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- PDB-6o06: Extracellular factors prime enterovirus particles for uncoating -

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Basic information

Entry
Database: PDB / ID: 6o06
TitleExtracellular factors prime enterovirus particles for uncoating
Components
  • VP1
  • VP2
  • VP3
KeywordsVIRUS / expanded particle
Function / homology
Function and homology information


caveolin-mediated endocytosis of virus by host cell / suppression by virus of host translation initiation factor activity / positive stranded viral RNA replication / ec:3.4.22.29: / pore-mediated entry of viral genome into host cell / suppression by virus of host mRNA export from nucleus / suppression by virus of host RIG-I activity / ec:3.4.22.28: / RNA-protein covalent cross-linking / T=pseudo3 icosahedral viral capsid ...caveolin-mediated endocytosis of virus by host cell / suppression by virus of host translation initiation factor activity / positive stranded viral RNA replication / ec:3.4.22.29: / pore-mediated entry of viral genome into host cell / suppression by virus of host mRNA export from nucleus / suppression by virus of host RIG-I activity / ec:3.4.22.28: / RNA-protein covalent cross-linking / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / pore formation by virus in membrane of host cell / integral to membrane of host cell / RNA helicase activity / ec:3.6.1.15: / suppression by virus of host gene expression / ec:2.7.7.48: / induction by virus of host autophagy / ion channel activity / protein complex oligomerization / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-directed 5'-3' RNA polymerase activity / DNA replication / transcription, DNA-templated / virion attachment to host cell / structural molecule activity / RNA binding / membrane / ATP binding
Viral coat protein subunit / 3C cysteine protease (picornain 3C) / Peptidase C3A/C3B, picornaviral / Helicase, superfamily 3, single-stranded DNA/RNA virus / RNA-directed RNA polymerase, C-terminal domain / Picornavirus coat protein VP4 superfamily / Picornavirus/Calicivirus coat protein / Picornavirus capsid / Picornavirus 2B protein / Picornavirus coat protein VP4 ...Viral coat protein subunit / 3C cysteine protease (picornain 3C) / Peptidase C3A/C3B, picornaviral / Helicase, superfamily 3, single-stranded DNA/RNA virus / RNA-directed RNA polymerase, C-terminal domain / Picornavirus coat protein VP4 superfamily / Picornavirus/Calicivirus coat protein / Picornavirus capsid / Picornavirus 2B protein / Picornavirus coat protein VP4 / AAA+ ATPase domain / RNA-directed RNA polymerase, catalytic domain / picornavirus capsid protein / Peptidase S1, PA clan / Poliovirus core protein 3a, soluble domain / RNA dependent RNA polymerase / RNA helicase / Picornavirus core protein 2A / Picornavirus 2B protein / Picornavirus coat protein (VP4) / Poliovirus 3A protein like / RdRp of positive ssRNA viruses catalytic domain profile. / Superfamily 3 helicase of positive ssRNA viruses domain profile. / Helicase, superfamily 3, single-stranded RNA virus / Picornavirales 3C/3C-like protease domain profile. / Poliovirus 3A protein-like / P-loop containing nucleoside triphosphate hydrolase / Peptidase C3, picornavirus core protein 2A
Genome polyprotein
Biological speciesEchovirus E1
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsDomanska, A. / Ruokolainen, V. / Pelliccia, M. / Laajala, M. / Butcher, S.J. / Marjomaki, V.S.
Funding supportFinland , 4件
OrganizationGrant numberCountry
Academy of Finland275199Finland
Academy of Finland315950Finland
Sigrid Juselius FoundationFinland
Jane and Aatos Erkko FoundationFinland
CitationJournal: J. Virol. / Year: 2019
Title: Extracellular albumin and endosomal ions prime enterovirus particles for uncoating that can be prevented by fatty acid saturation.
Authors: Visa Ruokolainen / Aušra Domanska / Mira Laajala / Maria Pelliccia / Sarah J Butcher / Varpu Marjomäki /
Abstract: There is limited information about the molecular triggers leading to the uncoating of enteroviruses in physiological conditions. Using real-time spectroscopy and sucrose gradients with ...There is limited information about the molecular triggers leading to the uncoating of enteroviruses in physiological conditions. Using real-time spectroscopy and sucrose gradients with radioactively-labeled virus we show at 37 °C, formation of a low amount of albumin-triggered, metastable, uncoating intermediate of echovirus 1 without receptor engagement. This conversion was blocked by saturating the albumin with fatty acids. High potassium but low sodium and calcium concentrations, mimicking the endosomal environment, also induced the formation of a metastable uncoating intermediate of echovirus 1. Together, these factors boosted the formation of the uncoating intermediate and infectivity of this intermediate was retained, as judged by end-point titration. Cryo-electron microscopy reconstruction of the virions treated with albumin and high potassium, low sodium and low calcium concentrations resulted in a 3.6 Å resolution model revealing a fenestrated capsid showing 4 % expansion and loss of the pocket factor, similarly to altered (A-) particles described for other enteroviruses. The dimer interface between VP2 molecules was opened, the VP1 N-termini disordered and most likely externalised. The RNA was clearly visible, anchored to the capsid. The results presented here suggest that extracellular albumin, partially saturated with fatty acids, likely leads to the formation of the infectious uncoating intermediate prior to the engagement with the cellular receptor. In addition, changes in mono- and divalent cations, likely occurring in endosomes, promote capsid opening and genome release.There is limited information about uncoating of enteroviruses in physiological conditions. Here, we focused on physiologically relevant factors that likely contribute to opening of echovirus 1 and other B-group enteroviruses. By combining biochemical and structural data, we show, that before entering cells, extracellular albumin is capable of priming the virus into a metastable, yet infectious intermediate state. The ionic changes that are suggested to occur in endosomes, can further contribute to uncoating and promote genome release, once the viral particle is endocytosed. Importantly, we provide a detailed high-resolution structure of a virion after treatment with albumin and a preset ion composition, showing pocket factor release, capsid expansion and fenestration, and the clearly visible genome still anchored to the capsid. This study provides valuable information about the physiological factors that contribute to the opening of B-group enteroviruses.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 15, 2019 / Release: Jun 12, 2019
RevisionDateData content typeProviderType
1.0Jun 12, 2019Structure modelrepositoryInitial release

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Structure visualization

Movie
  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: VP1
B: VP2
C: VP3


Theoretical massNumber of molelcules
Total (without water)86,2023
Polymers86,2023
Non-polymers00
Water0
1
A: VP1
B: VP2
C: VP3
x 60


Theoretical massNumber of molelcules
Total (without water)5,172,115180
Polymers5,172,115180
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
2


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
A: VP1
B: VP2
C: VP3
x 5


  • icosahedral pentamer
  • 431 kDa, 15 polymers
Theoretical massNumber of molelcules
Total (without water)431,01015
Polymers431,01015
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
A: VP1
B: VP2
C: VP3
x 6


  • icosahedral 23 hexamer
  • 517 kDa, 18 polymers
Theoretical massNumber of molelcules
Total (without water)517,21118
Polymers517,21118
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein/peptide VP1


Mass: 31604.373 Da / Num. of mol.: 1 / Fragment: UNP residues 570-850 / Source method: isolated from a natural source / Source: (natural) Echovirus E1 / References: UniProt: O91734
#2: Protein/peptide VP2


Mass: 28126.465 Da / Num. of mol.: 1 / Fragment: UNP residues 77-330 / Source method: isolated from a natural source / Source: (natural) Echovirus E1 / References: UniProt: O91734
#3: Protein/peptide VP3


Mass: 26471.074 Da / Num. of mol.: 1 / Fragment: UNP residues 331-569 / Source method: isolated from a natural source / Source: (natural) Echovirus E1 / References: UniProt: O91734

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Echovirus E1 / Type: VIRUS / Details: Echovirus 1 was purified from infected GMK cells / Entity ID: 1, 2, 3 / Source: NATURAL
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Echovirus E1
Details of virusEmpty: NO / Enveloped: NO / Isolate: OTHER / Type: VIRION
Natural hostOrganism: Homo sapiens
Virus shellName: icosahedralRegular icosahedron / Diameter: 300 nm / Triangulation number (T number): 1
Buffer solutionpH: 7.2
Details: 29 mM sodium chloride, 28 mM potassium ion, 0.145 mM magnesium chloride, 8 mM phosphate dibasic, 2 mM phosphate monobasic, 0.0093% faf-BSA
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R2/2
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 47.8 sec. / Electron dose: 30 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategory
4GctfCTF correction
7UCSF Chimera1.12model fitting
9RELION2.1initial Euler assignment
10RELION2.1final Euler assignment
11RELION2.1classification
12RELION2.13D reconstruction
13Coot0.8.8model refinement
14MDFFmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 14615 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 4JGY

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