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- EMDB-42344: The rotavirus VP5*/VP8* conformational transition permeabilizes m... -

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Basic information

Entry
Database: EMDB / ID: EMD-42344
TitleThe rotavirus VP5*/VP8* conformational transition permeabilizes membranes to Ca2+ (class 6 reconstruction)
Map dataLocal filter
Sample
  • Virus: Simian rotavirus A strain RRV
    • Protein or peptide: Outer capsid protein VP4
    • Protein or peptide: Outer capsid glycoprotein VP7
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM IONCalcium
KeywordsNon-enveloped virus / viral particle / entry / membrane-penetration / rotavirus / VP4 / VP5* / VP8* / calcium / Ca2+ / liposome / VIRAL PROTEIN
Function / homology
Function and homology information


host cell endoplasmic reticulum lumen / T=13 icosahedral viral capsid / host cell rough endoplasmic reticulum / host cytoskeleton / viral outer capsid / permeabilization of host organelle membrane involved in viral entry into host cell / symbiont entry into host cell via permeabilization of inner membrane / host cell endoplasmic reticulum-Golgi intermediate compartment / receptor-mediated virion attachment to host cell / virion attachment to host cell ...host cell endoplasmic reticulum lumen / T=13 icosahedral viral capsid / host cell rough endoplasmic reticulum / host cytoskeleton / viral outer capsid / permeabilization of host organelle membrane involved in viral entry into host cell / symbiont entry into host cell via permeabilization of inner membrane / host cell endoplasmic reticulum-Golgi intermediate compartment / receptor-mediated virion attachment to host cell / virion attachment to host cell / host cell plasma membrane / membrane / metal ion binding
Similarity search - Function
Glycoprotein VP7 / Glycoprotein VP7, domain 1 / Glycoprotein VP7, domain 2 / Glycoprotein VP7 / Haemagglutinin outer capsid protein VP4, concanavalin-like domain / Outer Capsid protein VP4 (Hemagglutinin) Concanavalin-like domain / Rotavirus VP4 helical domain / Rotavirus VP4 helical domain / Outer capsid protein VP4 / Rotavirus VP4, membrane interaction domain superfamily ...Glycoprotein VP7 / Glycoprotein VP7, domain 1 / Glycoprotein VP7, domain 2 / Glycoprotein VP7 / Haemagglutinin outer capsid protein VP4, concanavalin-like domain / Outer Capsid protein VP4 (Hemagglutinin) Concanavalin-like domain / Rotavirus VP4 helical domain / Rotavirus VP4 helical domain / Outer capsid protein VP4 / Rotavirus VP4, membrane interaction domain superfamily / Rotavirus VP4, membrane interaction domain / Rotavirus VP4 membrane interaction domain / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
Outer capsid protein VP4 / Outer capsid glycoprotein VP7
Similarity search - Component
Biological speciesSimian rotavirus A strain RRV
Methodsingle particle reconstruction / cryo EM / Resolution: 8.0 Å
AuthorsDe Sautu M / Herrmann T / Jenni S / Harrison SC
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA13202 United States
CitationJournal: PLoS Pathog / Year: 2024
Title: The rotavirus VP5*/VP8* conformational transition permeabilizes membranes to Ca2.
Authors: Marilina de Sautu / Tobias Herrmann / Gustavo Scanavachi / Simon Jenni / Stephen C Harrison /
Abstract: Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which ...Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca2+-stabilized trimer; loss of Ca2+ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca2+ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.
History
DepositionOct 12, 2023-
Header (metadata) releaseMar 27, 2024-
Map releaseMar 27, 2024-
UpdateApr 17, 2024-
Current statusApr 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42344.png

Downloads & links

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Map

FileDownload / File: emd_42344.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal filter
Voxel sizeX=Y=Z: 1.2375 Å
Density
Contour LevelBy AUTHOR: 0.002
Minimum - Maximum-0.011874346 - 0.029682962
Average (Standard dev.)-0.0000043350633 (±0.0023387885)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 316.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Full map, unmodified

Fileemd_42344_additional_1.map
AnnotationFull map, unmodified
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2, unmodified

Fileemd_42344_half_map_1.map
AnnotationHalf map 2, unmodified
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1, unmodified

Fileemd_42344_half_map_2.map
AnnotationHalf map 1, unmodified
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Simian rotavirus A strain RRV

EntireName: Simian rotavirus A strain RRV
Components
  • Virus: Simian rotavirus A strain RRV
    • Protein or peptide: Outer capsid protein VP4
    • Protein or peptide: Outer capsid glycoprotein VP7
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: Simian rotavirus A strain RRV

SupramoleculeName: Simian rotavirus A strain RRV / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / NCBI-ID: 444185 / Sci species name: Simian rotavirus A strain RRV / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No

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Macromolecule #1: Outer capsid protein VP4

MacromoleculeName: Outer capsid protein VP4 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Simian rotavirus A strain RRV
Molecular weightTheoretical: 86.655586 KDa
Recombinant expressionOrganism: Chlorocebus aethiops (grivet)
SequenceString: MASLIYRQLL TNSYTVDLSD EIQEIGSTKT QNVTINLGPF AQTGYAPVNW GPGETNDSTT VEPVLDGPYQ PTTFNPPVDY WMLLAPTAA GVVVEGTNNT DRWLATILVE PNVTSETRSY TLFGTQEQIT IANASQTQWK FIDVVKTTQN GSYSQYGPLQ S TPKLYAVM ...String:
MASLIYRQLL TNSYTVDLSD EIQEIGSTKT QNVTINLGPF AQTGYAPVNW GPGETNDSTT VEPVLDGPYQ PTTFNPPVDY WMLLAPTAA GVVVEGTNNT DRWLATILVE PNVTSETRSY TLFGTQEQIT IANASQTQWK FIDVVKTTQN GSYSQYGPLQ S TPKLYAVM KHNGKIYTYN GETPNVTTKY YSTTNYDSVN MTAFCDFYII PREEESTCTE YINNGLPPIQ NTRNIVPLAL SA RNIISHR AQANEDIVVS KTSLWKEMQY NRDITIRFKF ASSIVKSGGL GYKWSEISFK PANYQYTYTR DGEEVTAHTT CSV NGMNDF NFNGGSLPTD FVISRYEVIK ENSYVYVDYW DDSQAFRNMV YVRSLAANLN SVICTGGDYS FALPVGQWPV MTGG AVSLH SAGVTLSTQF TDFVSLNSLR FRFRLTVEEP SFSITRTRVS RLYGLPAANP NNGKEYYEVA GRFSLISLVP SNDDY QTPI TNSVTVRQDL ERQLGELREE FNALSQEIAM SQLIDLALLP LDMFSMFSGI KSTIDAAKSM ATSVMKKFKK SGLANS VST LTDSLSDAAS SISRGASIRS VGSSASAWTD VSTQITDVSS SVSSISTQTS TISRRLRLKE MATQTEGMNF DDISAAV LK TKIDRSTQIS PNTLPDIVTE ASEKFIPNRA YRVINNDEVF EAGTDGRFFA YRVETFDEIP FDVQKFADLV TDSPVISA I IDFKTLKNLN DNYGISRQQA FNLLRSDPRV LREFINQDNP IIRNRIEQLI MQCRL

UniProtKB: Outer capsid protein VP4

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Macromolecule #2: Outer capsid glycoprotein VP7

MacromoleculeName: Outer capsid glycoprotein VP7 / type: protein_or_peptide / ID: 2 / Number of copies: 18 / Enantiomer: LEVO
Source (natural)Organism: Simian rotavirus A strain RRV
Molecular weightTheoretical: 37.136531 KDa
Recombinant expressionOrganism: Chlorocebus aethiops (grivet)
SequenceString: MYGIEYTTVL TFLISLILLN YILKSLTRMM DFIIYRFLFI VVILSPLLKA QNYGINLPIT GSMDTAYANS TQEETFLTST LCLYYPTEA ATEINDNSWK DTLSQLFLTK GWPTGSVYFK EYTDIASFSV DPQLYCDYNV VLMKYDATLQ LDMSELADLI L NEWLCNPM ...String:
MYGIEYTTVL TFLISLILLN YILKSLTRMM DFIIYRFLFI VVILSPLLKA QNYGINLPIT GSMDTAYANS TQEETFLTST LCLYYPTEA ATEINDNSWK DTLSQLFLTK GWPTGSVYFK EYTDIASFSV DPQLYCDYNV VLMKYDATLQ LDMSELADLI L NEWLCNPM DITLYYYQQT DEANKWISMG SSCTIKVCPL NTQTLGIGCL TTDTATFEEV ATAEKLVITD VVDGVNHKLD VT TATCTIR NCKKLGPREN VAVIQVGGSD VLDITADPTT APQTERMMRI NWKKWWQVFY TVVDYVNQII QAMSKRSRSL NSA AFYYRI

UniProtKB: Outer capsid glycoprotein VP7

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 18 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 54 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: OTHER / Number images used: 70018
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6wxg


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-8uk3:
The rotavirus VP5*/VP8* conformational transition permeabilizes membranes to Ca2+ (class 6 reconstruction)

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