While structures of a subset of conformations and parts of the fusion machinery have been characterized, the nature and sequence of membrane deformations during fusion have largely eluded characterization.
Building upon studies that focused on early stages of HA-mediated membrane remodeling, here cryo-electron tomography cryo-ET was used to image the three-dimensional organization of intact influenza virions at different stages of fusion with liposomes, leading all the way to completion of the fusion reaction.
By monitoring the evolution of fusion intermediate populations over the course of acid-induced fusion, we identified the progression of membrane reorganization that leads to efficient fusion by an enveloped virus.
All Rights Reserved. In this work the impact of cholesterol and its analogs and various sphingolipids were examined and found to strongly influence the growth of pores.
Open questions also exist about the composition of the fusion pore. Bonnafous et al. The role of lipid rafts has been suggested to be important for organizing viral proteins prior to budding, but less is known about the role of rafts in viral entry. Among the 16 different HA proteins known, there are marked variations in binding and fusion properties. New experimental approaches may enable characterization of entry processes in ways that were not possible previously because binding and fusion could not be completely decoupled.
Characterization of the fusion kinetics of different strains of influenza is beneficial for understanding the fusion process and assessing strain to strain variations [ ].
These include determining if strain variation is manifested as changes in fusion and if these changes correlate to increased infectivity, assessing the importance of the fusion step in pandemic strain emergence, determining the influence of membrane chemistry on fusion and evaluating its connection to systemic infection, and establishing the efficacy of newly developed anti-fusogenic antibodies and anti-viral drugs. In vitro fusion assays employed to characterize virus fusion should mimic the in vivo endosomal environment as closely as possible.
Ideally intact virions should be used to preserve the natural features of the virus, which may play as yet unknown supporting roles in fusion. In addition, assays with fast triggering and data acquisition capabilities with suitable time resolution that does not obscure or influence the processes under study is paramount to acquiring the most quantitative data [ ].
To begin this section, we first review traditional fusion assays and their limitations, and then describe the state-of-the-art fusion assays enabled by modern technologies. The first in vitro strategies for studying virus fusion to membranes were ensemble or bulk assays [ ]. These initial assays reported either membrane mixing between the virus and the cell membrane or content release of the virus; but not both at the same time [ ].
Content mixing assays typically employed vesicles with encapsulated dye that upon fusion with either intact virions or reconstituted viral envelopes release their contents and give rise to a change in fluorescent signal. Two common approaches are the release of a calcium indicator dye that fluoresces when exposed to the surrounding buffer or changes in fluorescence due to dequenching of internal dye or fluorescence energy transfer FRET.
In membrane mixing assays, virus fusion is typically reported by changes in fluorescence resulting from fluorescence dequenching within the membrane upon fusion [ ], or FRET between fluorophore pairs residing in the membrane [ ]. In the most direct measurement of membrane mixing and associated kinetics, intact viral membranes are first labeled with fluorophores until the fluorescence signal is quenched. Then, the labeled virus is mixed in a cuvette with unlabeled host cell mimics containing the viral receptor, such as liposomes or ghost cells, and a baseline florescence signal is obtained in a fluorimeter.
To initiate fusion, a small amount of acid is added to the cuvette while the sample is rapidly mixed. The temporal change in the fluorescence signal is collected as the viruses fuse with the host membranes and the fluorophores originally in the viral membranes become diluted and dequench. Alternatively, a FRET method can be used to avoid labeling the virus itself by creating liposomes containing both fluorophores of a FRET pair that separate when virus fuses to vesicle.
From the change in the fluorescence signal in either approach, some information about the kinetics of virus fusion can be obtained. These approaches characterize the overall rate from the binding to the hemifusion step, determined over an ensemble population of virions within the cuvette.
Many studies of virus fusion to date have been conducted using this type of assay [ 96 , , , , , , ] and a great deal of what is known about virus fusion has been learned using this ensemble approach. However, there are several limitations that have restricted the amount of information that can be collected from these assays. First, virus fusion is stochastic and thus only averaged kinetic information is obtained from these assays, which can obscure intermediate steps [ ].
Second, since individual events cannot be observed in this assay, viral binding and fusion events cannot be distinguished visually making it difficult to study either processes individually. To circumvent this limitation, these assays can be conducted at cold temperatures to bind viruses first and then trigger with acidic buffer to decouple binding and fusion processes kinetically from each other [ 81 ].
An instantaneous pH change from neutral to acidic is ideal to trigger virus fusion at the same time point at a uniform pH value. Asynchronous triggering of events masks the magnitude of the pH dependence of fusion [ 92 ], which may be an important criterion when assessing infectivity. Therefore, due to the finite volume of the cuvette, rapid mixing of contents is required to quickly distribute the acid throughout the cuvette, but this rapid mixing leads to shearing, which can interrupt virus binding and does not mimic the quiescent environment inside an endosome.
A third important consideration is that the curvature of the two opposing membranes is opposite of that inside the endosome. It is unclear if this non-native geometry could result in membrane bending energies that alter the kinetics or pathway of fusion of the membranes. Finally, monitoring pore-opening kinetics is difficult to conduct simultaneously with membrane hemifusion in this ensemble approach. Many of these drawbacks can be overcome using IVI approaches and alternative cell membrane mimics with planar geometry.
Recognizing ensemble assay limitations, investigators moved to studying single event virus fusion using direct imaging of virions, reconstituted viral envelopes virosomes , or HA-expressing cells interacting with other cells or cell membrane mimics.
Several fusion studies of intact virus to erythrocytes [ ] or individual human erythrocytes to fibroblasts expressing the influenza virus hemagglutinin were reported [ , ]. These assays employed a flow chamber mounted to a microscope stage. Fusion was triggered by rapid acidification of the flow chamber and fusion monitored by a fluorescence increase due to redistribution of fluorescent dyes between either membrane or cytoplasmic compartments of fusing cells. Significant heterogeneity in lag times for events was reported, which could be in part due to asynchronous initiation of fusion, a point we will return to later.
Niles and Cohen [ ], used a video-epifluorescence microscope setup to study individual virions fusing to a planar BLM formed across the orifice of a Teflon support [ ] positioned within the field of view of a microscope.
In the execution of this assay, fluorescently-labeled, quenched viruses were loaded into a micropipette tip which was positioned in one side of the Teflon chamber already at the desired fusion pH. To coordinate the triggering of fusion, virus solution at neutral pH inside the micropipette was gently expelling near the acidified BLM surface. Virions contacting the BLM, either immediately bound to it or fell out of the field of view quickly.
Bound viruses could then undergo fusion with the BLM. A video camera recorded the fluorescent images of individual fusion events, detected as single dequenching events. These images were later processed to obtain hemifusion kinetic parameters. The Niles and Cohen [ 92 ] individual virion fusion technique showed that receptor binding alters fusion kinetics. In the presence of receptor, the kinetics followed Markovian behavior characteristic of Poisson process described by a rate constant defining the jump between distinct states.
Fusion triggered in the absence of receptor followed non-Markovian behavior with no characteristic rate parameter. This clear distinction in fusion kinetics was made possible by 1 decoupling of binding and fusion processes; 2 temporal synchronization of fusion initiation; and 3 statistical analysis of the individual fusion events.
While it was known from previous ensemble studies that gangliosides increase fusion rate [ , , ], the single virion approach of Niles and Cohen could quantitatively assess changes in rate and provide additional information about the kinetic pathway [ 92 , 93 ]. These measurements were corroborated by electrical conductance measurements [ ].
Later imaging assays that combined lipid mixing, contents mixing, and electrical conductance measurements in one assay provided important experimental details on the intermediate steps leading to pore formation and that it might proceed by a series of small pores forming initially [ ]. Importantly, these first experiments pioneered the approach of direct visualization of individual influenza fusion events and using planar bilayers in the form of BLMs as the cell membrane mimic.
However, the limited sensitivity of the equipment and the fragility of BLMs restricted the broader applicability of this method at the time. Nonetheless, these studies ushered in a new stochastic approach for studying virus fusion, which laid the critical groundwork for IVI assays of today.
Building upon the previous assays and with the advent of increasingly better technology for implementing single particle studies, fusion studies involving cell-cell fusion, virosome-cell fusion, and virus-cell fusion at the individual event level continued, targeting a variety of viruses [ 86 , , ]. Studies using reconstituted vesicles of HA allowed researchers to assess the impact of HA density on fusion. One study examined the impact of various forms of HA on fusion, reconstituting both HA 0 nonfusogenic form and HA 1,2 fusogenic form into vesicles [ 86 ].
This is an important feature of these assays, as it is not entirely clear that HA-expressing cells of reconstituted virosomes would behave the same way that a whole virus with its intact viral membrane and secondary proteins does. Recent work using intact virions in stochastic assays has created a wealth of new knowledge about fusion kinetics by providing information on the kinetics of intermediate steps of the fusion mechanism [ ] and information about the rate of acidification of the internal contents of the virus during fusion [ ].
These new assays employ total internal reflection fluorescence microscopy TIRF [ ], a surface-specific technique that illuminates about a nm thick layer from the interface of a change in refractive index, such as between glass and aqueous solution, and greatly facilitates distinguishing bound viruses from those in the bulk solution. Because TIRF is a surface-specific technique, it is compatible with planar platforms like microfluidic devices.
Microfluidic devices that have their walls coated with solid-supported lipid bilayers have been employed for numerous bio-analytical applications aimed at mimicking the cell surface. Supported lipid bilayers are robust materials that are easy to fabricate, their physico-chemical properties can be tailored, and the incorporation of membrane receptor molecules is straightforward [ , , ].
The spacing between the bilayer and the solid support can be tuned using cushions as well [ , , ]. These biomimetic TIRF platforms lend themselves well to single particle studies of vesicle rupture at surfaces [ ]; two-dimensional diffusion of phospholipids [ , ], proteins [ ], and virus-like particles [ ] in planar bilayers; vesicle fusion mediated by SNARE proteins [ , , ] and DNA hybridization [ ]; and virus fusion [ , ], as illustrated in Figure 4.
Total internal reflection fluorescence microscopy integrated with a microfluidic device. A supported lipid bilayer adsorbed to the walls of the microfluidic device will reside within this evanescent wave. Fluorescently-labeled viruses bound to the supported bilayer will be excited and emit a red signal. The emitted light is sent back to a sensitive camera for imaging. The addition of a second laser line or more allows multiple fluorophores to be monitored simultaneously, for example, one to mark the viral membrane and another to mark the internal contents.
Note that the size of the virus with respect to the bilayer is not drawn to scale. Inset Upon acidification of the microfluidic channel, virus fusion commences. A two color virus labeling scheme distinguished the hemifusion step green from pore formation red. Acidification to initiate virus fusion in this platform is achieved by flowing an acidic solution through the microfluidic channel to exchange with the neutral buffer initially in the device.
Intermediate fusion steps can be distinguished using a multi-color labeling procedure where the viral membrane is labeled with one color and the internal viral contents are labeled with another color [ ]. This strategy enables one to monitor the hemifusion process separately from the pore formation process within a single virion and to examine their relative timescales.
Hemifusion detection proceeds through a dequenching strategy, as was employed in previous ensemble assays, but here clearly marks the onset of the hemifusion event for an individual virion. Pore formation within the same virion is visibly detectable in this assay, through the release and subsequent diffusion of the internal fluorescent dye away from the fusion site.
Each individual fusion event occurs independently. From each fusion event, a number of critical pieces of information can be obtained. First, the lag time between acidification and hemifusion for that individual virion is marked by the time point at which the dequenching spike occurs. Second, information about the diffusion of the lipids at the fusion site can be obtained by monitoring the radial spread of the fluorescence following the dequenching spike.
Third, the lag time between hemifusion and pore formation for the same virion can be easily determined when using a two-color labeling strategy.
Cataloging the lag times for each individual fusion event yields statistical data that can then be analyzed to determine the kinetics of fusion for a population.
Within each fusion event, a number of intermediate steps occur between the initial conformational change of HA triggered by acidification and the creation of the fusion pore. The overall fusion process is a convolution of the various intermediate steps, each of which can be described as a Poisson process. The distribution of the lag times of a population can be modeled by a gamma distribution to obtain a shape, N , and rate parameter, k. When examining the hemifusion lag time distribution, k defines the hemifusion rate constant and N is interpreted to be the number of HA trimers working concertedly to bend and merge the membranes, as described in detail by Floyd et al.
In this work, analysis of pore formation lag times for X yielded a single exponential decay, indicating a one step process from the hemifusion state to the formation of the pore. While acidification by buffer flow exchange in microfluidic devices works reasonably well to trigger the conformational change of HA, a no-slip boundary condition at the membrane surface creates shear on viruses bound to the membrane.
This requirement creates an upper bound to how fast exchange can occur. The dynamics of the protein conformational changes for some HA proteins are known to be on the millisecond timescale [ ], so slow acidification by buffer exchange may present limitations in resolution of the technique. Slow acidification can also temporally spread initiation of fusion events across the field of view and possibly impart local pH gradients. These effects can limit the resolution of the data that can be obtained.
In the future, more information could be garnered by labeling the viral contents, for example the viral RNA, so that unpacking of the viral caspid and release of the viral genome is confirmed. Such an advance would not only aid in learning more about the fusion process, but allow for studies of RNA unpacking upon release and assist in identifying other potential targets for future anti-viral compounds.
With the establishment of these new approaches to study fusion, we anticipate that future studies will focus on a range of factors in virus fusion that have been difficult to examine in the past, continuing to fill in fundamental information that will inform the rational design of antiviral therapeutics.
In additional to fundamental fusion studies, these new tools offer better ways to characterize variations in fusion behavior among various virus strains, lab-adapted varieties, and clinical isolates, and the action of anti-fusogenic compounds acting on them.
Novel therapeutics such as protease and fusion inhibitors and cross-neutralizing antibodies that impede the fusion process may be directly studied in these platforms as well. Furthermore, it may be possible to also study endosome-specific factors in fusion, such as the impact of various lipids and enzymatic reactions that modify lipid species as the endosome matures, by replicating these reactions within the in vitro platform.
National Center for Biotechnology Information , U. Journal List Viruses v. Published online Jul Brian S. Hamilton , 1 Gary R. Gary R. Author information Article notes Copyright and License information Disclaimer. This article has been cited by other articles in PMC.
Abstract Hemagglutinin HA is the viral protein that facilitates the entry of influenza viruses into host cells. Keywords: influenza virus, hemagglutinin, membrane fusion, protease, virus entry. Introduction Influenza virus is a member of the Orthomyxoviridae family and is classified into three types; influenza A, influenza B, and influenza C [ 1 , 2 ]. Figure 1. Open in a separate window. Proteolytic Cleavage of HA 2.
Figure 2. Molecular Determinants of HA Cleavage The molecular determinants of HA cleavage are still largely unclear beyond the understanding that the proteases involved are most likely trypsin-like, serine proteases. Influenza Entry Processes 3. Overview of the Roles of HA in Viral Entry Proteolytic cleavage of HA is an important precursory step in the infection cycle that imparts new influenza viruses with the capacity to infect other cells.
HA Mediated Fusion Mechanism The actual transfer of viral genetic material to the cytosol requires the fusion of the viral membrane with the endosomal membrane and the formation of a fusion pore, through which the genetic material exits the viral capsid. Open Questions in Fusion While membrane fusion mediated by HA has been a well-studied subject, a number of key questions still remain unanswered. Figure 3. Studying HA Fusion Kinetics 4. Overview Among the 16 different HA proteins known, there are marked variations in binding and fusion properties.
In Vitro Ensemble Fusion Assays The first in vitro strategies for studying virus fusion to membranes were ensemble or bulk assays [ ]. Early Individual Virion Imaging of Virus Fusion to Cell Membrane Mimics Recognizing ensemble assay limitations, investigators moved to studying single event virus fusion using direct imaging of virions, reconstituted viral envelopes virosomes , or HA-expressing cells interacting with other cells or cell membrane mimics.
Stochastic Fusion Assays Building upon the previous assays and with the advent of increasingly better technology for implementing single particle studies, fusion studies involving cell-cell fusion, virosome-cell fusion, and virus-cell fusion at the individual event level continued, targeting a variety of viruses [ 86 , , ].
Figure 4. Data Treatment and Stochastic Analysis Each individual fusion event occurs independently. Limitations of Current IVI Assays and Considerations for Future Improvements While acidification by buffer flow exchange in microfluidic devices works reasonably well to trigger the conformational change of HA, a no-slip boundary condition at the membrane surface creates shear on viruses bound to the membrane.
Looking Ahead With the establishment of these new approaches to study fusion, we anticipate that future studies will focus on a range of factors in virus fusion that have been difficult to examine in the past, continuing to fill in fundamental information that will inform the rational design of antiviral therapeutics.
Conflict of Interest The authors declare no conflict of interest. References 1. Horimoto T. Influenza: Lessons from past pandemics, warnings from current incidents. Palese P. Orthomyxoviridae: The Viruses and Their Replication. In: Knipe D. Fields Virology. Open in a separate window. In the case of herpesviruses, cell type differences have been seen.
A need for low pH for fusion of some paramyxoviruses with cells is under investigation see text. For others e. Some coronavirus S precursors are e. For these latter coronaviruses S proteins as well as for Ebola virus GP and Hendra and Nipah virus F, post synthetic cleavage by extracellular or intracellular e. Bovine herpesvirus gB and human cytomegalovirus gBs are processed, but processing is not needed for cell entry Kopp et al.
The structure of the presumed pre-fusion trimer is not yet known. It is thought that their respective membrane embedded prehairpins are trimers.
Low pH Low pH is the sole known fusion trigger for orthomyxo-, rhabdo-, alpha-, flavi-, bunya-, and arenaviruses.
Binding of Host Cell Receptors The fusion proteins of most paramyxo-, retro-, and herpesviruses, as well as some coronaviruses, are activated by interactions with host cell receptors at neutral pH Bossart and Broder, in press ; Earp et al. Receptor Binding to an Independent Viral Receptor Binding Protein For the majority of paramyxoviruses and for herpesviruses, an envelope glycoprotein spike that is separate from the fusion protein mediates binding to host cell receptors Table 1.
Paramyxoviruses In the case of paramyxoviruses, host cell receptors bind to a viral receptor binding glycoprotein also referred to as an attachment protein and this information is somehow relayed to the fusion protein, which is then triggered to undergo dramatic conformational changes Connolly et al.
Influenza HA Influenza virus HA is the founding member of the large group of Class I fusion proteins and remains one of its best characterized. Paramyxovirus F Fusion of paramyxoviruses with target cells is mediated by the fusion protein, F. Fusion Peptides and Fusion Loops Some, but not all, fusion peptides can be preliminarily identified by sequence analysis as regions of intermediate hydrophobicity with predicted membrane binding potential.
Cytoplasmic Tails CTs The cytoplasmic tails CTs of viral fusion proteins have a variety of functions that differ both among and within virus families. SUMMARY In conclusion, we hope that we have given the reader an appreciation for how highly diverse fusion proteins mediate a common pathway of membrane fusion.
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This mutation was originally identified in isolates from oseltamivir-treated individuals infected with H1N1 virus [42] , and more recently, in patients infected with HPAI of H5N1 background, a troubling observation given the pandemic potential and extremely virulent nature of this IFV strain [43] — [45].
Perhaps more alarming was the finding that seasonal H1N1 viruses, carrying the oseltamivir resistance-conferring H mutation, emerged simultaneously in several countries in —, including countries where oseltamivir is not prescribed [10] , [12] — [14].
This in vitro finding indicates that DAS may be active against currently circulating oseltamivir-resistant IFV and further suggests that if novel strains, such as the pandemic influenza A H1N1 viruses, attain the HY mutation, DAS may be active against these new isolates as well. While the pandemic IFV A H1N1 and the oseltamivir-resistant seasonal IFV of — currently exhibit low case fatality rates, these patterns are potentially unstable.
Given the IFV' propensity for rapid evolution, the constant threat of an emerging highly virulent drug-resistant strain is concerning and supports the need to monitor evolution of the A H1N1 IFV strains and simultaneously develop specific vaccines and novel antivirals. Growth and differentiation of primary human bronchial epithelial cells to produce HAE culture were performed as described previously [46] , [47]. Briefly, primary human bronchial cells Cell Applications, San Diego, CA expanded to passage 3 were seeded in porous membrane inserts 4.
Three days after seeding the cells, the medium from the apical side was removed and the confluent monolayers were cultured at an air-liquid interface. The medium from the basal compartment was replaced daily, and the in vitro differentiation of primary cells was achieved after 4—6 weeks. Fixed cell monolayers were immunostained with primary anti-influenza A nucleoprotein NP mouse monoclonal antibody kind gift of Dr.
Robert Webster, St. Louis, MO. Cell clusters foci expressing viral antigen NP were counted using a fluorescent microscope Axiovert , Zeiss, Germany and viral titers were calculated. Virus yield in supernatants was determined as described above. The MDCK experiments were performed twice, yielding comparable results.
The HAE experiment was performed once. Analysis of total viral yield in bronchi tissue homogenate was performed as described previously [49]. In brief, total nucleic acid was extracted from the bronchi tissue using NucliSens easyMAG extraction system bioMerieux, Netherlands according to manufacturer's instructions.
All M-gene quantities are expressed normalized to B-actin level. A series of dilutions were prepared to generate calibration curves and run in parallel with the test samples. At the end of the assay, PCR products were subjected to a melting curve analysis to determine the specificity of the assay.
Treatments were repeated daily for a total of 5 treatments q. Ten mice per group were observed daily for morbidity, as measured by weight loss, or mortality, for 14 days post-infection p. Body weight is represented as percent change from day 0 body weight immediately prior to experiment. For viral titer analysis, whole lungs were collected from euthanized animals on day 3 and day 6 p. Lung tissues were collected and titrated for virus as described previously [52]. The mouse experiment was performed twice with comparable results.
All data was graphed with Prism 4. We thank Alexander Klimov for generously providing viruses and for valuable discussion of the data. We thank Mang Yu for critical evaluation of this manuscript. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field.
Abstract Background The recent emergence of a novel pandemic influenza A H1N1 strain in humans exemplifies the rapid and unpredictable nature of influenza virus evolution and the need for effective therapeutics and vaccines to control such outbreaks. Methods and Findings The activity of DAS against several pandemic influenza A H1N1 virus isolates was examined in MDCK cells, differentiated primary human respiratory tract culture, ex-vivo human bronchi tissue and mice.
Stoddart, University of California San Francisco, United States of America Received: June 10, ; Accepted: October 13, ; Published: November 6, This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Introduction In the United States alone influenza virus IFV causes over , hospitalizations annually and is responsible for approximately 36, deaths every year [1] , [2]. Download: PPT. Figure 1. Table 1. Table 2. Figure 2. Figure 3.
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