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. 2019 Feb 5;93(4):e01597-18.
doi: 10.1128/JVI.01597-18. Print 2019 Feb 15.

A 2.8-Angstrom-Resolution Cryo-Electron Microscopy Structure of Human Parechovirus 3 in Complex with Fab from a Neutralizing Antibody

Aušra Domanska  1   2 , Justin W Flatt  3   2 , Joonas J J Jukonen  3   2 , James A Geraets  3   2 , Sarah J Butcher  1   2
Affiliations

A 2.8-Angstrom-Resolution Cryo-Electron Microscopy Structure of Human Parechovirus 3 in Complex with Fab from a Neutralizing Antibody

Aušra Domanska et al. J Virol. .

Abstract

Human parechovirus 3 (HPeV3) infection is associated with sepsis characterized by significant immune activation and subsequent tissue damage in neonates. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and the lack of a vaccine or specific antiviral therapy. Toward the latter, we present a 2.8-Å-resolution structure of HPeV3 in complex with fragments from a neutralizing human monoclonal antibody, AT12-015, using cryo-electron microscopy (cryo-EM) and image reconstruction. Modeling revealed that the epitope extends across neighboring asymmetric units with contributions from capsid proteins VP0, VP1, and VP3. Antibody decoration was found to block binding of HPeV3 to cultured cells. Additionally, at high resolution, it was possible to model a stretch of RNA inside the virion and, from this, identify the key features that drive and stabilize protein-RNA association during assembly.IMPORTANCE Human parechovirus 3 (HPeV3) is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, for which, despite the severity of disease, there are no effective treatments available. Structural and molecular insights into virus neutralization are urgently needed, especially as clinical cases are on the rise. Toward this goal, we present the first structure of HPeV3 in complex with fragments from a neutralizing monoclonal antibody. At high resolution, it was possible to precisely define the epitope that, when targeted, prevents virions from binding to cells. Such an atomic-level description is useful for understanding host-pathogen interactions and viral pathogenesis mechanisms and for finding potential cures for infection and disease.

Keywords: cryo-EM; genome packaging; human parechovirus 3; neutralizing antibodies; picornavirus.

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Figures

FIG 1
FIG 1
Resolution assessment for the HPeV3-Fab complex. (A) “Gold standard” Fourier shell correlation (FSC) curves showing an overall nominal resolution at 2.8 Å. (B) Central cross-section of the three-dimensional density map alongside the structure colored according to local resolution (Å); 2-, 3-, and 5-fold symmetry axes are labeled by the oval, triangle, and pentagon, respectively.
FIG 2
FIG 2
Visualization of Fab-decorated HPeV3. Virus-Fab reconstruction shown radially color coded (purple, 100 Å; yellow, 120 Å; light blue, 140 Å; blue, 145 Å; and orange, 150 Å). (A) Surface view down a 2-fold axis of symmetry. Propeller regions can be seen as a dark blue triangles on the capsid surface, the Fabs are orange. (B) Cutaway view showing RNA (purple) at 5-fold vertices inside the viral capsid. (C) Central cross-section with well-defined layers of density corresponding to RNA, capsid, and Fab. (D) Side chains for viral coat proteins VP0 (blue), VP1 (green), and VP3 (light blue), as well as Fab heavy and light chains, VH (orange) and VL (light brown), respectively. (E) Modeled asymmetric unit with a Fab molecule bound. (F) High resolution at the Fab AT12-015-HPeV3 interface. (G) RNA anchoring on the inner surface of the virus is mediated by a tryptophan (Trp 24) residue from VP3.
FIG 3
FIG 3
Interactions between Fab AT12-015 and HPeV3. (A) Fab binds to an epitope extended across neighboring asymmetric units in the assembled virion. Viral capsid residues that participate in Fab heavy (VH) and light (VL) chain binding are highlighted in yellow and are reported in the accompanying table. (B) Stabilizing interactions at the interface. Residues that form hydrogen bonds or a salt bridge are highlighted in yellow on the left, and colored by chain on the enlarged inset on the right. In the inset, hydrogen bonds are shown as black dashed lines along with a centrally located salt bridge highlighted in red.
FIG 4
FIG 4
Antibody AT12-015 blocks virus binding to HT29 cells. (A) Representative fluorescence images of HPeV3 incubated in the presence or absence of various amounts of AT12-015 antibody. Cell nuclei were visualized using a Hoechst stain (blue), and bound virus was scored by measuring Alexa Fluor 488 intensity (green). Pictures were acquired using a 20× objective. Scale bar, 50 μm. (B) Effect of preincubation of HPeV3 with different amounts of human monoclonal antibody AT12-015. The results are the averages from three repeats of the cold binding assay. The error bars represent the standard errors of the means (SEMs). (C) AM28 had no effect on HPeV3 binding to HT29 cells. Representative fluorescence images of HPeV3 incubated in the presence (3 μg/ml) or absence (virus no AM28 preincubation) of AM28 antibody and added to the cells for binding. Noninfected cells (mock and AM28 no virus) served as the controls. Stained as in panel A and visualized with a 10× objective. Scale bar, 50 μm.
FIG 5
FIG 5
Ordered RNA inside the HPeV3 virion. (A) RNA density segmented from within the virion seen along an icosahedral 5-fold axis. The boxed segment of the density is enlarged in panel B. (B) Eight nucleotides fit to their corresponding density before reaching the helix-coil transition. (C) VP3 tails bridge two adjacent loops of RNA to promote efficient packaging and assembly. Small portion of the capsid VP3 sequence (amino acids Leu 16 to Arg 26) is shown to clarify the VP3-RNA network on the inner surface of the viral capsid. (D) The binding pocket for RNA on the inside of the capsid involves residues from VP1 and VP3. One RNA loop is stabilized by residues from a single VP1 chain (green) and three VP3 chains (light blue designated by ′, ′′, and ′′′).
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