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- EMDB-75345: A549 cell late endosome containing influenza A virus Pt52 -

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Basic information

Entry
Database: EMDB / ID: EMD-75345
TitleA549 cell late endosome containing influenza A virus Pt52
Map dataA549 cell late endosome containing influenza A virus
Sample
  • Cell: A549 cell
KeywordsIFITM3 / transmembrane protein / viral restriction factor / IAV / ANTIVIRAL PROTEIN
Biological speciesHomo sapiens (human)
Methodelectron tomography / negative staining
AuthorsButh SA / Melikian GB
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R37 AI150453 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 AI190198 United States
CitationJournal: J Virol / Year: 2026
Title: Cyclosporine A rescues the influenza virus fusion with IFITM3-expressing cells by relocating the restriction factor to intraluminal vesicles of multivesicular bodies.
Authors: Sergey A Buth / Mariana Marin / You Zhang / Ori Avinoam / Gregory B Melikyan /
Abstract: Interferon-induced transmembrane protein 3 (IFITM3) blocks fusion of many enveloped viruses with endosomes, likely by rigidifying the cell membrane. We and others have previously reported that ...Interferon-induced transmembrane protein 3 (IFITM3) blocks fusion of many enveloped viruses with endosomes, likely by rigidifying the cell membrane. We and others have previously reported that cyclosporine A (CsA) treatment rescues the ability of the influenza A virus (IAV) to fuse with and infect IFITM3-expressing cells, but the mechanism of such rescue remained controversial. Here, we employed correlative light-electron microscopy (CLEM) and on-section electron tomography workflow to visualize the IAV fusion intermediates arrested by IFITM3. Most common intermediates were tight IAV contacts with the limiting membrane and intraluminal vesicles of late endosomes. A hemifusion intermediate was also detected. We further visualized the effect of CsA on IFITM3 localization by immunoelectron microscopy (immuno-EM), which revealed a marked relocation of IFITM3 from the limiting membrane to intraluminal vesicles of endosomes. This CsA-induced IFITM3 sequestration in intraluminal vesicles likely relieves the block for IAV fusion with the limiting membrane of endosomes that culminates in a release of the viral nucleoprotein complex into the cytoplasm.IMPORTANCEThe mechanism by which interferon-induced transmembrane protein 3 (IFITM3) inhibits the fusion of diverse enveloped viruses with endosomes is not fully understood. Using correlative light-electron microscopy (CLEM) and on-section electron tomography, we detected IFITM3-arrested influenza A virus fusion intermediates with the limiting membrane and intraluminal vesicles of late endosomes. The most common arrested intermediate was a tight contact between the viral membrane and the limiting membrane of late endosomes, which formed more frequently compared to tight contacts with intraluminal vesicles. Immunoelectron microscopy (immuno-EM) revealed that pretreatment of IFITM3-expressing cells with cyclosporine A induced relocation of IFITM3 from the limiting membrane to intraluminal vesicles of endosomes and rescued the influenza A virus fusion. Collectively, these findings imply that the influenza virus fuses with the limiting membrane of endosomes, but not with intraluminal vesicles, leading to productive infection, and that IFITM3 enrichment at these sites is critical for blocking viral fusion.
History
DepositionJan 29, 2026-
Header (metadata) releaseMar 25, 2026-
Map releaseMar 25, 2026-
UpdateOct 7, 2026-
Current statusOct 7, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_75345.png

Downloads & links

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Map

FileDownload / File: emd_75345.map.gz / Format: CCP4 / Size: 282.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationA549 cell late endosome containing influenza A virus
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
18.93 Å/pix.
x 101 pix.
= 1911.93 Å		18.93 Å/pix.
x 837 pix.
= 15844.41 Å		18.93 Å/pix.
x 875 pix.
= 16563.75 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 18.93 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum1078.606199999999944 - 10300.27599999999984
Average (Standard dev.)7071.916000000000167 (±561.361599999999953)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin166450-60
Dimensions837875101
Spacing875837101
CellA: 16563.75 Å / B: 15844.41 Å / C: 1911.93 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : A549 cell

EntireName: A549 cell
Components
  • Cell: A549 cell

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Supramolecule #1: A549 cell

SupramoleculeName: A549 cell / type: cell / ID: 1 / Parent: 0
Details: A549 cell expressing IFITM3 transmembrane protein infected with influenza A virus at MOI 1.5
Source (natural)Organism: Homo sapiens (human) / Organ: lung

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Experimental details

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Structure determination

Methodnegative staining
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.2 / Details: DMEM media + 10% FSB
StainingType: POSITIVE / Material: Uranyl Acetate / Details: 0.1% UA in FS medium
Sugar embeddingMaterial: HM20
GridModel: EMS Formvar Carbon / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS
DetailsIAV infected cells were high pressure frozen, freeze-substituted and stained with 0.1% uranyl acetate, infiltrated with HM20 resin, and polymerized by UV Blocks were sectioned at 200-250nm.
High pressure freezingInstrument: BAL-TEC HPM 010
Details: The sapphire disc containing cell monolayer was sandwiched between two type B HPF planchettes and frozen according to manufacturer's procedure. Hexadecene-1 was used as a filler..
Cryo protectantHexadecene-1
SectioningUltramicrotomy - Instrument: Leica EM UC6 / Ultramicrotomy - Temperature: 293 K / Ultramicrotomy - Final thickness: 250

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Electron microscopy

MicroscopeJEOL 2200FS
Image recordingFilm or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Detector mode: OTHER / Average exposure time: 1.0 sec. / Average electron dose: 13.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 60.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 8.0 µm / Nominal defocus min: 8.0 µm / Nominal magnification: 10000
Sample stageSpecimen holder model: JEOL

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Image processing

DetailsDark subtracted and gain normalized, frame alignment and summing using SerialEM on-the-fly implementation
Final reconstructionSoftware - Name: eTomo / Number images used: 242
CTF correctionType: NONE

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