Structure and immunogenicity of pre-fusion-stabilized human metapneumovirus F glycoprotein

Battles, Michael B.; Más, Vicente; Olmedillas, Eduardo; Cano, Olga; Vázquez, Mónica; Laura, Rodriguez; Melero, José A.; McLellan, Jason S.

doi:10.1038/s41467-017-01708-9

Cited by 98 publications

(199 citation statements)

References 70 publications

(106 reference statements)

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“…A common approach to achieve prefusion stabilization has been through introduction of one or more proline residues within the loop of a helix-loop-helix motif that refolds into a continuous alpha helix in the postfusion conformation. This strategy has been used successfully to stabilize several class I viral fusion glycoproteins including RSV F, MPV F, influenza HA, HIV Env, and coronavirus Spike [41,[57][58][59][60]. Proline-based stabilization utilizes the restricted phi-psi angles of proline residues and disruption of the alpha-helix hydrogen bond network to inhibit the hinge motion required of the helix-loop-helix motif and the subsequent coil-to-helix structural transition required for refolding into the postfusion conformation.…”

Section: Discussionmentioning

confidence: 99%

Alternative conformations of a major antigenic site on RSV F

et al. 2019

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The respiratory syncytial virus (RSV) fusion (F) glycoprotein is a major target of neutralizing antibodies arising from natural infection, and antibodies that specifically bind to the prefusion conformation of RSV F generally demonstrate the greatest neutralization potency. Prefusion-stabilized RSV F variants have been engineered as vaccine antigens, but crystal structures of these variants have revealed conformational differences in a key antigenic site located at the apex of the trimer, referred to as antigenic site Ø. Currently, it is unclear if flexibility in this region is an inherent property of prefusion RSV F or if it is related to inadequate stabilization of site Ø in the engineered variants. Therefore, we set out to investigate the conformational flexibility of antigenic site Ø, as well as the ability of the human immune system to recognize alternative conformations of this site, by determining crystal structures of prefusion RSV F bound to neutralizing human-derived antibodies AM22 and RSD5. Both antibodies bound with high affinity and were specific for the prefusion conformation of RSV F. Crystal structures of the complexes revealed that the antibodies recognized distinct conformations of antigenic site Ø, each diverging at a conserved proline residue located in the middle of an α-helix. These data suggest that antigenic site Ø exists as an ensemble of conformations, with individual antibodies recognizing discrete states. Collectively, these results have implications for the refolding of pneumovirus and paramyxovirus fusion proteins and should inform development of prefusion-stabilized RSV F vaccine candidates.

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Section: Discussionmentioning

confidence: 99%

Alternative conformations of a major antigenic site on RSV F

et al. 2019

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“…Reflecting major efforts to identify a cost-effective alternative to high-price passive immunization with anti-RSV nAbs, a number of compounds have entered advanced preclinical development and clinical testing in recent years (Table 1). Breakthroughs in the structural and functional characterization of the viral entry machinery and polymerase complexes in the past decade [12][13][14][15][16][17][18][19][20][21] have furthermore created a novel opportunity for structure-informed mechanistic characterization and ligand optimization.…”

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confidence: 99%

Structural Insight into Paramyxovirus and Pneumovirus Entry Inhibition

Aggarwal

Plemper

2020

Viruses

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Paramyxoviruses and pneumoviruses infect cells through fusion (F) protein-mediated merger of the viral envelope with target membranes. Members of these families include a range of major human and animal pathogens, such as respiratory syncytial virus (RSV), measles virus (MeV), human parainfluenza viruses (HPIVs), and highly pathogenic Nipah virus (NiV). High-resolution F protein structures in both the metastable pre-and the postfusion conformation have been solved for several members of the families and a number of F-targeting entry inhibitors have progressed to advanced development or clinical testing. However, small-molecule RSV entry inhibitors have overall disappointed in clinical trials and viral resistance developed rapidly in experimental settings and patients, raising the question of whether the available structural information may provide a path to counteract viral escape through proactive inhibitor engineering. This article will summarize current mechanistic insight into F-mediated membrane fusion and examine the contribution of structural information to the development of small-molecule F inhibitors. Implications are outlined for future drug target selection and rational drug engineering strategies.Viruses 2020, 12, 342 2 of 16 from its natural bat host to domestic animals, resulting in case/fatality rates reaching from 40% to over 90% [9]. Continued spillover into the human population must be expected, and human-to-human transmission through respiratory secretions, urine, and saliva has been documented [10].Of the pneumoviruses, RSV infects almost all children before two years of age and is responsible for over 100,000 hospitalizations yearly in the United States alone. The monoclonal antibody palivizumab has been approved for immunoprophylaxis against RSV infection [11], however, use is restricted to high-risk patients due to the high-cost and need for prophylactic administration.Given the health, medical and economic burden associated with paramyxo-and pneumovirus infections, a major and currently unmet clinical need exists to expedite the development of novel safe and effective therapeutics for improved disease management and outbreak control. Current direct-acting therapeutics predominantly focus on preventing viral entry through neutralizing antibodies (nAbs) and on small-molecules targeting the envelope glycoproteins or inhibiting the viral RNA-dependent RNA polymerase (RdRP) complex. Reflecting major efforts to identify a cost-effective alternative to high-price passive immunization with anti-RSV nAbs, a number of compounds have entered advanced preclinical development and clinical testing in recent years (Table 1). Breakthroughs in the structural and functional characterization of the viral entry machinery and polymerase complexes in the past decade [12][13][14][15][16][17][18][19][20][21] have furthermore created a novel opportunity for structure-informed mechanistic characterization and ligand optimization.

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“…The syncytins are also reminiscent of the paramyxoviruses and pneumoviruses, which also induce fusion of the viral envelope with the plasma membranes. These viral fusion proteins all required mutagenesis to maintain them in the pre-fusion form to maintain conformational epitopes to use as immunogens and to carry out structural studies [49,50]. An initial structure of the pre-fusion form, however, guided those trials.…”

Section: Discussionmentioning

confidence: 99%

“…explored the effect of introducing helix-breaking residues (prolines) along the whole length of the Nhelix, with the goal of identifying positions that may not interfere with folding of the syncytins in their prefusion conformation. Such an approach was applied successfully to stabilize the pre-fusion state of HIV-1 Env [28] and later of other class I proteins, such as the spike protein of the Middle East respiratory syndromeerelated coronavirus [48] or the fusion (F) protein of the human respiratory syncytial virus as well as the human metapneumovirus [49,50].…”

Section: Potential Break Points Of the N-helix In The Prefusion Formmentioning

confidence: 99%

X-ray Structures of the Post-fusion 6-Helix Bundle of the Human Syncytins and their Functional Implications

Ruigrok

Vaney

Buchrieser

et al. 2019

Journal of Molecular Biology

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The retroviral envelope-derived proteins syncytin-1 and syncytin-2 (syn1 and syn2) drive placentation in humans by forming a syncytiotophoblast, a structure allowing for an exchange interface between maternal and fetal blood during pregnancy. Despite their essential role, little is known about the molecular mechanism underlying the syncytins' function. We report here the X-ray structures of the syn1 and syn2 transmembrane subunit ectodomains, featuring a 6-helix bundle (6HB) typical of the post-fusion state of gamma-retrovirus and filovirus fusion proteins. Contrary to the filoviruses, for which the fusion glycoprotein was crystallized both in the post-fusion and in the spring-loaded pre-fusion form, the highly unstable nature of the syncytins' prefusion form has precluded structural studies. We undertook a proline-scanning approach searching for regions in the syn1 6HB central helix that tolerate the introduction of helix-breaker residues and still fold correctly in the pre-fusion form. We found that there is indeed such a region, located two a-helical turns downstream a stutter in the central coiled-coil helix -precisely where the breaks of the spring-loaded helix of the filoviruses map. These mutants were fusion-inactive as they cannot form the 6HB, similar to the "SOSIP" mutant of HIV Env that allowed the high-resolution structural characterization of its labile pre-fusion form.These results now open a new window of opportunity to engineer more stable variants of the elusive pre-fusion trimer of the syncytins and other gamma-retroviruses envelope proteins for structural characterization.

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Structure and immunogenicity of pre-fusion-stabilized human metapneumovirus F glycoprotein

Cited by 98 publications

References 70 publications

Alternative conformations of a major antigenic site on RSV F

Alternative conformations of a major antigenic site on RSV F

Structural Insight into Paramyxovirus and Pneumovirus Entry Inhibition

X-ray Structures of the Post-fusion 6-Helix Bundle of the Human Syncytins and their Functional Implications

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