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- PDB-8at5: native Coxsackievirus A9 -

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Basic information

Entry
Database: PDB / ID: 8at5
Titlenative Coxsackievirus A9
Components(Capsid protein ...Capsid) x 4
KeywordsVIRUS / icosahedral symmetry / intact virion / picornavirus
Function / homology
Function and homology information


symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of RIG-I activity / picornain 2A / symbiont-mediated suppression of host mRNA export from nucleus / symbiont genome entry into host cell via pore formation in plasma membrane / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / endocytosis involved in viral entry into host cell / : / nucleoside-triphosphate phosphatase ...symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of RIG-I activity / picornain 2A / symbiont-mediated suppression of host mRNA export from nucleus / symbiont genome entry into host cell via pore formation in plasma membrane / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / endocytosis involved in viral entry into host cell / : / nucleoside-triphosphate phosphatase / protein complex oligomerization / monoatomic ion channel activity / RNA helicase activity / DNA replication / induction by virus of host autophagy / RNA-directed RNA polymerase / symbiont-mediated suppression of host gene expression / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / DNA-templated transcription / host cell nucleus / virion attachment to host cell / structural molecule activity / ATP hydrolysis activity / proteolysis / RNA binding / ATP binding / membrane / metal ion binding
Similarity search - Function
Picornavirus coat protein VP4 superfamily / Poliovirus 3A protein-like / Poliovirus 3A protein like / Picornavirus 2B protein / Poliovirus core protein 3a, soluble domain / Picornavirus 2B protein / Peptidase C3, picornavirus core protein 2A / Picornavirus core protein 2A / Picornavirus coat protein VP4 / Picornavirus coat protein (VP4) ...Picornavirus coat protein VP4 superfamily / Poliovirus 3A protein-like / Poliovirus 3A protein like / Picornavirus 2B protein / Poliovirus core protein 3a, soluble domain / Picornavirus 2B protein / Peptidase C3, picornavirus core protein 2A / Picornavirus core protein 2A / Picornavirus coat protein VP4 / Picornavirus coat protein (VP4) / 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. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / 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
MYRISTIC ACID / PALMITIC ACID / Genome polyprotein
Similarity search - Component
Biological speciesHuman coxsackievirus A9
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsDomanska, A. / Plavec, Z. / Ruokolainen, V. / Marjomaki, V.S. / Butcher, S.J.
Funding support Finland, 1items
OrganizationGrant numberCountry
Academy of Finland315950 Finland
CitationJournal: J Virol / Year: 2022
Title: Structural Studies Reveal that Endosomal Cations Promote Formation of Infectious Coxsackievirus A9 A-Particles, Facilitating RNA and VP4 Release.
Authors: Aušra Domanska / Zlatka Plavec / Visa Ruokolainen / Benita Löflund / Varpu Marjomäki / Sarah J Butcher /
Abstract: Coxsackievirus A9 (CVA9), an enterovirus, is a common cause of pediatric aseptic meningitis and neonatal sepsis. During cell entry, enterovirus capsids undergo conformational changes leading to ...Coxsackievirus A9 (CVA9), an enterovirus, is a common cause of pediatric aseptic meningitis and neonatal sepsis. During cell entry, enterovirus capsids undergo conformational changes leading to expansion, formation of large pores, externalization of VP1 N termini, and loss of the lipid factor from VP1. Factors such as receptor binding, heat, and acidic pH can trigger capsid expansion in some enteroviruses. Here, we show that fatty acid-free bovine serum albumin or neutral endosomal ionic conditions can independently prime CVA9 for expansion and genome release. Our results showed that CVA9 treatment with albumin or endosomal ions generated a heterogeneous population of virions, which could be physically separated by asymmetric flow field flow fractionation and computationally by cryo-electron microscopy (cryo-EM) and image processing. We report cryo-EM structures of CVA9 A-particles obtained by albumin or endosomal ion treatment and a control nonexpanded virion to 3.5, 3.3, and 2.9 Å resolution, respectively. Whereas albumin promoted stable expanded virions, the endosomal ionic concentrations induced unstable CVA9 virions which easily disintegrated, losing their genome. Loss of most of the VP4 molecules and exposure of negatively charged amino acid residues in the capsid's interior after expansion created a repulsive viral RNA-capsid interface, aiding genome release. Coxsackievirus A9 (CVA9) is a common cause of meningitis and neonatal sepsis. The triggers and mode of action of RNA release into the cell unusually do not require receptor interaction. Rather, a slow process in the endosome, independent of low pH, is required. Here, we show by biophysical separation, cryogenic electron microscopy, and image reconstruction that albumin and buffers mimicking the endosomal ion composition can separately and together expand and prime CVA9 for uncoating. Furthermore, we show in these expanded particles that VP4 is present at only ~10% of the occupancy found in the virion, VP1 is externalized, and the genome is repelled by the negatively charged, repulsive inner surface of the capsid that occurs due to the expansion. Thus, we can now link observations from cell biology of infection with the physical processes that occur in the capsid to promote genome uncoating.
History
DepositionAug 22, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 16, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 1, 2023Group: Database references / Refinement description
Category: citation / citation_author ...citation / citation_author / pdbx_initial_refinement_model / pdbx_related_exp_data_set
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: Capsid protein VP4
A: Capsid protein VP1
B: Capsid protein VP2
C: Capsid protein VP3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)96,9266
Polymers96,4424
Non-polymers4852
Water0
1
D: Capsid protein VP4
A: Capsid protein VP1
B: Capsid protein VP2
C: Capsid protein VP3
hetero molecules
x 60


Theoretical massNumber of molelcules
Total (without water)5,815,587360
Polymers5,786,499240
Non-polymers29,088120
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59

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Components

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Capsid protein ... , 4 types, 4 molecules DABC

#1: Protein Capsid protein VP4 / / P1A / Virion protein 4


Mass: 7352.039 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Viral protein 4, VP4 / Source: (natural) Human coxsackievirus A9 (strain Griggs) / Strain: Griggs / References: UniProt: P21404
#2: Protein Capsid protein VP1 / / P1D / Virion protein 1


Mass: 33869.020 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Viral protein 1, VP1 / Source: (natural) Human coxsackievirus A9 (strain Griggs) / Cell line: GMK / Organ: kidney / Strain: Griggs / References: UniProt: P21404
#3: Protein Capsid protein VP2 / / P1B / Virion protein 2


Mass: 28885.518 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Viral protein 2, VP2 / Source: (natural) Human coxsackievirus A9 (strain Griggs) / Strain: Griggs / References: UniProt: P21404
#4: Protein Capsid protein VP3 / / P1C / Virion protein 3


Mass: 26335.072 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Viral protein 3, VP3 / Source: (natural) Human coxsackievirus A9 (strain Griggs) / Strain: Griggs / References: UniProt: P21404

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Non-polymers , 2 types, 2 molecules

#5: Chemical ChemComp-MYR / MYRISTIC ACID / Myristic acid


Mass: 228.371 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H28O2
#6: Chemical ChemComp-PLM / PALMITIC ACID / Palmitic acid


Mass: 256.424 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H32O2

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Details

Has ligand of interestN

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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: Coxsackievirus A9 / Type: VIRUS / Entity ID: #2-#4, #1 / Source: NATURAL
Molecular weightValue: 8 MDa / Experimental value: NO
Source (natural)Organism: Coxsackievirus A9
Details of virusEmpty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRION
Natural hostOrganism: Homo sapiens
Virus shellName: icosahedralIcosahedron / Diameter: 300 nm / Triangulation number (T number): 1
Buffer solutionpH: 7.25 / Details: PBS containing 2mM MgCl2, pH 7.2
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: this sample was monodisperse
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
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 / Nominal magnification: 150000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refinerefinement
PHENIXrefinement
UCSF ChimeraX1.3/v9model building
EM software
IDNameVersionCategoryDetails
1Xmipp3particle selection2-step
2EPUimage acquisition
4CTFFIND4CTF correction
7UCSF Chimera1.15rcmodel fitting
9RELION2initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13Coot0.9.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 28818
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 21365 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 1D4M

1d4m

RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 73.2 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00396683
ELECTRON MICROSCOPYf_angle_d0.51449101
ELECTRON MICROSCOPYf_chiral_restr0.04371008
ELECTRON MICROSCOPYf_plane_restr0.00411176
ELECTRON MICROSCOPYf_dihedral_angle_d5.4211916

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