Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers.

Danieli, Tsafi; Pelletier, Sandra; Henis, Yoav I.; White, Judith M.

doi:10.1083/jcb.133.3.559

Cited by 328 publications

(382 citation statements)

References 69 publications

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“…It may represent the time required to form a fusion pore and deliver the luciferase into the cell cytoplasm and then access its substrates. Indeed, the data are consistent with the relatively slow formation of the fusion pore seen for other enveloped viruses such as influenza A virus and VSV, which can take many minutes to grow large enough to release the virus capsid (8,21). However, the lag may also indicate that cellular signaling, reorganization, or trafficking events may be required for entry, as suggested for HIV (13).…”

supporting

confidence: 74%

“…This is principally because, for these viruses, it is possible to induce an en masse fusion event by dropping the pH of the medium. Under these circumstances, fluorescence dequenching, or FRET (fluorescence resonance energy transfer), assays effectively measure the kinetics of fusion and have been used to understand the effects of mutations and antiviral drugs (3,8). For this, fluorescent probes incorporated into the virus membrane mix with and become diluted into the target cell or liposome membrane.…”

mentioning

confidence: 99%

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Rapid and Sensitive Detection of Retrovirus Entry by Using a Novel Luciferase-Based Content-Mixing Assay

Kolokoltsov

Davey

2004

J Virol

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We describe a novel assay that permits measurement of entry of murine leukemia virus and pseudotypes with greater sensitivity and more rapidly than previously possible. To achieve this, we encapsulated a sensitive reporter enzyme, luciferase, directly into fully infectious, intact viral particles. The enzyme is specifically targeted to the viral lumen, as a C-terminal fusion on the viral envelope protein. Only when the incorporated luciferase is released from the viral lumen and gains access to its substrates is light emitted and readily detected. When cells are perfused with luciferin, quantitative measurements of entry can be made in real time on live cells. Uniquely, the amount of cell-bound virus can be determined in the same assay by addition of detergent to expose the luciferase. We demonstrate that virus carrying a mutation in the fusion peptide binds normally to cells but is unable to infect them and gives no entry signal. Using this assay, we show that inhibitors of endosomal acidification inhibit signal from vesicular stomatitis virus pseudotypes but not murine leukemia virus, consistent with a pH-independent mode of entry for the latter virus. Additionally, the fusion kinetics are rapid, with a half-life of 25 min after a delay of 10 to 15 min. The future use of this assay will permit a detailed examination of the entry mechanism of viruses and provide a convenient platform to discover novel entry inhibitors. The design also permits packaging of potential therapeutic protein cargoes into functional virus particles and their specific delivery to cellular targets.

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supporting

confidence: 74%

mentioning

confidence: 99%

Rapid and Sensitive Detection of Retrovirus Entry by Using a Novel Luciferase-Based Content-Mixing Assay

Kolokoltsov

Davey

2004

J Virol

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“…Furthermore, using polypeptide cross-linking agents and sulfhydryl group reagents, Markovic et al (1998) demonstrated that GP64 forms transient, higher-order complexes of trimers upon application of low pH buffer (only in the presence of target membrane) at the same time that fusion is catalyzed. The ring hypothesis is further supported by work with influenza HA, which suggests that HA trimers function cooperatively and inhibit lipid dye transmigration during the initial fusion phases (Ellens et al, 1990;Clague et al, 1991;Tse et al, 1993;Zimmerberg et al, 1994;Blumenthal et al, 1996;Danieli et al, 1996;Chernomordik et al, 1998).…”

Section: The Mechanism Of Membrane Fusionmentioning

confidence: 59%

A Discrete Stage of Baculovirus GP64-mediated Membrane Fusion

Kingsley

Behbahani

Rashtian

et al. 1999

MBoC

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Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH-induced conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific peptide sequences of GP64 that are adjacent to and include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing. INTRODUCTIONThe mechanism of membrane fusion is currently a field of intense investigation, both for intracellular trafficking and enveloped viral infection. For viral fusion protein function, we have previously suggested that fusion protein oligomers form transient and cooperative higher-order structures or rings . These transient ring structures are proposed to facilitate conversion of kinetic energy, released during protein conformational change, into work on the target and host membranes required for membrane fusion. Indeed, biochemical evidence for baculovirus GP64 aggregates during membrane fusion has recently been reported (Markovic et al. 1998). Whether and how viral fusion proteins might function in a coordinated and cooperative manner is a major question in the field of membrane fusion. Because hydrophobic 4-3 heptad repeats are observed...

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“…A working model for the process of SARS CoV virion/ cell fusion can be extrapolated from this work and from work in several other RNA virus systems (10,22,24,29,42,46,82). After the fusion protein binds to the cellular receptor, the fusion protein undergoes a series of rearrangements, including the release of the receptor-binding subunit (S1) and trimerization and extension of the N helices of the S2 subunit toward the target membrane.…”

Section: Discussionmentioning

confidence: 99%

The Aromatic Domain of the Coronavirus Class I Viral Fusion Protein Induces Membrane Permeabilization: Putative Role during Viral Entry

et al. 2004

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Coronavirus (CoV) entry is mediated by the viral spike (S) glycoprotein, a class I viral fusion protein. During viral and target cell membrane fusion, the heptad repeat (HR) regions of the S2 subunit assume a trimer-of-hairpins structure, positioning the fusion peptide in close proximity to the C-terminal region of the ectodomain. The formation of this structure appears to drive apposition and subsequent fusion of viral and target cell membranes; however, the exact mechanism is unclear. Here, we characterize an aromatic amino acid rich region within the ectodomain of the S2 subunit that both partitions into lipid membranes and has the capacity to perturb lipid vesicle integrity. Circular dichroism analysis indicated that peptides analogous to the aromatic domains of the severe acute respiratory syndrome (SARS)-CoV, mouse hepatitis virus (MHV) and the human CoV OC43 S2 subunits, did not have a propensity for a defined secondary structure. These peptides strongly partitioned into lipid membranes and induced lipid vesicle permeabilization at peptide/lipid ratios of 1:100 in two independent leakage assays. Thus, partitioning of the peptides into the lipid interface is sufficient to disorganize membrane integrity. Our study of the S2 aromatic domain of three CoVs provides supportive evidence for a functional role of this region. We propose that, when aligned with the fusion peptide and transmembrane domains during membrane apposition, the aromatic domain of the CoV S protein functions to perturb the target cell membrane and provides a continuous track of hydrophobic surface, resulting in lipid-membrane fusion and subsequent viral nucleocapsid entry.

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Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers.

Cited by 328 publications

References 69 publications

Rapid and Sensitive Detection of Retrovirus Entry by Using a Novel Luciferase-Based Content-Mixing Assay

Rapid and Sensitive Detection of Retrovirus Entry by Using a Novel Luciferase-Based Content-Mixing Assay

A Discrete Stage of Baculovirus GP64-mediated Membrane Fusion

The Aromatic Domain of the Coronavirus Class I Viral Fusion Protein Induces Membrane Permeabilization: Putative Role during Viral Entry

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