scholarly journals During entry of alphaviruses, the E1 glycoprotein molecules probably form two separate populations that generate either a fusion pore or ion-permeable pores

2004 ◽  
Vol 85 (6) ◽  
pp. 1695-1701 ◽  
Author(s):  
Gerd Wengler ◽  
Andreas Koschinski ◽  
Gisela Wengler ◽  
Holger Repp
Keyword(s):  
Fusion Protein ◽  
Semliki Forest Virus ◽  
Fusion Pore ◽  
Viral Membrane ◽  
Endosomal Membrane ◽  
Target Membrane ◽  
Membrane Reaction ◽  
Fusion Pores

Studies using the alphavirus Semliki Forest virus have indicated that the viral E1 fusion protein forms two types of pore: fusion pores and ion-permeable pores. The formation of ion-permeable pores has not been generally accepted, partly because it was not evident how the protein might form these different pores. Here it is proposed that the choice of the target membrane determines whether a fusion pore or ion-permeable pores are formed. The fusion protein is activated in the endosome and for steric reasons only a fraction of the activated molecules can interact with the endosomal membrane. This target membrane reaction forms the fusion pore. It is proposed that the rest of the activated molecules interact with the membrane in which the protein is anchored and that this self-membrane reaction leads to formation of ion-permeable pores, which can be detected in the target membrane after fusion of the viral membrane into the target membrane.

scholarly journals The fusion kinetics of influenza hemagglutinin expressing cells to planar bilayer membranes is affected by HA density and host cell surface.

1995 ◽  
Vol 106 (5) ◽  
pp. 783-802 ◽  
Author(s):  
G B Melikyan ◽  
W D Niles ◽  
F S Cohen
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Pore Formation ◽  
Surface Proteins ◽  
Fusion Pore ◽  
Planar Bilayer ◽  
Bilayer Membranes ◽  
Pore Evolution ◽  
Fusion Pores ◽  
Kinetics Of

Time-resolved admittance measurements were used to follow formation of individual fusion pores connecting influenza virus hemagglutinin (HA)-expressing cells to planar bilayer membranes. By measuring in-phase, out-of-phase, and dc components of currents, pore conductances were resolved with millisecond time resolution. Fusion pores developed in stages, from small pores flickering open and closed, to small successful pores that remained open until enlarging their lumens to sizes greater than those of viral nucleocapsids. The kinetics of fusion and the properties of fusion pores were studied as functions of density of the fusion protein HA. The consequences of treating cell surfaces with proteases that do not affect HA were also investigated. Fusion kinetics were described by waiting time distributions from triggering fusion, by lowering pH, to the moment of pore formation. The kinetics of pore formation became faster as the density of active HA was made greater or when cell surface proteins were extensively cleaved with proteases. In accord with this faster kinetics, the intervals between transient pore openings within the flickering stage were shorter for higher HA density and more extensive cell surface treatment. Whereas the kinetics of fusion depended on HA density, the lifetimes of open fusion pores were independent of HA density. However, the lifetimes of open pores were affected by the proteolytic treatment of the cells. Faster fusion kinetics correlated with shorter pore openings. We conclude that the density of fusion protein strongly affects the kinetics of fusion pore formation, but that once formed, pore evolution is not under control of fusion proteins but rather under the influence of mechanical forces, such as membrane bending and tension.

scholarly journals Functions of the Stem Region of the Semliki Forest Virus Fusion Protein during Virus Fusion and Assembly

2006 ◽  
Vol 80 (22) ◽  
pp. 11362-11369 ◽  
Author(s):  
Maofu Liao ◽  
Margaret Kielian
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Virus Assembly ◽  
Protein A ◽  
Stem Length ◽  
Membrane Fusion Protein ◽  
Virus Fusion ◽  
Target Membrane ◽  
Domain Iii

ABSTRACT Membrane fusion of the alphaviruses is mediated by the E1 protein, a class II virus membrane fusion protein. During fusion, E1 dissociates from its heterodimer interaction with the E2 protein and forms a target membrane-inserted E1 homotrimer. The structure of the homotrimer is that of a trimeric hairpin in which E1 domain III and the stem region fold back toward the target membrane-inserted fusion peptide loop. The E1 stem region has a strictly conserved length and several highly conserved residues, suggesting the possibility of specific stem interactions along the trimer core and an important role in driving membrane fusion. Mutagenesis studies of the alphavirus Semliki Forest virus (SFV) here demonstrated that there was a strong requirement for the E1 stem in virus assembly and budding, probably reflecting its importance in lateral interactions of the envelope proteins. Surprisingly, however, neither the conserved length nor any specific residues of the stem were required for membrane fusion. Although the highest fusion activity was observed with wild-type E1, efficient fusion was mediated by stem mutants containing a variety of substitutions or deletions. A minimal stem length was required but could be conferred by a series of alanine residues. The lack of a specific stem sequence requirement during SFV fusion suggests that the interaction of domain III with the trimer core can provide sufficient driving force to mediate membrane merger.

scholarly journals The role of the C terminus of the SNARE protein SNAP-25 in fusion pore opening and a model for fusion pore mechanics

2008 ◽  
Vol 105 (40) ◽  
pp. 15388-15392 ◽  
Author(s):  
Qinghua Fang ◽  
Khajak Berberian ◽  
Liang-Wei Gong ◽  
Ismail Hafez ◽  
Jakob B. Sørensen ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Mechanistic Model ◽  
Snare Complex ◽  
Fusion Pore ◽  
Snare Protein ◽  
C Terminus ◽  
Target Membrane ◽  
Pore Opening ◽  
Fusion Pores

Formation of a fusion pore between a vesicle and its target membrane is thought to involve the so-called SNARE protein complex. However, there is no mechanistic model explaining how the fusion pore is opened by conformational changes in the SNARE complex. It has been suggested that C-terminal zipping triggers fusion pore opening. A SNAP-25 mutant named SNAP-25Δ9 (lacking the last nine C-terminal residues) should lead to a less-tight C-terminal zipping. Single exocytotic events in chromaffin cells expressing this mutant were characterized by carbon fiber amperometry and cell-attached patch capacitance measurements. Cells expressing SNAP-25Δ9 displayed smaller amperometric “foot-current” currents, reduced fusion pore conductances, and lower fusion pore expansion rates. We propose that SNARE/lipid complexes form proteolipid fusion pores. Fusion pores involving the SNAP-25Δ9 mutant will be less tightly zipped and may lead to a longer fusion pore structure, consistent with the observed decrease of fusion pore conductance.

scholarly journals Visualization of the Target-Membrane-Inserted Fusion Protein of Semliki Forest Virus by Combined Electron Microscopy and Crystallography

Cell ◽  
2003 ◽  
Vol 114 (5) ◽  
pp. 573-583 ◽  
Author(s):  
Don L Gibbons ◽  
Inge Erk ◽  
Brigid Reilly ◽  
Jorge Navaza ◽  
Margaret Kielian ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fusion Protein ◽  
Semliki Forest Virus ◽  
Target Membrane

scholarly journals Reversible Acid-Induced Inactivation of the Membrane Fusion Protein of Semliki Forest Virus

2005 ◽  
Vol 79 (12) ◽  
pp. 7942-7948 ◽  
Author(s):  
Barry-Lee Waarts ◽  
Jolanda M. Smit ◽  
Onwuchekwa J. C. Aneke ◽  
Gerald M. McInerney ◽  
Peter Liljeström ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Semliki Forest Virus ◽  
Membrane Lipids ◽  
Model System ◽  
Low Ph ◽  
Complete Loss ◽  
Viral Fusion ◽  
Membrane Fusion Protein ◽  
Target Membrane ◽  
Partial Reversion

ABSTRACT Previously, it has been shown that the exposure of Semliki Forest virus (SFV) to a mildly acidic environment induces a rapid and complete loss of the ability of the virus to bind and fuse to target membranes added subsequently. In the present study, incubation of SFV at low pH followed by a specific reneutralization step resulted in a partial reversion of this loss of viral fusion capacity, as assessed in a liposomal model system. Also, the ability of the viral E1 fusion protein to undergo liposome-stimulated trimerization was restored. Furthermore, acid-treated and neutralized SFV largely retained infectivity. Exposure of SFV to low pH induced dissociation of the E1/E2 heterodimer, which was not reversed upon neutralization. It is concluded that the SFV E1 fusion protein, after acid-induced dissociation from E2, rapidly adopts an intermediate, nontrimeric conformation in which it is no longer able to interact with target membrane lipids. Neutralization restores the ability of E1 to interact with membranes. This interaction, however, remains strictly dependent on low pH.

scholarly journals Reversible Merger of Membranes at the Early Stage of Influenza Hemagglutinin-mediated Fusion

2000 ◽  
Vol 11 (7) ◽  
pp. 2359-2371 ◽  
Author(s):  
Eugenia Leikina ◽  
Leonid V. Chernomordik
Keyword(s):  
Fusion Protein ◽  
Surface Density ◽  
Early Stage ◽  
Low Ph ◽  
Fusion Pore ◽  
Complete Fusion ◽  
Physiological Temperature ◽  
Influenza Hemagglutinin ◽  
Pore Opening ◽  
Fusion Pores

Fusion mediated by influenza hemagglutinin (HA), a prototype fusion protein, is commonly detected as lipid and content mixing between fusing cells. Decreasing the surface density of fusion-competent HA inhibited these advanced fusion phenotypes and allowed us to identify an early stage of fusion at physiological temperature. Although lipid flow between membranes was restricted, the contacting membrane monolayers were apparently transiently connected, as detected by the transformation of this fusion intermediate into complete fusion after treatments known to destabilize hemifusion diaphragms. These reversible connections disappeared within 10–20 min after application of low pH, indicating that after the energy released by HA refolding dissipated, the final low pH conformation of HA did not support membrane merger. Although the dynamic character and the lack of lipid mixing at 37°C distinguish the newly identified fusion intermediate from the intermediate arrested at 4°C described previously, both intermediates apparently belong to the same family of restricted hemifusion (RH) structures. Because the formation of transient RH structures at physiological temperatures was as fast as fusion pore opening and required less HA, we hypothesize that fusion starts with the formation of multiple RH sites, only a few of which then evolve to become expanding fusion pores.

scholarly journals Nascent fusion pore opening monitored at single-SNAREpin resolution

2021 ◽  
Vol 118 (5) ◽  
pp. e2024922118
Author(s):  
Paul Heo ◽  
Jeff Coleman ◽  
Jean-Baptiste Fleury ◽  
James E. Rothman ◽  
Frederic Pincet
Keyword(s):  
Energy Landscape ◽  
Three Dimensional ◽  
Fusion Pore ◽  
Fusion Event ◽  
Direct Estimate ◽  
Target Membrane ◽  
Pore Opening ◽  
Two Peaks ◽  
Fusion Pores ◽  
Second Peak

Vesicle fusion with a target membrane is a key event in cellular trafficking and ensures cargo transport within the cell and between cells. The formation of a protein complex, called SNAREpin, provides the energy necessary for the fusion process. In a three-dimensional microfluidic chip, we monitored the fusion of small vesicles with a suspended asymmetric lipid bilayer. Adding ion channels into the vesicles, our setup allows the observation of a single fusion event by electrophysiology with 10-μs precision. Intriguingly, we identified that small transient fusion pores of discrete sizes reversibly opened with a characteristic lifetime of ∼350 ms. The distribution of their apparent diameters displayed two peaks, at 0.4 ± 0.1 nm and 0.8 ± 0.2 nm. Varying the number of SNAREpins, we demonstrated that the first peak corresponds to fusion pores induced by a single SNAREpin and the second peak is associated with pores involving two SNAREpins acting simultaneously. The pore size fluctuations provide a direct estimate of the energy landscape of the pore. By extrapolation, the energy landscape for three SNAREpins does not exhibit any thermally significant energy barrier, showing that pores larger than 1.5 nm are spontaneously produced by three or more SNAREpins acting simultaneously, and expand indefinitely. Our results quantitatively explain why one SNAREpin is sufficient to open a fusion pore and more than three SNAREpins are required for cargo release. Finally, they also explain why a machinery that synchronizes three SNAREpins, or more, is mandatory to ensure fast neurotransmitter release during synaptic transmission.

scholarly journals Probing the Flavivirus Membrane Fusion Mechanism by Using Monoclonal Antibodies

2007 ◽  
Vol 81 (20) ◽  
pp. 11526-11531 ◽  
Author(s):  
Karin Stiasny ◽  
Samantha Brandler ◽  
Christian Kössl ◽  
Franz X. Heinz
Keyword(s):  
Monoclonal Antibodies ◽  
Fusion Protein ◽  
Binding Sites ◽  
Fusion Inhibition ◽  
Tick Borne Encephalitis ◽  
Target Membrane ◽  
Initial Interaction ◽  
Tick Borne Encephalitis Virus ◽  
Late Stages

ABSTRACT In this study, we investigated in a flavivirus model (tick-borne encephalitis virus) the mechanisms of fusion inhibition by monoclonal antibodies directed to the different domains of the fusion protein (E) and to different sites within each of the domains by using in vitro fusion assays. Our data indicate that, depending on the location of their binding sites, the monoclonal antibodies impaired early or late stages of the fusion process, by blocking the initial interaction with the target membrane or by interfering with the proper formation of the postfusion structure of E, respectively. These data provide new insights into the mechanisms of flavivirus fusion inhibition by antibodies and their possible contribution to virus neutralization.

Sign in / Sign up

Export Citation Format

Share Document