Rapid Membrane Fusion of Individual Virus Particles with Supported Lipid Bilayers

Wessels, Laura; Elting, Mary Williard; Scimeca, Dominic; Weninger, Keith

doi:10.1529/biophysj.106.097485

Cited by 78 publications

(102 citation statements)

References 90 publications

(145 reference statements)

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“…short-lived intermediate states cannot be discriminated. To overcome this population averaging and obtain more kinetic detail, we designed a single-particle assay based on earlier single-particle work by our group (Floyd et al, 2008) and others (Hinterdorfer et al, 1994;Imai et al, 2006;Ivanovic et al, 2013;Melikyan et al, 2005;Niles & Cohen, 1991;Wessels et al, 2007) (Fig. 3).…”

Section: Results Ph-dependent Fusion Of Chikv With Liposomesmentioning

confidence: 99%

Chikungunya virus fusion properties elucidated by single-particle and bulk approaches

Duijl-Richter

Blijleven

Oijen

et al. 2015

Journal of General Virology

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Chikungunya virus (CHIKV) is a rapidly spreading, enveloped alphavirus causing fever, rash and debilitating polyarthritis. No specific treatment or vaccines are available to treat or prevent infection. For the rational design of vaccines and antiviral drugs, it is imperative to understand the molecular mechanisms involved in CHIKV infection. A critical step in the life cycle of CHIKV is fusion of the viral membrane with a host cell membrane. Here, we elucidate this process using ensemble-averaging liposome-virus fusion studies, in which the fusion behaviour of a large virus population is measured, and a newly developed microscopy-based single-particle assay, in which the fusion kinetics of an individual particle can be visualised. The combination of these approaches allowed us to obtain detailed insight into the kinetics, lipid dependency and pH dependency of hemifusion. We found that CHIKV fusion is strictly dependent on low pH, with a threshold of pH 6.2 and optimal fusion efficiency below pH 5.6. At this pH, CHIKV fuses rapidly with target membranes, with typically half of the fusion occurring within 2 s after acidification. Cholesterol and sphingomyelin in the target membrane were found to strongly enhance the fusion process. By analysing our single-particle data using kinetic models, we were able to deduce that the number of rate-limiting steps occurring before hemifusion equals about three. To explain these data, we propose a mechanistic model in which multiple E1 fusion trimers are involved in initiating the fusion process.

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Section: Results Ph-dependent Fusion Of Chikv With Liposomesmentioning

confidence: 99%

Chikungunya virus fusion properties elucidated by single-particle and bulk approaches

Duijl-Richter

Blijleven

Oijen

et al. 2015

Journal of General Virology

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“…A planar target bilayer of controlled lipid composition is formed on a glass support and can be designed to incorporate lipid or proteinaceous receptors and a lipid-coupled pH-sensitive fluorescent probe. Synchronous acidification is achieved in a microfluidic channel by flowing in low-pH buffer [96], by light-induced liberation of caged protons [97] or by pre-mixing [98]. Using TIRF-M, low-background and high-contrast fluorescence signals are extracted to monitor particles rolling along the bilayer and to visualize arrest, hemifusion and opening of a pore ( Figure 3A).…”

Section: Experimental Design Of Single-particle Viral Fusion Assaysmentioning

confidence: 99%

Mechanisms of influenza viral membrane fusion

Blijleven

Boonstra

Onck

et al. 2016

Seminars in Cell & Developmental Biology

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Influenza viral particles are enveloped by a lipid bilayer. A major step in infection is fusion of the viral and host cellular membranes, a process with large kinetic barriers. Influenza membrane fusion is catalyzed by hemagglutinin (HA), a class I viral fusion protein activated by low pH. The exact nature of the HA conformational changes that deliver the energy required for fusion remains poorly understood. This review summarizes our current knowledge of HA structure and dynamics, describes recent single-particle experiments and modeling studies, and discusses their role in understanding how multiple HAs mediate fusion. These approaches provide a mechanistic picture in which HAs independently and stochastically insert into the target membrane, forming a cluster of HAs that is collectively able to overcome the barrier to membrane fusion. The new experimental and modeling approaches described in this review hold promise for a more complete understanding of other viral fusion systems and the protein systems responsible for cellular fusion. Disciplines Medicine and Health Sciences | Social and Behavioral Sciences Publication DetailsBlijleven, J. S., Boonstra, S., Onck, P. R., van AbstractInfluenza viral particles are enveloped by a lipid bilayer. A major step in infection is fusion of the viral and host cellular membranes, a process with large kinetic barriers. Influenza membrane fusion is catalyzed by hemagglutinin (HA), a class I viral fusion protein activated by low pH. The exact nature of the HA conformational changes that deliver the energy required for fusion remains poorly understood. This review summarizes our current knowledge of HA structure and dynamics, describes recent single-particle experiments and modeling studies, and discusses their role in understanding how multiple HAs mediate fusion. These approaches provide a mechanistic picture in which HAs independently and stochastically insert into the target membrane, forming a cluster of HAs that is collectively able to overcome the barrier to membrane fusion. The new experimental and modeling approaches described in this review hold promise for a more complete understanding of other viral fusion systems and the protein systems responsible for cellular fusion.

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“…The characterization of hemifusion and pore formation by individual retrovirus particles demonstrates the strength of single-particle observations for extracting detailed kinetic information on fusion intermediates (18). Similar approaches have enabled visualization of hemifusion by individual influenza virions (19,20), but direct observation of the entire influenza fusion process and its kinetic characterization at the singleparticle level has been difficult to achieve. We report here the development of a method for real-time detection of both hemifusion and pore formation for individual, intact influenza virus particles fusing with a target lipid bilayer.…”

mentioning

confidence: 99%

Single-particle kinetics of influenza virus membrane fusion

Floyd¹,

Ragains²,

Skehel³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

262

389

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Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays are generally limited to observation of ensembles of multiple fusion events, confounding more detailed analysis of the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support. Analysis of lipid-and content-mixing trajectories on a particle-by-particle basis provides evidence for multiple, long-lived kinetic intermediates leading to hemifusion, followed by a single, rate-limiting step to pore formation. We interpret the series of intermediates preceding hemifusion as a result of the requirement that multiple copies of the trimeric hemagglutinin fusion protein be activated to initiate the fusion process.enveloped viruses ͉ lipid bilayer ͉ single molecule ͉ virus entry

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Rapid Membrane Fusion of Individual Virus Particles with Supported Lipid Bilayers

Cited by 78 publications

References 90 publications

Chikungunya virus fusion properties elucidated by single-particle and bulk approaches

Chikungunya virus fusion properties elucidated by single-particle and bulk approaches

Mechanisms of influenza viral membrane fusion

Single-particle kinetics of influenza virus membrane fusion

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