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- EMDB-50321: sub-tomogram averaging map of CHIKV replication organelle connect... -

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Basic information

Entry
Database: EMDB / ID: EMD-50321
Titlesub-tomogram averaging map of CHIKV replication organelle connection to the plasma membrane
Map data
Sample
  • Cell: Chikungunya virus complex at the base of replication organelles
KeywordsChikungunya / Alphavirus / Replication organelle / cryogenic-electron tomography / VIRUS
Biological speciesChikungunya virus
Methodsubtomogram averaging / cryo EM / Resolution: 31.0 Å
AuthorsLe Bihan O / Girard J / Briant L / Bron P
Funding support France, 1 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-18-CE11-002 France
CitationJournal: J Virol / Year: 2024
Title: fate of Chikungunya virus replication organelles.
Authors: Justine Girard / Olivier Le Bihan / Joséphine Lai-Kee-Him / Maria Girleanu / Eric Bernard / Cedric Castellarin / Matthew Chee / Aymeric Neyret / Danièle Spehner / Xavier Holy / Anne-Laure ...Authors: Justine Girard / Olivier Le Bihan / Joséphine Lai-Kee-Him / Maria Girleanu / Eric Bernard / Cedric Castellarin / Matthew Chee / Aymeric Neyret / Danièle Spehner / Xavier Holy / Anne-Laure Favier / Laurence Briant / Patrick Bron /
Abstract: Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for an acute musculoskeletal disease in humans. Replication of the viral RNA genome occurs in specialized membranous replication ...Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for an acute musculoskeletal disease in humans. Replication of the viral RNA genome occurs in specialized membranous replication organelles (ROs) or spherules, which contain the viral replication complex. Initially generated by RNA synthesis-associated plasma membrane deformation, alphavirus ROs are generally rapidly endocytosed to produce type I cytopathic vacuoles (CPV-I), from which nascent RNAs are extruded for cytoplasmic translation. By contrast, CHIKV ROs are poorly internalized, raising the question of their fate and functionality at the late stage of infection. Here, using cryogenic-electron microscopy approaches, we investigate the outcome of CHIKV ROs and associated replication machinery in infected human cells. We evidence the late persistence of CHIKV ROs at the plasma membrane with a crowned protein complex at the spherule neck similar to the recently resolved replication complex. The unexpectedly heterogeneous and large diameter of these compartments suggests a continuous, dynamic growth of these organelles beyond the replication of a single RNA genome. Ultrastructural analysis of surrounding cytoplasmic regions supports that outgrown CHIKV ROs remain dynamically active in viral RNA synthesis and export to the cell cytosol for protein translation. Interestingly, rare ROs with a homogeneous diameter are also marginally internalized in CPV-I near honeycomb-like arrangements of unknown function, which are absent in uninfected controls, thereby suggesting a temporal regulation of this internalization. Altogether, this study sheds new light on the dynamic pattern of CHIKV ROs and associated viral replication at the interface with cell membranes in infected cells.IMPORTANCEThe Chikungunya virus (CHIKV) is a positive-stranded RNA virus that requires specialized membranous replication organelles (ROs) for its genome replication. Our knowledge of this viral cycle stage is still incomplete, notably regarding the fate and functional dynamics of CHIKV ROs in infected cells. Here, we show that CHIKV ROs are maintained at the plasma membrane beyond the first viral cycle, continuing to grow and be dynamically active both in viral RNA replication and in its export to the cell cytosol, where translation occurs in proximity to ROs. This contrasts with the homogeneous diameter of ROs during internalization in cytoplasmic vacuoles, which are often associated with honeycomb-like arrangements of unknown function, suggesting a regulated mechanism. This study sheds new light on the dynamics and fate of CHIKV ROs in human cells and, consequently, on our understanding of the Chikungunya viral cycle.
History
DepositionMay 15, 2024-
Header (metadata) releaseJun 26, 2024-
Map releaseJun 26, 2024-
UpdateJul 31, 2024-
Current statusJul 31, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50321.png

Downloads & links

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Map

FileDownload / File: emd_50321.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
5.86 Å/pix.
x 128 pix.
= 750.336 Å		5.86 Å/pix.
x 128 pix.
= 750.336 Å		5.86 Å/pix.
x 128 pix.
= 750.336 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 5.862 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.67
Minimum - Maximum-14.620279999999999 - 19.78032
Average (Standard dev.)0.074125394 (±1.0002098)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 750.336 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50321_msk_1.map
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Half map: #1

Fileemd_50321_half_map_1.map
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Half map: #2

Fileemd_50321_half_map_2.map
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Sample components

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Entire : Chikungunya virus complex at the base of replication organelles

EntireName: Chikungunya virus complex at the base of replication organelles
Components
  • Cell: Chikungunya virus complex at the base of replication organelles

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Supramolecule #1: Chikungunya virus complex at the base of replication organelles

SupramoleculeName: Chikungunya virus complex at the base of replication organelles
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Chikungunya virus

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil R2/1 / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
Details: 3 microliters of 10 nm BSA-treated fiducial gold particles were added on both sides of the grid before blotting.
DetailsHEK293T cells were infected at the MOI of 50 for 17h in the growing medium and then fixed with 4% paraformaldehyde before freezing.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average exposure time: 1.3 sec. / Average electron dose: 1.1 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 10.0 µm / Calibrated defocus min: 4.0 µm / Calibrated magnification: 29000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 10.0 µm / Nominal defocus min: 4.0 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 31.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2 (ver. 2.99) / Number subtomograms used: 98
ExtractionNumber tomograms: 38 / Number images used: 463 / Method: manual / Software - Name: EMAN2 (ver. 2.99)
Final 3D classificationNumber classes: 4 / Avg.num./class: 41 / Software - Name: EMAN2 (ver. 2.99)
Final angle assignmentType: OTHER / Software - Name: EMAN2 (ver. 2.99) / Details: Subtomogram alignment
FSC plot (resolution estimation)

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