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- EMDB-7879: Infectious exosomes from poliovirus-infected HeLa cells -

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Basic information

Entry
Database: EMDB / ID: EMD-7879
TitleInfectious exosomes from poliovirus-infected HeLa cells
Map dataInfectious exosomes from poliovirus-infected HeLa cells
Sample
  • Virus: Human poliovirus 1 Mahoney
Biological speciesHuman poliovirus 1 Mahoney
Methodelectron tomography / cryo EM
AuthorsYang JE
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesR01 GM102474 United States
National Institutes of Health/National Institute of General Medical SciencesU24GM116787 United States
CitationJournal: Sci Rep / Year: 2020
Title: Complexity and ultrastructure of infectious extracellular vesicles from cells infected by non-enveloped virus.
Authors: Jie E Yang / Evan D Rossignol / Deborah Chang / Joseph Zaia / Isaac Forrester / Kiran Raja / Holly Winbigler / Daniela Nicastro / William T Jackson / Esther Bullitt /
Abstract: Enteroviruses support cell-to-cell viral transmission prior to their canonical lytic spread of virus. Poliovirus (PV), a prototype for human pathogenic positive-sense RNA enteroviruses, and ...Enteroviruses support cell-to-cell viral transmission prior to their canonical lytic spread of virus. Poliovirus (PV), a prototype for human pathogenic positive-sense RNA enteroviruses, and picornaviruses in general, transport multiple virions en bloc via infectious extracellular vesicles, 100~1000 nm in diameter, secreted from host cells. Using biochemical and biophysical methods we identify multiple components in secreted microvesicles, including mature PV virions; positive-sense genomic and negative-sense replicative, template viral RNA; essential viral replication proteins; and cellular proteins. Using cryo-electron tomography, we visualize the near-native three-dimensional architecture of secreted infectious microvesicles containing both virions and a unique morphological component that we describe as a mat-like structure. While the composition of these mat-like structures is not yet known, based on our biochemical data they are expected to be comprised of unencapsidated RNA and proteins. In addition to infectious microvesicles, CD9-positive exosomes released from PV-infected cells are also infectious and transport virions. Thus, our data show that, prior to cell lysis, non-enveloped viruses are secreted within infectious vesicles that also transport viral unencapsidated RNAs, viral and host proteins. Understanding the structure and function of these infectious particles helps elucidate the mechanism by which extracellular vesicles contribute to the spread of non-enveloped virus infection.
History
DepositionMay 15, 2018-
Header (metadata) releaseJul 25, 2018-
Map releaseMay 15, 2019-
UpdateMay 27, 2020-
Current statusMay 27, 2020Processing site: RCSB / Status: Released

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Structure visualization

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Structure viewerEM map:
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Supplemental images

emd_7879.png

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Map

FileDownload / File: emd_7879.map.gz / Format: CCP4 / Size: 1 GB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationInfectious exosomes from poliovirus-infected HeLa cells
Voxel sizeX=Y=Z: 8.2 Å
Density
Minimum - Maximum-128 - 119
Average (Standard dev.)3.0909173 (±8.271279)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-65-81201
Dimensions19841622340
Spacing16221984340
CellA: 13300.399 Å / B: 16268.8 Å / C: 2788.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z8.19999938347728.28.2
M x/y/z16221984340
origin x/y/z0.0000.0000.000
length x/y/z13300.39916268.8002788.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ280280280
MAP C/R/S123
start NC/NR/NS-81-65201
NC/NR/NS16221984340
D min/max/mean-128.000119.0003.091

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Supplemental data

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Sample components

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Entire : Human poliovirus 1 Mahoney

EntireName: Human poliovirus 1 Mahoney
Components
  • Virus: Human poliovirus 1 Mahoney

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Supramolecule #1: Human poliovirus 1 Mahoney

SupramoleculeName: Human poliovirus 1 Mahoney / type: virus / ID: 1 / Parent: 0
Details: virion-containing microvesicles collected from supernatant of poliovirus-infected cells at 8 hours post-infection
NCBI-ID: 12081 / Sci species name: Human poliovirus 1 Mahoney / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

Concentration1.5 mg/mL
BufferpH: 7 / Details: 1% glutaraldehyde in 1x PBS
GridMaterial: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Details: The grid was coated with an extra layer of carbon (holes not covered) to provide extra support.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK II
DetailsThe microvesicles from poliovirus-infected cells were collected through a series of centrifugations to enrich 100-1000 nm diameter membrane particles, followed by an annexin-V column purification to enrich phosphatidylserine-containing microvesicles.
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: TED PELLA INC / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 160 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 4.8 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: eTomo / Number images used: 47

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