Elevated temperature triggers human respiratory syncytial virus F protein six-helix bundle formation

Yunus, Abdul S.; Jackson, Trent P.; Crisafi, Katherine; Burimski, Irina; Kilgore, Nicole; Zoumplis, Dorian; Allaway, Graham P.; Wild, Carl; Salzwedel, Karl

doi:10.1016/j.virol.2009.10.040

Cited by 37 publications

(35 citation statements)

References 64 publications

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“…The addition of heat has been reported to be a surrogate trigger for many full-length paramyxovirus fusion proteins (11,29,34,42,45). The mechanism probably involves thermal destabilization of the metastable F protein, initiating its conformational change.…”

Section: Discussionmentioning

confidence: 99%

“…The mechanism probably involves thermal destabilization of the metastable F protein, initiating its conformational change. Yunis et al (45) found that heating RSVinfected cells to 45 to 55°C enhanced antibody recognition of the membrane-anchored F protein by antiserum specific for the posttriggered 6-HB structure, indicating that elevated temperature can act as a surrogate trigger for the transmembrane RSV F protein. Raising the temperature to 60°C has also been shown to act as a surrogate trigger for the PIV5 sF protein, resulting in liposome association (11).…”

Section: Discussionmentioning

confidence: 99%

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Soluble Respiratory Syncytial Virus Fusion Protein in the Fully Cleaved, Pretriggered State Is Triggered by Exposure to Low-Molarity Buffer

Chaiwatpongsakorn

Epand

Collins

et al. 2011

J Virol

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The paramyxovirus fusion (F) glycoprotein is anchored in the virion membrane in a metastable, pretriggered form. Once triggered, the F protein undergoes a dramatic conformational extension that inserts its hydrophobic fusion peptide into the target cell membrane, then folds back on itself to bring the membranes together and initiate fusion. Unlike most other paramyxoviruses, the respiratory syncytial virus (RSV) F protein alone is sufficient to mediate membrane fusion and virus infection. To study the triggering mechanism of the RSV F protein, we have generated a soluble F (sF) protein by replacing the transmembrane and cytoplasmic tail domains with a 6His tag. The sF protein is secreted efficiently from 293T cells in a fully cleaved form. It is recognized by neutralizing monoclonal antibodies, appears spherical by electron microscopic analysis, and is not aggregated, all consistent with a native, pretriggered trimer. The sF protein was purified on a Ni ؉2 column and eluted with 50 mM phosphate buffer containing 500 mM NaCl and 250 mM imidazole. Dialysis against 10 mM buffer caused the sF protein to trigger, forming "hat pin"-shaped molecules that aggregated as rosettes, characteristic of the posttriggered form. Further dialysis experiments indicated that the efficiency of triggering correlated well with the reduction of buffer molarity. Reduction of buffer molarity by dilution also resulted in exposure of the fusion peptide, as detected by liposome association, confirming sF protein triggering. Mutation of the furin cleavage site adjacent to the fusion peptide prevented liposome association, further confirming that association is via the fusion peptide.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Soluble Respiratory Syncytial Virus Fusion Protein in the Fully Cleaved, Pretriggered State Is Triggered by Exposure to Low-Molarity Buffer

Chaiwatpongsakorn

Epand

Collins

et al. 2011

J Virol

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“…This refolding can also occur spontaneously on virions and cell surfaces 11 , and it can be stimulated with heat 12 , indicating that prefusion RSV F is metastable. During the refolding, the hydrophobic fusion peptide at the N terminus of the F 1 subunit pulls out from the central cavity of the prefusion trimer and inserts into the host-cell membrane 7 .…”

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confidence: 98%

Molecular mechanism of respiratory syncytial virus fusion inhibitors

Battles

Langedijk

Furmanova-Hollenstein

et al. 2015

Nat Chem Biol

128

171

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Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. Here we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitors or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors.

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“…This transition is irreversible under physiological conditions and renders the fusion protein inactive. Heat, low-molarity buffer, and freeze-thaw cycles have all been shown to cause this unidirectional conformation change (17,19,20). Recently, a number of conformation-specific antibodies have been characterized.…”

Section: Fig 2 Expression and Processing Of Hrc Mutations Transientlmentioning

confidence: 99%

The Heptad Repeat C Domain of the Respiratory Syncytial Virus Fusion Protein Plays a Key Role in Membrane Fusion

Bermingham

Chappell

Watterson

et al. 2018

J Virol

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Respiratory syncytial virus (RSV) mediates host cell entry through the fusion (F) protein, which undergoes a conformational change to facilitate the merger of viral and host lipid membrane envelopes. The RSV F protein comprises a trimer of disulfide-bonded F 1 and F 2 subunits that is present on the virion surface in a metastable prefusion state. This prefusion form is readily triggered to undergo refolding to bring two heptad repeats (heptad repeat A [HRA] and HRB) into close proximity to form a six-helix bundle that stabilizes the postfusion form and provides the free energy required for membrane fusion. This process can be triggered independently of other proteins. Here, we have performed a comprehensive analysis of a third heptad repeat region, HRC (amino acids 75 to 97), an amphipathic ␣-helix that lies at the interface of the prefusion F trimer and is a major structural feature of the F 2 subunit. We performed alanine scanning mutagenesis from Lys-75 to Met-97 and assessed all mutations in transient cell culture for expression, proteolytic processing, cell surface localization, protein conformation, and membrane fusion. Functional characterization revealed a striking distribution of activity in which fusion-increasing mutations localized to one side of the helical face, while fusion-decreasing mutations clustered on the opposing face. Here, we propose a model in which HRC plays a stabilizing role within the globular head for the prefusion F trimer and is potentially involved in the early events of triggering, prompting fusion peptide release and transition into the postfusion state.IMPORTANCE RSV is recognized as the most important viral pathogen among pediatric populations worldwide, yet no vaccine or widely available therapeutic treatment is available. The F protein is critical for the viral replication process and is the major target for neutralizing antibodies. Recent years have seen the development of prefusion stabilized F protein-based approaches to vaccine design. A detailed understanding of the specific domains and residues that contribute to protein stability and fusion function is fundamental to such efforts. Here, we present a comprehensive mutagenesis-based study of a region of the RSV F 2 subunit (amino acids 75 to 97), referred to as HRC, and propose a role for this helical region in maintaining the delicate stability of the prefusion form.KEYWORDS respiratory syncytial virus, fusion, membrane fusion, mutagenesis, heptad repeat R espiratory syncytial virus (RSV) is widely considered the most significant viral pediatric pathogen worldwide. Belonging to the Pneumovirinae family, RSV causes substantial morbidity and mortality worldwide, with an estimated 33.8 million acute lower respiratory tract infections per year in children under 5 years of age, and has been linked to almost 200,000 deaths annually, 99% of which are in developing countries (1, 2). RSV is also recognized as an important pathogen of high-risk adult and elderly populations (3). Despite this, no targeted drug or vaccin...

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Elevated temperature triggers human respiratory syncytial virus F protein six-helix bundle formation

Cited by 37 publications

References 64 publications

Soluble Respiratory Syncytial Virus Fusion Protein in the Fully Cleaved, Pretriggered State Is Triggered by Exposure to Low-Molarity Buffer

Soluble Respiratory Syncytial Virus Fusion Protein in the Fully Cleaved, Pretriggered State Is Triggered by Exposure to Low-Molarity Buffer

Molecular mechanism of respiratory syncytial virus fusion inhibitors

The Heptad Repeat C Domain of the Respiratory Syncytial Virus Fusion Protein Plays a Key Role in Membrane Fusion

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