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- PDB-8eqs: Structure of SARS-CoV-1 Orf3a in late endosome/lysosome-like envi... -

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Basic information

Entry
Database: PDB / ID: 8eqs
TitleStructure of SARS-CoV-1 Orf3a in late endosome/lysosome-like environment, MSP1D1 nanodisc
Components
  • Apolipoprotein A-I
  • ORF3a protein
KeywordsVIRAL PROTEIN / Membrane protein / SARS-CoV / SARS-CoV-2
Function / homology
Function and homology information


symbiont-mediated activation of host inflammasome-mediated signal transduction / Maturation of protein 3a / Defective ABCA1 causes TGD / high-density lipoprotein particle receptor binding / peptidyl-methionine modification / HDL clearance / spherical high-density lipoprotein particle / Scavenging by Class B Receptors / negative regulation of response to cytokine stimulus / protein oxidation ...symbiont-mediated activation of host inflammasome-mediated signal transduction / Maturation of protein 3a / Defective ABCA1 causes TGD / high-density lipoprotein particle receptor binding / peptidyl-methionine modification / HDL clearance / spherical high-density lipoprotein particle / Scavenging by Class B Receptors / negative regulation of response to cytokine stimulus / protein oxidation / regulation of intestinal cholesterol absorption / vitamin transport / blood vessel endothelial cell migration / cholesterol import / negative regulation of heterotypic cell-cell adhesion / ABC transporters in lipid homeostasis / apolipoprotein A-I receptor binding / apolipoprotein receptor binding / high-density lipoprotein particle binding / negative regulation of cell adhesion molecule production / Microbial modulation of RIPK1-mediated regulated necrosis / negative regulation of cytokine production involved in immune response / HDL assembly / negative regulation of very-low-density lipoprotein particle remodeling / phosphatidylcholine biosynthetic process / glucocorticoid metabolic process / acylglycerol homeostasis / Translation of Structural Proteins / Virion Assembly and Release / phosphatidylcholine-sterol O-acyltransferase activator activity / positive regulation of phospholipid efflux / Chylomicron remodeling / SARS-CoV-1-mediated effects on programmed cell death / cellular response to lipoprotein particle stimulus / Chylomicron assembly / high-density lipoprotein particle clearance / phospholipid efflux / chylomicron / high-density lipoprotein particle remodeling / positive regulation of cholesterol metabolic process / reverse cholesterol transport / lipid storage / phospholipid homeostasis / high-density lipoprotein particle assembly / chemorepellent activity / low-density lipoprotein particle / lipoprotein biosynthetic process / cholesterol transfer activity / cholesterol transport / high-density lipoprotein particle / very-low-density lipoprotein particle / endothelial cell proliferation / regulation of Cdc42 protein signal transduction / HDL remodeling / : / cholesterol efflux / Scavenging by Class A Receptors / triglyceride homeostasis / voltage-gated calcium channel complex / adrenal gland development / negative regulation of interleukin-1 beta production / negative chemotaxis / cholesterol binding / cholesterol biosynthetic process / amyloid-beta formation / positive regulation of Rho protein signal transduction / monoatomic ion channel activity / host cell Golgi membrane / endocytic vesicle / positive regulation of cholesterol efflux / negative regulation of tumor necrosis factor-mediated signaling pathway / Scavenging of heme from plasma / cholesterol metabolic process / Retinoid metabolism and transport / SARS-CoV-1 targets host intracellular signalling and regulatory pathways / positive regulation of stress fiber assembly / Attachment and Entry / heat shock protein binding / voltage-gated potassium channel complex / endocytic vesicle lumen / positive regulation of substrate adhesion-dependent cell spreading / positive regulation of phagocytosis / cholesterol homeostasis / integrin-mediated signaling pathway / Post-translational protein phosphorylation / Heme signaling / PPARA activates gene expression / phospholipid binding / negative regulation of inflammatory response / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / SARS-CoV-1 activates/modulates innate immune responses / Platelet degranulation / extracellular vesicle / : / amyloid-beta binding / cytoplasmic vesicle / secretory granule lumen / blood microparticle / early endosome / protein stabilization
Similarity search - Function
Protein 3a, betacoronavirus / 3a-like viroporin, transmembrane domain, alpha/betacoronavirus / 3a-like viroporin, cytosolic domain, alpha/betacoronavirus / Betacoronavirus viroporin / Coronavirus (CoV) 3a-like viroporin trans-membrane (TM) domain profile. / Coronavirus (CoV) 3a-like viroporin cytosolic (CD) domain profile. / Apolipoprotein A/E / : / Apolipoprotein A1/A4/E domain
Similarity search - Domain/homology
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / Apolipoprotein A-I / ORF3a protein
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsMiller, A.N. / Houlihan, P.R. / Matamala, E. / Cabezas-Bratesco, D. / Lee, G.Y. / Cristofori-Armstrong, B. / Dilan, T.L. / Sanchez-Martinez, S. / Matthies, D. / Yan, R. ...Miller, A.N. / Houlihan, P.R. / Matamala, E. / Cabezas-Bratesco, D. / Lee, G.Y. / Cristofori-Armstrong, B. / Dilan, T.L. / Sanchez-Martinez, S. / Matthies, D. / Yan, R. / Yu, Z. / Ren, D. / Brauchi, S.E. / Clapham, D.E.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
Citation
Journal: Elife / Year: 2023
Title: The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins.
Authors: Alexandria N Miller / Patrick R Houlihan / Ella Matamala / Deny Cabezas-Bratesco / Gi Young Lee / Ben Cristofori-Armstrong / Tanya L Dilan / Silvia Sanchez-Martinez / Doreen Matthies / Rui ...Authors: Alexandria N Miller / Patrick R Houlihan / Ella Matamala / Deny Cabezas-Bratesco / Gi Young Lee / Ben Cristofori-Armstrong / Tanya L Dilan / Silvia Sanchez-Martinez / Doreen Matthies / Rui Yan / Zhiheng Yu / Dejian Ren / Sebastian E Brauchi / David E Clapham /
Abstract: The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi ...The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as viroporins. Here, we show that neither SARS-CoV-2 nor SARS-CoV-1 Orf3a form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a positively charged aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a's ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.
#1: Journal: bioRxiv / Year: 2022
Title: The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins.
Authors: Alexandria N Miller / Patrick R Houlihan / Ella Matamala / Deny Cabezas-Bratesco / Gi Young Lee / Ben Cristofori-Armstrong / Tanya L Dilan / Silvia Sanchez-Martinez / Doreen Matthies / Rui ...Authors: Alexandria N Miller / Patrick R Houlihan / Ella Matamala / Deny Cabezas-Bratesco / Gi Young Lee / Ben Cristofori-Armstrong / Tanya L Dilan / Silvia Sanchez-Martinez / Doreen Matthies / Rui Yan / Zhiheng Yu / Dejian Ren / Sebastian E Brauchi / David E Clapham
Abstract: The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi ...The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as a viroporin. Here we show that neither SARS-CoV-2 nor SARS-CoV-1 form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a basic aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.
History
DepositionOct 9, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 8, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ORF3a protein
B: ORF3a protein
C: Apolipoprotein A-I
D: Apolipoprotein A-I
hetero molecules


Theoretical massNumber of molelcules
Total (without water)123,4346
Polymers121,9464
Non-polymers1,4882
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ORF3a protein / Accessory protein 3a / Protein 3a / Protein U274 / Protein X1


Mass: 36268.219 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus
Gene: 3a / Production host: Homo sapiens (human) / References: UniProt: P59632
#2: Protein Apolipoprotein A-I / Apo-AI / ApoA-I / Apolipoprotein A1 / MSP1D1 scaffold protein


Mass: 24704.729 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: APOA1 / Production host: Escherichia coli (E. coli) / References: UniProt: P02647
#3: Chemical ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE


Mass: 744.034 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C41H78NO8P / Comment: DOPE, phospholipid*YM
Has ligand of interestN

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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

ComponentName: Structure of SARS-CoV-1 Orf3a in late endosome/lysosome-like environment, MSP1D1 nanodisc
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13970
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

EM software
IDNameVersionCategory
1Topaz0.9particle selection
4CTFFIND4CTF correction
9cryoSPARC3initial Euler assignment
10RELION3.1final Euler assignment
12RELION3.13D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 162607 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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