Probing the paramyxovirus fusion (F) protein-refolding event from pre- to postfusion by oxidative footprinting

Poor, Taylor A.; Jones, Lisa M.; Sood, Amika; Leser, George P.; Plasencia, Manolo; Rempel, Don L.; Jardetzky, Theodore S.; Woods, Robert J.; Gross, Michael L.; Lamb, Robert A.

doi:10.1073/pnas.1408983111

Cited by 48 publications

(50 citation statements)

References 78 publications

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“…Increased flexibility in this region could potentiate premature triggering, with HRC playing a critical role in the initiation of the pre-to-postfusion transition. Of interest, a recent study showed that the N-terminal region of HRC in parainfluenza virus 5 (PIV5) is among the first sections of the protein head to move in the prefusion structure along with the fusion peptide, indicating that HRC movement may be one of the earliest events in the fusion process (31). These findings, in combination with results presented here, may point to a conserved role for this helix across the Paramyxoviridae family.…”

Section: Discussionsupporting

confidence: 72%

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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Section: Discussionsupporting

confidence: 72%

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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“…Secondly, FPOP holds promise for examining short-lived protein folding intermediates [33]. FPOP has been adopted by various researchers, and it has been applied to a range of protein systems [34][35][36][37][38][39][40][41][42][43][44][45].…”

Section: Introductionmentioning

confidence: 99%

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

Vahidi

Konermann

2016

J. Am. Soc. Mass Spectrom.

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Time (s)0Bleaching of the reporter dye cyanine-5 (Cy5) served as readout of the timedependent radical milieu. Surprisingly, Cy5 oxidation extends over tens of milliseconds. This time range is four orders of magnitude longer than expected from the FPOP literature. We demonstrate that the glutamine scavenger generates metastable secondary radicals in the FPOP solution, and that these radicals lengthen the time frame of Cy5 oxidation. Cy5 and similar dyes are widely used for monitoring the radical dose experienced by proteins in solution. The measured Cy5 kinetics thus strongly suggest that protein oxidation in FPOP extends over a much longer time window than previously thought (i.e., many milliseconds instead of one microsecond). The optical approach developed here should be suitable for assessing the performance of future FPOP-like techniques with improved temporal labeling characteristics.

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“…The attachment protein's F-activating region then interacts with an exposed hydrophobic loop in the IgG-like domain of F to provide the energy needed to promote F refolding and membrane fusion (4,5). This energy barrier to the activation of F can also be overcome by increased temperature (6)(7)(8)(9)(10) or by the introduction of destabilizing point mutations (11)(12)(13). The paramyxovirus parainfluenza virus 5 (PIV5) WR isolate differs in sequence from the W3A isolate by only three residues: the L22, P443, and A516 in WR are P22, S443, and V516 in W3A (14).…”

mentioning

confidence: 99%

On the Stability of Parainfluenza Virus 5 F Proteins

Poor

Song

Welch

et al. 2015

J Virol

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The crystal structure of the F protein (prefusion form) of the paramyxovirus parainfluenza virus 5 (PIV5) WR isolate was determined. We investigated the basis by which point mutations affect fusion in PIV5 isolates W3A and WR, which differ by two residues in the F ectodomain. The P22 stabilizing site acts through a local conformational change and a hydrophobic pocket interaction, whereas the S443 destabilizing site appears sensitive to both conformational effects and amino acid charge/polarity changes.

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Probing the paramyxovirus fusion (F) protein-refolding event from pre- to postfusion by oxidative footprinting

Cited by 48 publications

References 78 publications

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

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

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

On the Stability of Parainfluenza Virus 5 F Proteins

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