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- PDB-9mxc: Cryo-EM Structure of Human Enterovirus D68 USA/IL/14-18952 in Com... -

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Basic information

Entry
Database: PDB / ID: 9mxc
TitleCryo-EM Structure of Human Enterovirus D68 USA/IL/14-18952 in Complex with Fc-MFSD6(L3)
Components
  • (viral protein ...) x 4
  • Major facilitator superfamily domain-containing protein 6
KeywordsVIRUS / Glycan / MFSD6 / Receptor
Function / homology
Function and homology information


symbiont-mediated suppression of cytoplasmic pattern recognition receptor signaling pathway / antigen processing and presentation of exogenous peptide antigen via MHC class I / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MDA-5 activity / protein sequestering 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 ...symbiont-mediated suppression of cytoplasmic pattern recognition receptor signaling pathway / antigen processing and presentation of exogenous peptide antigen via MHC class I / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MDA-5 activity / protein sequestering 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 / nucleoside-triphosphate phosphatase / channel activity / symbiont-mediated suppression of host toll-like receptor signaling pathway / monoatomic ion transmembrane transport / symbiont-mediated suppression of host NF-kappaB cascade / RNA helicase activity / symbiont-mediated suppression of host innate immune response / endocytosis involved in viral entry into host cell / symbiont-mediated activation of host autophagy / RNA-directed RNA polymerase / cysteine-type endopeptidase activity / viral RNA genome replication / RNA-directed RNA polymerase activity / DNA-templated transcription / virion attachment to host cell / host cell nucleus / structural molecule activity / ATP hydrolysis activity / proteolysis / RNA binding / zinc ion binding / ATP binding / membrane / plasma membrane / cytoplasm
Similarity search - Function
Major facilitator superfamily associated domain / : / MFS_1 like family / MFS transporter superfamily / Picornavirus coat protein / Poliovirus 3A protein-like / Poliovirus 3A protein like / Picornavirus 2B protein / Poliovirus core protein 3a, soluble domain / Picornavirus 2B protein ...Major facilitator superfamily associated domain / : / MFS_1 like family / MFS transporter superfamily / Picornavirus coat protein / 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
Genome polyprotein / Genome polyprotein / Genome polyprotein / Genome polyprotein / Major facilitator superfamily domain-containing protein 6
Similarity search - Component
Biological speciesHomo sapiens (human)
enterovirus D68
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.1 Å
AuthorsXu, L. / Pintilie, G. / Varanese, L. / Carette, J.E. / Chiu, W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)DGE-1656518 United States
CitationJournal: Nature / Year: 2025
Title: MFSD6 is an entry receptor for enterovirus D68.
Authors: Lauren Varanese / Lily Xu / Christine E Peters / Grigore Pintilie / David S Roberts / Suyash Raj / Mengying Liu / Yaw Shin Ooi / Jonathan Diep / Wenjie Qiao / Christopher M Richards / Jeremy ...Authors: Lauren Varanese / Lily Xu / Christine E Peters / Grigore Pintilie / David S Roberts / Suyash Raj / Mengying Liu / Yaw Shin Ooi / Jonathan Diep / Wenjie Qiao / Christopher M Richards / Jeremy Callaway / Carolyn R Bertozzi / Sabrina Jabs / Erik de Vries / Frank J M van Kuppeveld / Claude M Nagamine / Wah Chiu / Jan E Carette /
Abstract: With the near eradication of poliovirus due to global vaccination campaigns, attention has shifted to other enteroviruses that can cause polio-like paralysis syndrome (now termed acute flaccid ...With the near eradication of poliovirus due to global vaccination campaigns, attention has shifted to other enteroviruses that can cause polio-like paralysis syndrome (now termed acute flaccid myelitis). In particular, enterovirus D68 (EV-D68) is believed to be the main driver of epidemic outbreaks of acute flaccid myelitis in recent years, yet not much is known about EV-D68 host interactions. EV-D68 is a respiratory virus but, in rare cases, can spread to the central nervous system to cause severe neuropathogenesis. Here we use genome-scale CRISPR screens to identify the poorly characterized multipass membrane transporter MFSD6 as a host entry factor for EV-D68. Knockout of MFSD6 expression abrogated EV-D68 infection in cell lines and primary cells corresponding to respiratory and neural cells. MFSD6 localized to the plasma membrane and was required for viral entry into host cells. MFSD6 bound directly to EV-D68 particles through its extracellular, third loop (L3). We determined the cryo-electron microscopy structure of EV-D68 in a complex with MFSD6 L3, revealing the interaction interface. A decoy receptor, engineered by fusing MFSD6 L3 to Fc, blocked EV-D68 infection of human primary lung epithelial cells and provided near-complete protection in a lethal mouse model of EV-D68 infection. Collectively, our results reveal MFSD6 as an entry receptor for EV-D68, and support the targeting of MFSD6 as a potential mechanism to combat infections by this emerging pathogen with pandemic potential.
History
DepositionJan 18, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 26, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Mar 26, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: viral protein 1
B: viral protein 2
C: viral protein 3
D: viral protein 4
E: Major facilitator superfamily domain-containing protein 6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)92,1389
Polymers91,0125
Non-polymers1,1264
Water2,288127
1
A: viral protein 1
B: viral protein 2
C: viral protein 3
D: viral protein 4
E: Major facilitator superfamily domain-containing protein 6
hetero molecules
x 60


Theoretical massNumber of molelcules
Total (without water)5,528,285540
Polymers5,460,729300
Non-polymers67,556240
Water5,405300
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
2


  • Idetical with deposited unit
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
A: viral protein 1
B: viral protein 2
C: viral protein 3
D: viral protein 4
E: Major facilitator superfamily domain-containing protein 6
hetero molecules
x 5


  • icosahedral pentamer
  • 461 kDa, 25 polymers
Theoretical massNumber of molelcules
Total (without water)460,69045
Polymers455,06125
Non-polymers5,63020
Water45025
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: viral protein 1
B: viral protein 2
C: viral protein 3
D: viral protein 4
E: Major facilitator superfamily domain-containing protein 6
hetero molecules
x 6


  • icosahedral 23 hexamer
  • 553 kDa, 30 polymers
Theoretical massNumber of molelcules
Total (without water)552,82854
Polymers546,07330
Non-polymers6,75624
Water54030
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

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Viral protein ... , 4 types, 4 molecules ABCD

#1: Protein viral protein 1


Mass: 32854.242 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) enterovirus D68 / Cell line: RD
References: UniProt: A0A1I9KHM1, picornain 2A, nucleoside-triphosphate phosphatase, picornain 3C, RNA-directed RNA polymerase
#2: Protein viral protein 2


Mass: 26527.137 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Residues 1-11 deleted / Source: (natural) enterovirus D68 / Cell line: RD
References: UniProt: A0A1B0T636, picornain 2A, nucleoside-triphosphate phosphatase, picornain 3C, RNA-directed RNA polymerase
#3: Protein viral protein 3


Mass: 27156.867 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) enterovirus D68 / Cell line: RD
References: UniProt: A0A097BW17, picornain 2A, nucleoside-triphosphate phosphatase, picornain 3C, RNA-directed RNA polymerase
#4: Protein/peptide viral protein 4 / P1A / Virion protein 4


Mass: 3401.665 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Residues 1-27 and 58-68 deleted / Source: (natural) enterovirus D68 / Cell line: RD / References: UniProt: Q68T42

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Protein/peptide / Sugars , 2 types, 2 molecules E

#5: Protein/peptide Major facilitator superfamily domain-containing protein 6 / Macrophage MHC class I receptor 2 homolog


Mass: 1072.236 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: IL2 secretion sequence (residues 1-20), MFSD6 loop 3 residues 153-285 (residues 21-153), linker (residues 154-155), and rabbit IgG tag (residues 156-378)
Source: (gene. exp.) Homo sapiens (human) / Cell line: HEK293T GALE/GALK2 KO / Gene: MFSD6, MMR2 / Cell line (production host): HEK293T GALE/GALK2 KO / Production host: Homo sapiens (human) / References: UniProt: Q6ZSS7
#6: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1056.964 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/4,6,5/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5][a1221m-1a_1-5]/1-1-2-3-3-4/a4-b1_a6-f1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}[(6+1)][a-L-Fucp]{}}LINUCSPDB-CARE

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Non-polymers , 2 types, 130 molecules

#7: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Na
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 127 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1enterovirus D68VIRUSEnterovirus D68 propagated and purified from infection of RD cells. MFSD6(L3)-Fc generated from transient transfection of HEK293T GALE/GALK2 KO cells.#1-#50MULTIPLE SOURCES
2MFSD6(L3)-Fc residues 200-208 with N-linked glycosylation site at N207COMPLEXN-linked glycosylated MFSD6(L3) sequence with rabbit IgG Fc tag#51RECOMBINANT
3Enterovirus D68COMPLEXEnterovirus D68 propagated and purified from infection of RD cells#1-#41NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21enterovirus D684278914-18952
32Homo sapiens (human)9606HEK293T GALE/GALK2 KO
43enterovirus D684278914-18952
Details of virusEmpty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRION
Natural host
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens9606
21enterovirus D6842789
33enterovirus D6842789
Buffer solution
IDSpecimen-IDpHDetails
11820 mM Tris-HCl (pH 8.0), 120 mM NaCl, and 1 mM EDTA (pH 8.0)
2281.2 mL of 100 mM Glycine buffer (pH 3.0) neutralized with 185 uL of 1 M Tris-HCl (pH 8.0), yielding a final concentration of 86.6 mM Glycine and 133.6 mM Tris-HCl.
338
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HCl, pH 8.0Tris-HCl1
2120 mMsodium chlorideNaCl1
31 mMEDTA, pH 8.0EDTA1
486.6 mMGlycine, pH 3.0C2H5NO22
5133.6 mMTris-HCl, pH 8.0Tris-HCl2
Specimen

Experiment-ID: 1 / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES

IDConc. (mg/ml)Details
10.089
20.505
3Purified virus samples with ligand, MFSD6(L3)-Fc generated from HEK293T GALE/GALK2 KO, were incubated in molar excess for 1 hr at 34C before freezing.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: Freezing carried out with 4 s blot time, 1 s wait time.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DARK FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.28 sec. / Electron dose: 49.99 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11869
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 15 eV

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Processing

EM software
IDNameCategory
1crYOLOparticle selection
2EPUimage acquisition
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
8UCSF ChimeraXmodel fitting
9ISOLDEmodel fitting
11RELIONinitial Euler assignment
12cryoSPARCinitial Euler assignment
13RELIONfinal Euler assignment
14cryoSPARCfinal Euler assignment
16RELION3D reconstruction
17cryoSPARC3D reconstruction
18ISOLDEmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 28888
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 8656 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Details: Initial rigid fitting was done using Chimera and then ISOLDE was used for flexible fitting.
Atomic model buildingSource name: AlphaFold / Type: in silico model

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