Influenza Virus-Membrane Fusion Triggered by Proton Uncaging for Single Particle Studies of Fusion Kinetics

Costello, Deirdre A.; Lee, Donald W.; Drewes, Jennifer; Vasquez, Kevin; Kisler, Kassandra; Wiesner, Ulrich; Pollack, Lois; Whittaker, Gary R.; Daniel, Susan

doi:10.1021/ac3006473

Cited by 41 publications

(53 citation statements)

References 51 publications

(132 reference statements)

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“…Both cholesterol and sphingomyelin in the host-cell membrane strongly supported CHIKV fusion activity. The single-particle assay indicated that multiple, parallel rate-limiting steps precede hemifusion, a phenomenon described earlier for other membrane-fusing viruses (Brandenberg et al, 2015;Costello et al, 2012;Danieli et al, 1996;Floyd et al, 2008). We propose that these steps arise from the parallel action of several fusion trimers.…”

Section: Introductionsupporting

confidence: 72%

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: Introductionsupporting

confidence: 72%

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%

“…Single-particle experiments on influenza viral fusion showed that the waiting times between decrease of the pH and the cessation of rolling, and hemifusion and pore formation events showed rise-and-decay behavior, suggesting that these processes involve multiple steps [61,88,96,97,100]. The powerful combination of single-particle experiments and analytical [101] and numerical [61] modeling has resulted in a picture in which fusion is the result of a number of HAs acting in a parallel, stochastic fashion, whose proximity allows their stochastic activity to result in a level of coordination needed to overcome the large energetic barriers associated with fusion.…”

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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“…TfRs were dialyzed overnight in PBS and concentrated using a 10-kDa Amicon Ultra-15 centrifugal filter unit (EMD Millipore). Purified TfR-eGFP was loaded on top of a supported lipid bilayer (SLB), formed inside a microfluidic device as described elsewhere (15,16). A detailed schematic view of the SPT experimental setup is shown in Fig.…”

mentioning

confidence: 99%

Single-Particle Tracking Shows that a Point Mutation in the Carnivore Parvovirus Capsid Switches Binding between Host-Specific Transferrin Receptors

Lee

Allison

Bacon

et al. 2016

J Virol

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bDetermining how viruses infect new hosts via receptor-binding mechanisms is important for understanding virus emergence. We studied the binding kinetics of canine parvovirus (CPV) variants isolated from raccoons-a newly recognized CPV host-to different carnivore transferrin receptors (TfRs) using single-particle tracking. Our data suggest that CPV may utilize adhesion-strengthening mechanisms during TfR binding and that a single mutation in the viral capsid at VP2 position 300 can profoundly alter receptor binding and infectivity. Canine parvovirus (CPV) is a pathogen of dogs that emerged and caused a pandemic of disease in the 1970s and is Ͼ99% identical in nucleotide sequence to feline panleukopenia virus (FPV), a parvovirus that infects cats and other carnivore hosts but not dogs (1-3). Although the emergence of CPV has been presumed to be the result of a direct transfer of FPV or a similar virus from domestic cats to dogs, we recently demonstrated that CPV exists endemically in sylvatic cycles in North America involving a number of wild carnivore hosts, most notably raccoons (4, 5). These recent findings, along with the lack of isolation or detection of intermediate viruses between FPV and CPV from domestic animals, suggest that parvoviruses transfer frequently between domestic and wild carnivores and that the events preceding the pandemic emergence of CPV were more complex than previously believed (6).Although raccoons have long been known to be susceptible to FPV infection (7), they have only recently been identified as an important host for viruses that are closely related to CPV (4, 8). While CPVs from dogs, wolves, and coyotes all contain a Gly at capsid (VP2) position 300, CPVs from raccoons contain an Asp at that position, suggesting that this mutation is important for the adaptation of CPV to raccoons and possibly other wild carnivore hosts (4, 6). Additionally, VP2 position 300 is the most variable residue in the capsid (9-11). Since FPV and CPV capsids can bind to the transferrin receptor type 1 (TfR), in part by involving the structural region surrounding VP2 position 300 (12), the variations observed at this position appear to be selected by the unique TfR structures of individual carnivore hosts. To examine this phenomenon and to better understand the receptor-binding mechanisms involved, we used single-particle tracking (SPT) techniques to characterize the binding of raccoon-derived CPVs, containing either a 300-Asp or 300-Gly VP2 residue, to dog and raccoon TfRs.The virus studied here was the prototype CPV isolated from raccoons (CPV/Raccoon/VA/118-A/07, GenBank sequence accession number JN867610), which contains an Asp at VP2 position 300 and cannot be propagated in dog cells (4, 6). We refer to this virus as Rac118-300D. However, a single point mutation of the VP2 300-Asp (codon GAT) to a Gly (codon GGT) results in efficient dog cell infec- Citation Lee DW, Allison AB, Bacon KB, Parrish CR, Daniel S. 2016. Single-particle tracking shows that a point mutation in the carnivore parvovirus caps...

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Influenza Virus-Membrane Fusion Triggered by Proton Uncaging for Single Particle Studies of Fusion Kinetics

Cited by 41 publications

References 51 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 Tracking Shows that a Point Mutation in the Carnivore Parvovirus Capsid Switches Binding between Host-Specific Transferrin Receptors

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