scholarly journals Vacuole membrane fusion

2003 ◽  
Vol 162 (2) ◽  
pp. 211-222 ◽  
Author(s):  
Martin J. Bayer ◽  
Christoph Reese ◽  
Susanne Bühler ◽  
Christopher Peters ◽  
Andreas Mayer
Keyword(s):  
Membrane Fusion ◽  
Proton Translocation ◽  
Integral Membrane Proteins ◽  
Fusion Pore ◽  
Fusion Partner ◽  
Fusion Activity ◽  
Vacuole Fusion ◽  
Tightly Coupled ◽  
Inhibitory Antibodies

Pore models of membrane fusion postulate that cylinders of integral membrane proteins can initiate a fusion pore after conformational rearrangement of pore subunits. In the fusion of yeast vacuoles, V-ATPase V0 sectors, which contain a central cylinder of membrane integral proteolipid subunits, associate to form a transcomplex that might resemble an intermediate postulated in some pore models. We tested the role of V0 sectors in vacuole fusion. V0 functions in fusion and proton translocation could be experimentally separated via the differential effects of mutations and inhibitory antibodies. Inactivation of the V0 subunit Vph1p blocked fusion in the terminal reaction stage that is independent of a proton gradient. Δvph1 mutants were capable of docking and trans-SNARE pairing and of subsequent release of lumenal Ca2+, but they did not fuse. The Ca2+-releasing channel appears to be tightly coupled to V0 because inactivation of Vph1p by antibodies blocked Ca2+ release. Vph1 deletion on only one fusion partner sufficed to severely reduce fusion activity. The functional requirement for Vph1p correlates to V0 transcomplex formation in that both occur after docking and Ca2+ release. These observations establish V0 as a crucial factor in vacuole fusion acting downstream of trans-SNARE pairing.

scholarly journals Membrane lysis during biological membrane fusion: collateral damage by misregulated fusion machines

2008 ◽  
Vol 183 (2) ◽  
pp. 181-186 ◽  
Author(s):  
Alex Engel ◽  
Peter Walter
Keyword(s):  
Membrane Fusion ◽  
Biological Membrane ◽  
Membrane Integrity ◽  
Fusion Pore ◽  
Mechanistic Models ◽  
Collateral Damage ◽  
Vacuole Fusion ◽  
Membrane Lysis

In the canonical model of membrane fusion, the integrity of the fusing membranes is never compromised, preserving the identity of fusing compartments. However, recent molecular simulations provided evidence for a pathway to fusion in which holes in the membrane evolve into a fusion pore. Additionally, two biological membrane fusion models—yeast cell mating and in vitro vacuole fusion—have shown that modifying the composition or altering the relative expression levels of membrane fusion complexes can result in membrane lysis. The convergence of these findings showing membrane integrity loss during biological membrane fusion suggests new mechanistic models for membrane fusion and the role of membrane fusion complexes.

The Role of the Transmembrane and of the Intraviral Domain of Glycoproteins in Membrane Fusion of Enveloped Viruses

2000 ◽  
Vol 20 (6) ◽  
pp. 571-595 ◽  
Author(s):  
Britta Schroth-Diez ◽  
Kai Ludwig ◽  
Bolormaa Baljinnyam ◽  
Christine Kozerski ◽  
Qiang Huang ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Fusion Proteins ◽  
Transmembrane Domain ◽  
Fusion Process ◽  
Fusion Pore ◽  
Minimum Length ◽  
Viral Fusion ◽  
Fusion Activity ◽  
Enveloped Viruses

Fusion of enveloped viruses with their target membrane is mediated by viral integral glycoproteins. A conformational change of their ectodomain triggers membrane fusion. Several studies suggest that an extended, triple-stranded rod-shaped α-helical coiled coil resembles a common structural and functional motif of the ectodomain of fusion proteins. From that, it is believed that essential features of the fusion process are conserved among the various enveloped viruses. However, this has not been established so far for the highly conserved transmembrane and intraviral sequences of fusion proteins. The article will focus on the role of both sequences in the fusion process. Recent studies from various enveloped viruses strongly imply that a transmembrane domain with a minimum length is required for later steps of membrane fusion, i.e., the formation and enlargement of the aqueous fusion pore. Although no specific sequence of the TM is necessary for pore formation, distinct properties and motifs of the domain may be obligatory to ascertain full fusion activity. However, with some exceptions, the intraviral domain seems to be not required for fusion activity of viral fusion proteins.

scholarly journals Regulatory Role of the Morbillivirus Attachment Protein Head-to-Stalk Linker Module in Membrane Fusion Triggering

2018 ◽  
Vol 92 (18) ◽  
Author(s):  
Michael Herren ◽  
Neeta Shrestha ◽  
Marianne Wyss ◽  
Andreas Zurbriggen ◽  
Philippe Plattet
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Surface ◽  
Membrane Fusion ◽  
Measles Virus ◽  
Cell Entry ◽  
Regulatory Role ◽  
Fusion Activity ◽  
Animal Pathogens

ABSTRACTMorbillivirus (e.g., measles virus [MeV] and canine distemper virus [CDV]) host cell entry is coordinated by two interacting envelope glycoproteins, namely, an attachment (H) protein and a fusion (F) protein. The ectodomain of H proteins consists of stalk, connector, and head domains that assemble into functional noncovalent dimer-of-dimers. The role of the C-terminal module of the H-stalk domain (termed linker) and the connector, although putatively able to assume flexible structures and allow receptor-induced structural rearrangements, remains largely unexplored. Here, we carried out a nonconservative mutagenesis scan analysis of the MeV and CDV H-linker/connector domains. Our data demonstrated that replacing isoleucine 146 in H-linker (H-I146) with any charged amino acids prevented virus-mediated membrane fusion activity, despite proper trafficking of the mutants to the cell surface and preserved binding efficiency to the SLAM/CD150 receptor. Nondenaturing electrophoresis revealed that these charged amino acid changes led to the formation of irregular covalent H tetramers rather than functional dimer-of-dimers formed when isoleucine or other hydrophobic amino acids were present at residue position 146. Remarkably, we next demonstrated that covalent H tetramerizationper sewas not the only mechanism preventing F activation. Indeed, the neutral glycine mutant (H-I146G), which exhibited strong covalent tetramerization propensity, maintained limited fusion promotion activity. Conversely, charged H-I146 mutants, which additionally carried alanine substitution of natural cysteines (H-C139A and H-C154A) and thus were unable to form covalently linked tetramers, were fusion activation defective. Our data suggest a dual regulatory role of the hydrophobic residue at position 146 of the morbillivirus head-to-stalk H-linker module: securing the assembly of productive dimer-of-dimers and contributing to receptor-induced F-triggering activity.IMPORTANCEMeV and CDV remain important human and animal pathogens. Development of antivirals may significantly support current global vaccination campaigns. Cell entry is orchestrated by two interacting glycoproteins (H and F). The current hypothesis postulates that tetrameric H ectodomains (composed of stalk, connector, and head domains) undergo receptor-induced rearrangements to productively trigger F; these conformational changes may be regulated by the H-stalk C-terminal module (linker) and the following connector domain. Mutagenesis scan analysis of both microdomains revealed that replacing amino acid 146 in the H-linker region with nonhydrophobic residues produced covalent H tetramers which were compromised in triggering membrane fusion activity. However, these mutant proteins retained their ability to traffic to the cell surface and to bind to the virus receptor. These data suggest that the morbillivirus linker module contributes to the folding of functional pre-F-triggering H tetramers. Furthermore, such structures might be critical to convert receptor engagement into F activation.

scholarly journals Third Helical Domain of the Nipah Virus Fusion Glycoprotein Modulates both Early and Late Steps in the Membrane Fusion Cascade

2020 ◽  
Vol 94 (19) ◽  
Author(s):  
J. Lizbeth Reyes Zamora ◽  
Victoria Ortega ◽  
Gunner P. Johnston ◽  
Jenny Li ◽  
Nicole M. André ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Host Range ◽  
Viral Entry ◽  
Nipah Virus ◽  
Fusion Process ◽  
Fusion Pore ◽  
Animal Pathogens ◽  
Fusion Glycoprotein ◽  
Helical Region

ABSTRACT Medically important paramyxoviruses, such as measles, mumps, parainfluenza, Nipah, and Hendra viruses, infect host cells by directing fusion of the viral and cellular plasma membranes. Upon infection, paramyxoviruses cause a second type of membrane fusion, cell-cell fusion (syncytium formation), which is linked to pathogenicity. Host cell receptor binding causes conformational changes in the attachment glycoprotein (HN, H, or G) that trigger a conformational cascade in the fusion (F) glycoprotein that mediates membrane fusion. F, a class I fusion protein, contains the archetypal heptad repeat regions 1 (HR1) and 2 (HR2). It is well established that binding of HR1 and HR2 is key to fusing viral and cellular membranes. In this study, we uncovered a novel fusion-modulatory role of a third structurally conserved helical region (HR3) in F. Based on its location within the F structure, and structural differences between its prefusion and postfusion conformations, we hypothesized that the HR3 modulates triggering of the F conformational cascade (still requiring G). We used the deadly Nipah virus (NiV) as an important paramyxoviral model to perform alanine scan mutagenesis and a series of multidisciplinary structural/functional analyses that dissect the various states of the membrane fusion cascade. Remarkably, we found that specific residues within the HR3 modulate not only early F-triggering but also late extensive fusion pore expansion steps in the membrane fusion cascade. Our results characterize these novel fusion-modulatory roles of the F HR3, improving our understanding of the membrane fusion process for NiV and likely for the related Henipavirus genus and possibly Paramyxoviridae family members. IMPORTANCE The Paramyxoviridae family includes important human and animal pathogens, such as measles, mumps, and parainfluenza viruses and the deadly henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviruses infect the respiratory tract and the central nervous system (CNS) and can be highly infectious. Most paramyxoviruses have a limited host range. However, the biosafety level 4 NiV and HeV are highly pathogenic and have a wide mammalian host range. Nipah viral infections result in acute respiratory syndrome and severe encephalitis in humans, leading to 40 to 100% mortality rates. The lack of licensed vaccines or therapeutic approaches against NiV and other important paramyxoviruses underscores the need to understand viral entry mechanisms. In this study, we uncovered a novel role of a third helical region (HR3) of the NiV fusion glycoprotein in the membrane fusion process that leads to viral entry. This discovery sets HR3 as a new candidate target for antiviral strategies for NiV and likely for related viruses.

scholarly journals Induction of Heterosubtypic Cross-Protection against Influenza by a Whole Inactivated Virus Vaccine: The Role of Viral Membrane Fusion Activity

PLoS ONE ◽  
2012 ◽  
Vol 7 (1) ◽  
pp. e30898 ◽  
Author(s):  
Natalija Budimir ◽  
Anke Huckriede ◽  
Tjarko Meijerhof ◽  
Louis Boon ◽  
Emma Gostick ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Virus Vaccine ◽  
Cross Protection ◽  
Fusion Activity ◽  
Viral Membrane ◽  
Viral Membrane Fusion

scholarly journals Roles of Cholesterol in Early and Late Steps of the Nipah Virus Membrane Fusion Cascade

2021 ◽  
Author(s):  
Erik M. Contreras ◽  
Gunner P. Johnston ◽  
David W. Buchholz ◽  
Victoria Ortega ◽  
I. Abrrey Monreal ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Viral Entry ◽  
Nipah Virus ◽  
Fusion Pore ◽  
Membrane Cholesterol ◽  
Enveloped Viruses ◽  
Cell Cell

Cholesterol has been implicated in various viral life cycle steps for different enveloped viruses, including viral entry into host cells, cell-cell fusion, and viral budding from infected cells. Enveloped viruses acquire their membranes from their host cells. Though cholesterol has been associated with binding and entry of various enveloped viruses into cells, cholesterol’s exact function in the viral-cell membrane fusion process remains largely elusive, particularly for the paramyxoviruses. Further, paramyxoviral fusion occurs at the host cell membrane and is essential for both virus entry (virus-cell fusion) and syncytia formation (cell-cell fusion), central to viral pathogenicity. Nipah virus (NiV) is a deadly member of the Paramyxoviridae family, which also includes Hendra, measles, mumps, human parainfluenza, and various veterinary viruses. The zoonotic NiV causes severe encephalitis, vasculopathy, and respiratory symptoms, leading to a high mortality rate in humans. We used NiV as a model to study the role of membrane cholesterol in paramyxoviral membrane fusion. We used a combination of methyl-beta cyclodextrin (MβCD), lovastatin, and cholesterol to deplete or enrich cell membrane cholesterol outside cytotoxic concentrations. We found that the levels of cellular membrane cholesterol directly correlated with the levels of cell-cell fusion induced. These phenotypes were paralleled using NiV/vesicular stomatitis virus (NiV/VSV) pseudotyped viral infection assays. Remarkably, our mechanistic studies revealed that cholesterol reduces an early F-triggering step but enhances a late fusion pore formation step in the NiV membrane fusion cascade. Thus, our results expand our mechanistic understanding of the paramyxoviral/henipaviral entry and cell-cell fusion processes. IMPORTANCE Cholesterol has been implicated in various steps of the viral life cycle for different enveloped viruses. Nipah virus (NiV) is a highly pathogenic enveloped virus in the Henipavirus genus within the Paramyxoviridae family, capable of causing a high mortality rate in humans and high morbidity in domestic and agriculturally important animals. The role of cholesterol for NiV or the henipaviruses is unknown. Here we show that the levels of cholesterol influence the levels of NiV-induced cell-cell membrane fusion during syncytia formation, and virus-cell membrane fusion during viral entry. Further, the specific role of cholesterol in membrane fusion is not well defined for the paramyxoviruses. We show that the levels of cholesterol affect an early F-triggering step and a late fusion pore formation step during the membrane fusion cascade. Thus, our results expand our mechanistic understanding of the viral entry and cell-cell fusion processes, which may aid the development of antivirals.

scholarly journals The Pre-Transmembrane Domain of the Autographa californica Multicapsid Nucleopolyhedrovirus GP64 Protein Is Critical for Membrane Fusion and Virus Infectivity

2009 ◽  
Vol 83 (21) ◽  
pp. 10993-11004 ◽  
Author(s):  
Zhaofei Li ◽  
Gary W. Blissard
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Aromatic Amino Acids ◽  
Autographa Californica ◽  
Fusion Pore ◽  
Virus Infectivity ◽  
Viral Fusion ◽  
Fusion Activity

ABSTRACT The envelope glycoprotein, GP64, of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is a class III viral fusion protein that mediates pH-triggered membrane fusion during virus entry. Viral fusion glycoproteins from many viruses contain a short region in the ectodomain and near the transmembrane domain, referred to as the pre-transmembrane (PTM) domain. In some cases, the PTM domain is rich in aromatic amino acids and plays an important role in membrane fusion. Although the 23-amino-acid (aa) PTM domain of AcMNPV GP64 lacks aromatic amino acids, we asked whether this region might also play a significant role in membrane fusion. We generated alanine scanning and single and multiple amino acid substitutions in the GP64 PTM domain. We specifically focused on amino acid positions conserved between baculovirus GP64 and thogotovirus GP75 proteins, as well as hydrophobic and charged amino acids. For each PTM-modified construct, we examined trimerization, cell surface localization, and membrane fusion activity. Membrane merger and pore formation were also examined. We identified eight aa positions that are important for membrane fusion activity. Critical positions were not clustered in the linear sequence but were distributed throughout the PTM domain. While charged residues were not critical or essential, three hydrophobic amino acids (L465, L476, and L480) played an important role in membrane fusion activity and appear to be involved in formation of the fusion pore. We also asked whether selected GP64 constructs were capable of rescuing a gp64null AcMNPV virus. These studies suggested that several conserved residues (T463, G460, G462, and G474) were not required for membrane fusion but were important for budding and viral infectivity.

scholarly journals Modification of the Cytoplasmic Domain of Influenza Virus Hemagglutinin Affects Enlargement of the Fusion Pore

2000 ◽  
Vol 74 (16) ◽  
pp. 7529-7537 ◽  
Author(s):  
Christine Kozerski ◽  
Evgeni Ponimaskin ◽  
Britta Schroth-Diez ◽  
Michael F. G. Schmidt ◽  
Andreas Herrmann
Keyword(s):  
Influenza Virus ◽  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Expression System ◽  
Fusion Pore ◽  
Target Cells ◽  
Fusion Activity ◽  
Wild Type ◽  
Influenza Virus Hemagglutinin ◽  
Pore Enlargement

ABSTRACT The fusion activity of chimeras of influenza virus hemagglutinin (HA) (from A/fpv/Rostock/34; subtype H7) with the transmembrane domain (TM) and/or cytoplasmic tail (CT) either from the nonviral, nonfusogenic T-cell surface protein CD4 or from the fusogenic Sendai virus F-protein was studied. Wild-type or chimeric HA was expressed in CV-1 cells by the transient T7-RNA-polymerase vaccinia virus expression system. Subsequently, the fusion activity of the expression products was monitored with red blood cells or ghosts as target cells. To assess the different steps of fusion, target cells were labeled with the fluorescent membrane label octadecyl rhodamine B-chloride (R18) (membrane fusion) and with the cytoplasmic fluorophores calcein (molecular weight [MW], 623; formation of small aqueous fusion pore) and tetramethylrhodamine-dextran (MW, 10,000; enlargement of fusion pore). All chimeric HA/F-proteins, as well as the chimera with the TM of CD4 and the CT of HA, were able to mediate the different steps of fusion very similarly to wild-type HA. Quite differently, chimeric proteins with the CT of CD4 were strongly impaired in mediating pore enlargement. However, membrane fusion and formation of small pores were similar to those of wild-type HA, indicating that the conformational change of the ectodomain and earlier fusion steps were not inhibited. Various properties of the CT which may affect pore enlargement are considered. We surmise that the hydrophobicity of the sequence adjacent to the transmembrane domain is important for pore dilation.

scholarly journals The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

2008 ◽  
Vol 19 (9) ◽  
pp. 3769-3781 ◽  
Author(s):  
Gábor Nagy ◽  
Ira Milosevic ◽  
Ralf Mohrmann ◽  
Katrin Wiederhold ◽  
Alexander M. Walter ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Fusion Rate ◽  
High Rate ◽  
Fusion Pore ◽  
Chimeric Proteins ◽  
Null Mouse ◽  
Charged Amino Acids ◽  
Protein Receptor ◽  
Sensitive Factor

The assembly of four soluble N-ethylmaleimide-sensitive factor attachment protein receptor domains into a complex is essential for membrane fusion. In most cases, the four SNARE-domains are encoded by separate membrane-targeted proteins. However, in the exocytotic pathway, two SNARE-domains are present in one protein, connected by a flexible linker. The significance of this arrangement is unknown. We characterized the role of the linker in SNAP-25, a neuronal SNARE, by using overexpression techniques in synaptosomal-associated protein of 25 kDa (SNAP-25) null mouse chromaffin cells and fast electrophysiological techniques. We confirm that the palmitoylated linker-cysteines are important for membrane association. A SNAP-25 mutant without cysteines supported exocytosis, but the fusion rate was slowed down and the fusion pore duration prolonged. Using chimeric proteins between SNAP-25 and its ubiquitous homologue SNAP-23, we show that the cysteine-containing part of the linkers is interchangeable. However, a stretch of 10 hydrophobic and charged amino acids in the C-terminal half of the SNAP-25 linker is required for fast exocytosis and in its absence the calcium dependence of exocytosis is shifted toward higher concentrations. The SNAP-25 linker therefore might have evolved as an adaptation toward calcium triggering and a high rate of execution of the fusion process, those features that distinguish exocytosis from other membrane fusion pathways.

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