Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers

Swanson, Kurtis J.; Settembre, Ethan C.; Shaw, Christine A.; Dey, Antu; Rappuoli, Rino; Mandl, Christian W.; Dormitzer, Philip R.; Carfı́, Andrea

doi:10.1073/pnas.1106536108

Cited by 247 publications

(272 citation statements)

References 39 publications

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“…This is most noticeable in E82A, L83A, V90A, L93A, and, to a lesser extent, in Y86A and L96A, where a larger portion of the protein present is in the 70-kDa F 0 form that is not cleaved at either of the two cleavage sites. (14,15). Two protomers are shown in two shades of gray with surface representation, while the third is shown as a ribbon with coloring as described for panel A. TM, transmembrane region.…”

Section: Resultsmentioning

confidence: 99%

“…While pre-and postfusion forms of F have been crystallized, many of the specific events that underlie the process are still unknown (14,15). Since HRA and HRB have been extensively studied due to their role in the formation of the critical 6HB, we shifted our focus to a heptad repeat in the F 2 subunit (aa 75 to 97), referred to herein as HRC, which has remained largely uncharacterized in RSV.…”

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confidence: 99%

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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: Resultsmentioning

confidence: 99%

mentioning

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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“…The search for new hRSV vaccines has been reinvigorated by recent advances in structure‐based design of soluble hRSV F proteins folded in either the prefusion (McLellan et al , 2013a; Krarup et al , 2015) or postfusion conformations (McLellan et al , 2011b; Swanson et al , 2011). Stabilized soluble forms of prefusion hRSV F were shown to induce higher levels of neutralizing antibodies than soluble postfusion hRSV F in mice, cotton rats, and non‐human primates (McLellan et al , 2013a; Krarup et al , 2015; Palomo et al , 2016).…”

Section: Introductionmentioning

confidence: 99%

“…Stabilized soluble forms of prefusion hRSV F were shown to induce higher levels of neutralizing antibodies than soluble postfusion hRSV F in mice, cotton rats, and non‐human primates (McLellan et al , 2013a; Krarup et al , 2015; Palomo et al , 2016). However, postfusion F, which has the advantage of being highly stable, can induce sizeable levels of neutralizing antibodies and afford protection against hRSV because it shares certain epitopes with prefusion F (Swanson et al , 2011). Recently, the structure of a soluble form of postfusion hMPV F was determined, revealing extensive similarity with postfusion hRSV F despite having only ~38% sequence identity (Mas et al , 2016).…”

Section: Introductionmentioning

confidence: 99%

Chimeric Pneumoviridae fusion proteins as immunogens to induce cross‐neutralizing antibody responses

et al. 2017

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Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV), two members of the Pneumoviridae family, account for the majority of severe lower respiratory tract infections worldwide in very young children. They are also a frequent cause of morbidity and mortality in the elderly and immunocompromised adults. High levels of neutralizing antibodies, mostly directed against the viral fusion (F) glycoprotein, correlate with protection against either hRSV or hMPV. However, no cross‐neutralization is observed in polyclonal antibody responses raised after virus infection or immunization with purified F proteins. Based on crystal structures of hRSV F and hMPV F, we designed chimeric F proteins in which certain residues of well‐characterized antigenic sites were swapped between the two antigens. The antigenic changes were monitored by ELISA with virus‐specific monoclonal antibodies. Inoculation of mice with these chimeras induced polyclonal cross‐neutralizing antibody responses, and mice were protected against challenge with the virus used for grafting of the heterologous antigenic site. These results provide a proof of principle for chimeric fusion proteins as single immunogens that can induce cross‐neutralizing antibody and protective responses against more than one human pneumovirus.

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“…High-resolution structures of several paramyxovirus F proteins have been solved in both the prefusion (8,18) and postfusion (19)(20)(21) conformations. In the prefusion form, the HR domain adjacent to the fusion peptide, HR-A, is broken up into multiple distinct sections forming the upper part of the prefusion Fprotein head structure, and the membrane-proximal HR-B domains assemble into a short, three-helix bundle, the prefusion F-protein stalk.…”

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confidence: 99%

Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain

Brindley¹,

Plattet²,

Plemper³

2014

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

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Enveloped viruses such as HIV and members of the paramyxovirus family use metastable, proteinaceous fusion machineries to merge the viral envelope with cellular membranes for infection. A hallmark of the fusogenic glycoproteins of these pathogens is refolding into a thermodynamically highly stable fusion core structure composed of six antiparallel α-helices, and this structure is considered instrumental for pore opening and/or enlargement. Using a paramyxovirus fusion (F) protein, we tested this paradigm by engineering covalently restricted F proteins that are predicted to be unable to close the six-helix bundle core structure fully. Several candidate bonds formed efficiently, resulting in F trimers and higher-order complexes containing covalently linked dimers. The engineered F complexes were incorporated into recombinant virions efficiently and were capable of refolding into a postfusion conformation without temporary or permanent disruption of the disulfide bonds. They efficiently formed fusion pores based on virus replication and quantitative cell-to-cell and virus-to-cell fusion assays. Complementation of these F mutants with a monomeric, fusion-inactive F variant enriched the F oligomers for heterotrimers containing a single disulfide bond, without affecting fusion complementation profiles compared with standard F protein. Our demonstration that complete closure of the fusion core does not drive paramyxovirus entry may aid the design of strategies for inhibiting virus entry. membrane fusion | measles virus M embrane fusion is an essential process in eukaryotic cell biology. Examples range from fusion of lipid vesicles to sustain intracellular transport along secretory and endocytotic pathways to cell fusion in fertilization and development and to the fusion of viral with cellular membranes resulting in viral infection. Because there are several high-energy barriers (1), lipid membranes do not merge spontaneously under physiological conditions and therefore require the aid of auxiliary proteins to induce membrane stress and lower the activation energy for lipid merger. For vesicular transport, for instance, soluble N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE) proteins resident in both the target and donor membrane mediate the process (2). Two fundamental differences distinguish cellular fusion machineries from viral fusogenic membrane glycoproteins (vFMGs), which mediate the entry of enveloped viruses by the fusion of the envelope with cellular membranes: (i) vFMGs do not require adapter proteins but insert a fusion peptide domain into the target membrane; and (ii) vFMGs fold into metastable prefusion conformations, obviating the dependence on external energy sources. Three distinct types of viral fusion (F) proteins have been classified: class I vFMGs are found, among others, on influenza virus, retroviruses, and paramyxoviruses; class II proteins are present on alpha-and flaviviruses; and class III proteins mediate herpes-and rhabdovirus entry (reviewed in ref.3).Once the fusion p...

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Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers

Cited by 247 publications

References 39 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

Chimeric Pneumoviridae fusion proteins as immunogens to induce cross‐neutralizing antibody responses

Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain

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