamin Regulates Specific Membrane Fusion Events Necessary for Acrosomal Exocytosis in Mouse Spermatozoa

Mammalian spermatozoa must complete an acrosome reaction prior to fertilizing an oocyte. The acrosome reaction is a unique exocytotic event involving a series of prolonged membrane fusions that ultimately result in the production of membrane vesicles and release of the acrosomal contents. This event requires the concerted action of a large number of fusion-competent signaling and scaffolding proteins. Here we show that two different members of the dynamin GTPase family localize to the developing acrosome of maturing mouse germ cells. Both dynamin 1 and 2 also remain within the periacrosomal region of mature mouse spermatozoa and are thus well positioned to regulate the acrosome reaction. Two pharmacological inhibitors of dynamin, dynasore and Dyngo-4a, blocked the in vitro induction of acrosomal exocytosis by progesterone, but not by the calcium ionophore A23187, and elicited a concomitant reduction of in vitro fertilization. In vivo treatment with these inhibitors also resulted in spermatozoa displaying reduced acrosome reaction potential. Dynamin 1 and 2 phosphorylation increased on progesterone treatment, and this was also selectively blocked by dynasore. On the basis of our collective data, we propose that dynamin could regulate specific membrane fusion events necessary for acrosomal exocytosis in mouse spermatozoa.     

TAT蛋白转导结构域介导融合蛋白在小鼠活体的跨膜递送作用

目的探讨跨膜递送短肽——TAT蛋白转导结构域(简称TAT)介导的与其融合的活性蛋白在活体的跨膜递送作用。方法以融合蛋白GST-TAT-GFP,GST-GFP-TAT和GST-GFP为研究模型蛋白,不经过蛋白质的变性处理、直接通过向小鼠腹腔注射和皮肤涂抹这两种含TAT的融合蛋白及作为对照的融合蛋白GST-GFP,一定时间作用后取体内器官和皮肤做冷冻切片,荧光显微镜检测这些融合蛋白的跨膜递送情况;并对分别融合在C端或者N端的TAT介导GFP在活体动物体内和皮肤的跨膜递送作用进行对比。结果腹腔注射实验结果表明,TAT可以介导不经过蛋白质的变性处理的融合蛋白GST-TAT-GFP和GST-GFP-TAT跨膜递送进入到小鼠的心脏、肝、肾、脾和肺,甚至脑组织;其中GST-GFP-TAT跨膜递送效率比GST-TAT-GFP更高。结构模拟分析提示GST-GFP-TAT与GST-TAT-GFP中的TAT的暴露情况不同可能是造成两种蛋白跨膜递送活性差异的重要因素。皮肤实验的结果则表明TAT不仅介导融合蛋白GST-TAT-GFP和GST-GFP-TAT进入小鼠表皮,而且使其进入小鼠皮肤的真皮层。结论 TAT可以跨膜递送不经过变性处理的融合蛋白进入小鼠皮肤和体内,递送效率可能与TAT的暴露程度相关;这些结果为在蛋白质疗法方面应用TAT提供了进一步的理论依据。     

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.     

Cathepsin B-mediated Autophagy Flux Facilitates the Anthrax Toxin Receptor 2-mediated Delivery of Anthrax Lethal Factor into the Cytoplasm

Anthrax lethal toxin (LeTx) is a virulence factor secreted by Bacillus anthracis and has direct cytotoxic effects on most cells once released into the cytoplasm. The cytoplasmic delivery of the proteolytically active component of LeTx, lethal factor (LF), is carried out by the transporter component, protective antigen, which interacts with either of two known surface receptors known as anthrax toxin receptor (ANTXR) 1 and 2. We found that the cytoplasmic delivery of LF by ANTXR2 was mediated by cathepsin B (CTSB) and required lysosomal fusion with LeTx-containing endosomes. Also, binding of protective antigen to ANXTR1 or -2 triggered autophagy, which facilitated the cytoplasmic delivery of ANTXR2-associated LF. We found that whereas cells treated with the membrane-permeable CTSB inhibitor CA074-Me- or CTSB-deficient cells had no defect in fusion of LC3-containing autophagic vacuoles with lysosomes, autophagic flux was significantly delayed. These results suggested that the ANTXR2-mediated cytoplasmic delivery of LF was enhanced by CTSB-dependent autophagic flux.     

Viral Vectors for Gene Delivery and Expression in the CNS

Viral vectors have emerged as an important tool for manipulating gene expression in the adult mammalian brain. The adult brain is composed largely of nondividing cells, and therefore DNA viruses have become the vehicle of choice for neurobiologists interested in somatic gene transfer. Recombinant viral vectors based upon adenovirus or herpes simplex virus have been created in which a gene essential for viral replication is removed and a gene of interest is inserted in the viral genome. While this eliminates pathogenicity due to viral replication, retention of viral genes and continued expression of these genes may limit the potential of the current generation of vectors. Defective viral vectors represent a different approach, in which only viral recognition signals are used to allow packaging of foreign DNA into a viral coat while eliminating the possibility of viral gene expression within target cells. The defective HSV vector has been used to transfer genes into the adult rat brain. This vector has also been used for analysis of the preproenkephalin promoterin vivo,and important regions of this promoter have been identified using this technique. A modification ofin situPCR has been developed as an adjunctive tool for sensitively documenting the presence of vector DNA within target cells duringin vivopromoter studies. Finally, the adenoassociated virus vector has been used as the first fully defective DNA viral vector, which also eliminates any contamination by helper viruses. This vector can transfer genes into the mammalian brain and has shown significant behavioral recovery in a rodent model of Parkinson's disease. Future work will undoubtedly result in still more diverse and improved vectors; however, these studies have documented the importance of viral vectors to both basic neurobiology and the potential treatment of neurologic disease.     

Dual Mutation Events in the Haemagglutinin-Esterase and Fusion Protein from an Infectious Salmon Anaemia Virus HPR0 Genotype Promote Viral Fusion and Activation by an Ubiquitous Host Protease

In Infectious salmon anaemia virus (ISAV), deletions in the highly polymorphic region (HPR) in the near membrane domain of the haemagglutinin-esterase (HE) stalk, influence viral fusion. It is suspected that selected mutations in the associated Fusion (F) protein may also be important in regulating fusion activity. To better understand the underlying mechanisms involved in ISAV fusion, several mutated F proteins were generated from the Scottish Nevis and Norwegian SK779/06 HPR0. Co-transfection with constructs encoding HE and F were performed, fusion activity assessed by content mixing assay and the degree of proteolytic cleavage by western blot. Substitutions in Nevis F demonstrated that K276 was the most likely cleavage site in the protein. Furthermore, amino acid substitutions at three sites and two insertions, all slightly upstream of K276, increased fusion activity. Co-expression with HE harbouring a full-length HPR produced high fusion activities when trypsin and low pH were applied. In comparison, under normal culture conditions, groups containing a mutated HE with an HPR deletion were able to generate moderate fusion levels, while those with a full length HPR HE could not induce fusion. This suggested that HPR length may influence how the HE primes the F protein and promotes fusion activation by an ubiquitous host protease and/or facilitate subsequent post-cleavage refolding steps. Variations in fusion activity through accumulated mutations on surface glycoproteins have also been reported in other orthomyxoviruses and paramyxoviruses. This may in part contribute to the different virulence and tissue tropism reported for HPR0 and HPR deleted ISAV genotypes.     

Tim23p Contains Separate and Distinct Signals for Targeting to Mitochondria and Insertion into the Inner Membrane

The Tim23 protein is an essential inner membrane (IM) component of the yeast mitochondrial protein import pathway. Tim23p does not carry an amino-terminal presequence; therefore, the targeting information resides within the mature protein. Tim23p is anchored in the IM via four transmembrane segments and has two positively charged loops facing the matrix. To identify the import signal for Tim23p, we have constructed several altered versions of the Tim23 protein and examined their function and import in yeast cells, as well as their import into isolated mitochondria. We replaced the positively charged amino acids in one or both loops with alanine residues and found that the positive charges are not required for import into mitochondria, but at least one positively charged loop is required for insertion into the IM. Furthermore, we find that the signal to target Tim23p to mitochondria is carried in at least two of the hydrophobic transmembrane segments. Our results suggest that Tim23p contains separate import signals: hydrophobic segments for targeting Tim23p to mitochondria, and positively charged loops for insertion into the IM. We therefore propose that Tim23p is imported into mitochondria in at least two distinct steps.     

The Plasma Membrane and Peripheral Cytoplasm of the Green Algal Flagellate, Gloeomonas kupfferi

A morphological analysis of the plasma membrane and peripheralendomembrane components of the unicellular chlamydomonad flagellate,Gloeomonas kupfferi, was performed. Conventional fixation, freezesubstitution, and rapid freeze-deep etch processing protocolsfor electron microscopic analyses revealed the following. Theplasma membrane is highlighted by distinct infoldings whichdo not appear to be sensitive to changes in osmotic or cellcycle conditions. These infoldings are irregularly-spaced androughly 200-225 nm apart. Each elliptical infolding is 400-440nm long and 90-115 nm wide. The exoplasmic face (EF) of eachinfolding is highlighted by an aggregation of 130-160 nm intramembranousparticles (imps) that are 9.1-10·5 nm in size. Theseinfoldings are associated with the peripheral endoplasmic reticulumnetwork and microtubular network internally and the inner walllayer externally. It is suggested that these infoldings maybe associated with cell wall maintenance.Copyright 1994, 1999Academic Press Gloeomonas, plasma membrane, infoldings, freeze fracture     

Distinct Palmitoylation Events at the Amino-terminal Conserved Cysteines of Env7 Direct Its Stability,Localization, and Vacuolar Fusion Regulation in S. cerevisiae

Palmitoylation at cysteine residues is the only known reversible form of lipidation and has been implicated in protein membrane association as well as function. Many palmitoylated proteins have regulatory roles in dynamic cellular processes, including membrane fusion. Recently, we identified Env7 as a conserved and palmitoylated protein kinase involved in negative regulation of membrane fusion at the lysosomal vacuole. Env7 contains a palmitoylation consensus sequence, and substitution of its three consecutive cysteines (Cys¹³–Cys¹⁵) results in a non-palmitoylated and cytoplasmic Env7. In this study, we further dissect and define the role(s) of individual cysteines of the consensus sequence in various properties of Env7 in vivo. Our results indicate that more than one of the cysteines serve as palmitoylation substrates, and any pairwise combination is essential and sufficient for near wild type levels of Env7 palmitoylation, membrane localization, and phosphorylation. Furthermore, individually, each cysteine can serve as a minimum requirement for distinct aspects of Env7 behavior and function in cells. Cys¹³ is sufficient for membrane association, Cys¹⁵ is essential for the fusion regulatory function of membrane-bound Env7, and Cys¹⁴ and Cys¹⁵ are redundantly essential for protection of membrane-bound Env7 from proteasomal degradation. A role for Cys¹⁴ and Cys¹⁵ in correct sorting at the membrane is also discussed. Thus, palmitoylation at the N-terminal cysteines of Env7 directs not only its membrane association but also its stability, phosphorylation, and cellular function.     

脂质膜体与线粒体内膜的融合

<正> 膜融合现象在细胞免疫、细胞识别和相互作用、病毒感架、细胞分泌以及细胞器之间相互关系等方面均有重要意义。利用人工脂质膜体(liposome)与细胞(器)膜之间的融合,不但为阐明上述问题的分子机理提供了一种模型,而且将为细胞生物工程研究提供手段。本文是我们实验室关于脂质膜体与鼠肝线粒体内膜体(mitoplast)融合的一系列研究结果的简要报道。