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- EMDB-15692: Expanded Coxsackievirus A9 after 0.01% faf-BSA treatment -

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Basic information

Entry
Database: EMDB / ID: EMD-15692
TitleExpanded Coxsackievirus A9 after 0.01% faf-BSA treatment
Map dataExpanded RNA containing CVA9 after 0.01% faf-BSA treatment
Sample
  • Virus: Coxsackievirus A9
    • Protein or peptide: Capsid protein VP1
    • Protein or peptide: Capsid protein VP2
    • Protein or peptide: Capsid protein VP3
Keywordsicosahedral symmetry / expanded virion / picornavirus / A-particle / VIRUS
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 / channel activity ...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 / channel activity / monoatomic ion transmembrane transport / DNA replication / RNA helicase activity / symbiont-mediated activation of host autophagy / RNA-directed RNA polymerase / 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) / Peptidase C3A/C3B, picornaviral / 3C cysteine protease (picornain 3C) / Picornavirales 3C/3C-like protease domain / Picornavirales 3C/3C-like protease domain profile. / 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
Biological speciesHuman coxsackievirus A9 (strain Griggs) / Coxsackievirus A9
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsDomanska A / Plavec Z / Ruokolainen V / Loflund B / Marjomaki VS / Butcher SJ
Funding support Finland, 4 items
OrganizationGrant numberCountry
Academy of Finland315950 Finland
Academy of Finland336471 Finland
Jane and Aatos Erkko Foundation Finland
Sigrid Juselius Foundation95-7202-38 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 29, 2022-
Header (metadata) releaseOct 26, 2022-
Map releaseOct 26, 2022-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15692.png

Downloads & links

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Map

FileDownload / File: emd_15692.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationExpanded RNA containing CVA9 after 0.01% faf-BSA treatment
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.97 Å/pix.
x 400 pix.
= 388. Å		0.97 Å/pix.
x 400 pix.
= 388. Å		0.97 Å/pix.
x 400 pix.
= 388. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.97 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0185
Minimum - Maximum-0.03760837 - 0.08049
Average (Standard dev.)0.0013763426 (±0.0059161847)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 388.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map 2 of expanded CVA9 after 0.01% faf-BSA treatment

Fileemd_15692_half_map_1.map
AnnotationHalf map 2 of expanded CVA9 after 0.01% faf-BSA treatment
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 of expanded CVA9 after 0.01% faf-BSA treatment

Fileemd_15692_half_map_2.map
AnnotationHalf map 1 of expanded CVA9 after 0.01% faf-BSA treatment
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Coxsackievirus A9

EntireName: Coxsackievirus A9
Components
  • Virus: Coxsackievirus A9
    • Protein or peptide: Capsid protein VP1
    • Protein or peptide: Capsid protein VP2
    • Protein or peptide: Capsid protein VP3

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Supramolecule #1: Coxsackievirus A9

SupramoleculeName: Coxsackievirus A9 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 12067 / Sci species name: Coxsackievirus A9 / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 8 MDa
Virus shellShell ID: 1 / Name: icosahedral / Diameter: 300.0 Å / T number (triangulation number): 1

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Macromolecule #1: Capsid protein VP1

MacromoleculeName: Capsid protein VP1 / type: protein_or_peptide / ID: 1 / Details: Viral protein VP1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human coxsackievirus A9 (strain Griggs) / Strain: Griggs
Molecular weightTheoretical: 33.86902 KDa
SequenceString: GDVEEAIERA VVHVADTMRS GPSNSASVPA LTAVETGHTS QVTPSDTMQT RHVKNYHSRS ESTVENFLGR SACVYMEEYK TTDNDVNKK FVAWPINTKQ MVQMRRKLEM FTYLRFDMEV TFVITSRQDP GTTLAQDMPV LTHQIMYVPP GGPIPAKVDD Y AWQTSTNP ...String:
GDVEEAIERA VVHVADTMRS GPSNSASVPA LTAVETGHTS QVTPSDTMQT RHVKNYHSRS ESTVENFLGR SACVYMEEYK TTDNDVNKK FVAWPINTKQ MVQMRRKLEM FTYLRFDMEV TFVITSRQDP GTTLAQDMPV LTHQIMYVPP GGPIPAKVDD Y AWQTSTNP SIFWTEGNAP ARMSIPFISI GNAYSNFYDG WSNFDQRGSY GYNTLNNLGH IYVRHVSGSS PHPITSTIRV YF KPKHTRA WVPRPPRLCQ YKKAFSVDFT PTPITDTRKD INTVTTVAQS RRRGDMSTLN TH

UniProtKB: Genome polyprotein

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Macromolecule #2: Capsid protein VP2

MacromoleculeName: Capsid protein VP2 / type: protein_or_peptide / ID: 2 / Details: Viral protein VP2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human coxsackievirus A9 (strain Griggs) / Strain: Griggs
Molecular weightTheoretical: 28.885518 KDa
SequenceString: SPTVEECGYS DRVRSITLGN STITTQECAN VVVGYGRWPT YLRDDEATAE DQPTQPDVAT CRFYTLDSIK WEKGSVGWWW KFPEALSDM GLFGQNMQYH YLGRAGYTIH VQCNASKFHQ GCLLVVCVPE AEMGGAVVGQ AFSATAMANG DKAYEFTSAT Q SDQTKVQT ...String:
SPTVEECGYS DRVRSITLGN STITTQECAN VVVGYGRWPT YLRDDEATAE DQPTQPDVAT CRFYTLDSIK WEKGSVGWWW KFPEALSDM GLFGQNMQYH YLGRAGYTIH VQCNASKFHQ GCLLVVCVPE AEMGGAVVGQ AFSATAMANG DKAYEFTSAT Q SDQTKVQT AIHNAGMGVG VGNLTIYPHQ WINLRTNNSA TIVMPYINSV PMDNMFRHYN FTLMVIPFVK LDYADTASTY VP ITVTVAP MCAEYNGLRL AQAQ

UniProtKB: Genome polyprotein

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Macromolecule #3: Capsid protein VP3

MacromoleculeName: Capsid protein VP3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human coxsackievirus A9 (strain Griggs) / Strain: Griggs
Molecular weightTheoretical: 26.335072 KDa
SequenceString: GLPTMNTPGS TQFLTSDDFQ SPCALPQFDV TPSMNIPGEV KNLMEIAEVD SVVPVNNVQD TTDQMEMFRI PVTINAPLQQ QVFGLRLQP GLDSVFKHTL LGEILNYYAH WSGSMKLTFV FCGSAMATGK FLIAYSPPGA NPPKTRKDAM LGTHIIWDIG L QSSCVLCV ...String:
GLPTMNTPGS TQFLTSDDFQ SPCALPQFDV TPSMNIPGEV KNLMEIAEVD SVVPVNNVQD TTDQMEMFRI PVTINAPLQQ QVFGLRLQP GLDSVFKHTL LGEILNYYAH WSGSMKLTFV FCGSAMATGK FLIAYSPPGA NPPKTRKDAM LGTHIIWDIG L QSSCVLCV PWISQTHYRL VQQDEYTSAG YVTCWYQTGM IVPPGTPNSS SIMCFASACN DFSVRMLRDT PFISQDNKLQ

UniProtKB: Genome polyprotein

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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

Concentration0.1 mg/mL
BufferpH: 7.25
Details: DPBS containing 0.01% faf-BSA, pH 7.25, incubated at 37 C for 1 h
GridModel: Quantifoil R1.2/1.3 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Detailsthis sample was monodisperse

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 150000
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 15179
Startup modelType of model: OTHER
Details: Initial model generated from the data using Relion 2 3D initial model protocol
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 5533
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2) / Details: Relion 2
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final 3D classificationSoftware - Name: RELION (ver. 3)

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Atomic model buiding 1

Initial modelPDB ID:

1d4m


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8aw6:
Expanded Coxsackievirus A9 after 0.01% faf-BSA treatment

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