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- EMDB-2761: Structural Basis of Human Parechovirus Neutralization by Human Mo... -

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Basic information

Entry
Database: EMDB / ID: EMD-2761
TitleStructural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies
Map dataReconstruction of human parechovirus 1 in complex with Fab fragments of AM28 human monoclonal antibody
Sample
  • Sample: Human parechovirus in complex with Fab fragment of human monoclonal antibody AM28
  • Virus: Human parechovirus 1
  • Protein or peptide: human monoclonal antibodyMonoclonal antibody
KeywordsHuman parechovirus 1 / AM28 / human monoclonal antibody / HPeV1-AM28 Fab
Function / homology
Function and homology information


host cell nucleolus / host cell Golgi membrane / ribonucleoside triphosphate phosphatase activity / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / : / nucleoside-triphosphate phosphatase / protein complex oligomerization / monoatomic ion channel activity ...host cell nucleolus / host cell Golgi membrane / ribonucleoside triphosphate phosphatase activity / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / : / nucleoside-triphosphate phosphatase / protein complex oligomerization / monoatomic ion channel activity / clathrin-dependent endocytosis of virus by host cell / RNA helicase activity / host cell endoplasmic reticulum membrane / induction by virus of host autophagy / RNA-directed RNA polymerase / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / DNA-templated transcription / structural molecule activity / virion attachment to host cell / proteolysis / RNA binding / ATP binding / membrane / metal ion binding
Similarity search - Function
Viral polyprotein, parechovirus P3B / Parechovirus Genome-linked protein / Viral polyprotein, parechovirus P3A / Picornaviridae P3A protein / LRAT domain profile. / LRAT domain / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / Picornavirales 3C/3C-like protease domain / Picornavirales 3C/3C-like protease domain profile. / Peptidase C3A/C3B, picornaviral ...Viral polyprotein, parechovirus P3B / Parechovirus Genome-linked protein / Viral polyprotein, parechovirus P3A / Picornaviridae P3A protein / LRAT domain profile. / LRAT domain / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / Picornavirales 3C/3C-like protease domain / Picornavirales 3C/3C-like protease domain profile. / Peptidase C3A/C3B, picornaviral / 3C cysteine protease (picornain 3C) / Picornavirus capsid / picornavirus capsid protein / Helicase, superfamily 3, single-stranded RNA virus / Superfamily 3 helicase of positive ssRNA viruses domain profile. / Helicase, superfamily 3, single-stranded DNA/RNA virus / RNA helicase / Picornavirus/Calicivirus coat protein / Viral coat protein subunit / RNA-directed RNA polymerase, C-terminal domain / Viral RNA-dependent RNA polymerase / Reverse transcriptase/Diguanylate cyclase domain / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesHomo sapiens (human) / Human parechovirus 1
Methodsingle particle reconstruction / cryo EM / Resolution: 19.76 Å
AuthorsShakeel S / Westerhuis BM / Ora A / Koen G / Bakker A / Claassen Y / Beaumont T / Wolthers K / Butcher SJ
CitationJournal: J Virol / Year: 2015
Title: Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies.
Authors: Shabih Shakeel / Brenda M Westerhuis / Ari Ora / Gerrit Koen / Arjen Q Bakker / Yvonne Claassen / Koen Wagner / Tim Beaumont / Katja C Wolthers / Sarah J Butcher /
Abstract: Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has ...Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases and has given moderate success. Direct inhibition of parechovirus infection using monoclonal antibodies is a potential treatment. We have developed two neutralizing monoclonal antibodies against HPeV1 and HPeV2, namely, AM18 and AM28, which also cross-neutralize other viruses. Here, we present the mapping of their epitopes using peptide scanning, surface plasmon resonance, fluorescence-based thermal shift assays, electron cryomicroscopy, and image reconstruction. We determined by peptide scanning and surface plasmon resonance that AM18 recognizes a linear epitope motif including the arginine-glycine-aspartic acid on the C terminus of capsid protein VP1. This epitope is normally used by the virus to attach to host cell surface integrins during entry and is found in 3 other viruses that AM18 neutralizes. Therefore, AM18 is likely to cause virus neutralization by aggregation and by blocking integrin binding to the capsid. Further, we show by electron cryomicroscopy, three-dimensional reconstruction, and pseudoatomic model fitting that ordered RNA interacts with HPeV1 VP1 and VP3. AM28 recognizes quaternary epitopes on the capsid composed of VP0 and VP3 loops from neighboring pentamers, thereby increasing the RNA accessibility temperature for the virus-AM28 complex compared to the virus alone. Thus, inhibition of RNA uncoating probably contributes to neutralization by AM28.
IMPORTANCE: Human parechoviruses can cause mild infections to severe diseases in young children, such as neonatal sepsis, encephalitis, and cardiomyopathy. Intravenous immunoglobulin treatment is the ...IMPORTANCE: Human parechoviruses can cause mild infections to severe diseases in young children, such as neonatal sepsis, encephalitis, and cardiomyopathy. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases. In order to develop more targeted treatment, we have searched for human monoclonal antibodies that would neutralize human parechoviruses 1 and 2, associated with mild infections such as gastroenteritis and severe infections of the central nervous system, and thus allow safe treatment. In the current study, we show how two such promising antibodies interact with the virus, modeling the atomic interactions between the virus and the antibody to propose how neutralization occurs. Both antibodies can cause aggregation; in addition, one antibody interferes with the virus recognizing its target cell, while the other, recognizing only the whole virus, inhibits the genome uncoating and replication in the cell.
History
DepositionAug 26, 2014-
Header (metadata) releaseOct 1, 2014-
Map releaseJul 22, 2015-
UpdateSep 9, 2015-
Current statusSep 9, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 7999
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 7999
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4udf
  • Surface level: 7999
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4udf
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2761.map.gz / Format: CCP4 / Size: 15.1 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationReconstruction of human parechovirus 1 in complex with Fab fragments of AM28 human monoclonal antibody
Voxel sizeX=Y=Z: 2.17 Å
Density
Contour LevelBy AUTHOR: 7999.0 / Movie #1: 7999
Minimum - Maximum-4448.0 - 32443.0
Average (Standard dev.)3666.095458979999876 (±7633.060058590000153)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-100
Dimensions201201201
Spacing201201201
CellA=B=C: 436.17 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z2.172.172.17
M x/y/z201201201
origin x/y/z0.0000.0000.000
length x/y/z436.170436.170436.170
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-100-100-100
NC/NR/NS201201201
D min/max/mean-4448.00032443.0003666.095

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Supplemental data

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Sample components

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Entire : Human parechovirus in complex with Fab fragment of human monoclon...

EntireName: Human parechovirus in complex with Fab fragment of human monoclonal antibody AM28
Components
  • Sample: Human parechovirus in complex with Fab fragment of human monoclonal antibody AM28
  • Virus: Human parechovirus 1
  • Protein or peptide: human monoclonal antibodyMonoclonal antibody

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Supramolecule #1000: Human parechovirus in complex with Fab fragment of human monoclon...

SupramoleculeName: Human parechovirus in complex with Fab fragment of human monoclonal antibody AM28
type: sample / ID: 1000 / Number unique components: 2

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Supramolecule #1: Human parechovirus 1

SupramoleculeName: Human parechovirus 1 / type: virus / ID: 1 / Name.synonym: HPeV1
Details: Fab fragments of human monoclonal antibody, AM28 were attached to the virus
NCBI-ID: 12063 / Sci species name: Human parechovirus 1 / Sci species strain: 1 / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No / Syn species name: HPeV1
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES

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Macromolecule #1: human monoclonal antibody

MacromoleculeName: human monoclonal antibody / type: protein_or_peptide / ID: 1 / Name.synonym: AM28
Details: Fab fragments of human monoclonal antibody, AM28 were attached to the virus
Number of copies: 60 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: 293T cells

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5 / Details: 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 4.06 µm / Nominal defocus min: 1.65 µm / Nominal magnification: 69000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
DateFeb 1, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 65
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 19.76 Å / Resolution method: OTHER / Software - Name: AUTO3DEM / Number images used: 270
DetailsParticles were selected using runethan. CTF was estimated using ctffind3 and image reconstruction done in AUTO3DEM
FSC plot (resolution estimation)

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