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

Basic information

• Entry: Database: PDB / ID: 8eqs
• Title: Structure of SARS-CoV-1 Orf3a in late endosome/lysosome-like environment, MSP1D1 nanodisc

Components

• Apolipoprotein A-IApolipoprotein AI
• ORF3a protein

• Keywords: VIRAL PROTEIN / Membrane protein / SARS-CoV / SARS-CoV-2

Function / homology

Function:

: / Maturation of protein 3a / Defective ABCA1 causes TGD / Scavenging by Class B Receptors / HDL clearance / high-density lipoprotein particle receptor binding / spherical high-density lipoprotein particle / positive regulation of hydrolase activity / negative regulation of response to cytokine stimulus / regulation of intestinal cholesterol absorption / protein oxidation / vitamin transport / cholesterol import / high-density lipoprotein particle binding / Microbial modulation of RIPK1-mediated regulated necrosis / ABC transporters in lipid homeostasis / blood vessel endothelial cell migration / negative regulation of heterotypic cell-cell adhesion / apolipoprotein receptor binding / apolipoprotein A-I receptor binding / negative regulation of cytokine production involved in immune response / negative regulation of cell adhesion molecule production / HDL assembly / peptidyl-methionine modification / negative regulation of very-low-density lipoprotein particle remodeling / phosphatidylcholine biosynthetic process / Translation of Structural Proteins / Virion Assembly and Release / glucocorticoid metabolic process / phosphatidylcholine metabolic process / Chylomicron remodeling / phosphatidylcholine-sterol O-acyltransferase activator activity / positive regulation of phospholipid efflux / lipid storage / Chylomicron assembly / positive regulation of cholesterol metabolic process / phospholipid homeostasis / high-density lipoprotein particle clearance / high-density lipoprotein particle remodeling / phospholipid efflux / cholesterol transfer activity / reverse cholesterol transport / chemorepellent activity / high-density lipoprotein particle assembly / very-low-density lipoprotein particle / cholesterol transport / positive regulation of CoA-transferase activity / lipoprotein biosynthetic process / high-density lipoprotein particle / endothelial cell proliferation / regulation of Cdc42 protein signal transduction / triglyceride homeostasis / inorganic cation transmembrane transport / HDL remodeling / negative regulation of interleukin-1 beta production / Scavenging by Class A Receptors / cholesterol efflux / cholesterol binding / voltage-gated calcium channel complex / negative chemotaxis / positive regulation of Rho protein signal transduction / adrenal gland development / host cell Golgi membrane / cholesterol biosynthetic process / endocytic vesicle / negative regulation of tumor necrosis factor-mediated signaling pathway / positive regulation of cholesterol efflux / Scavenging of heme from plasma / voltage-gated potassium channel complex / positive regulation of phagocytosis / Attachment and Entry / positive regulation of substrate adhesion-dependent cell spreading / Retinoid metabolism and transport / positive regulation of stress fiber assembly / endocytic vesicle lumen / heat shock protein binding / cholesterol metabolic process / cholesterol homeostasis / integrin-mediated signaling pathway / Post-translational protein phosphorylation / regulation of protein phosphorylation / : / Heme signaling / phospholipid binding / PPARA activates gene expression / negative regulation of inflammatory response / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / extracellular vesicle / Platelet degranulation / protein complex oligomerization / monoatomic ion channel activity / amyloid-beta binding / cytoplasmic vesicle / secretory granule lumen / collagen-containing extracellular matrix / blood microparticle / protein stabilization / early endosome / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / G protein-coupled receptor signaling pathway

Domain/homology:

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

Component:

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / Apolipoprotein A-I / ORF3a protein

• Biological species: Severe acute respiratory syndrome coronavirus; Homo sapiens (human)
• Method: ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
• Authors: 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

Organization	Grant number	Country
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 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 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 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 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

Deposition	Oct 9, 2022	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Feb 8, 2023	Provider: repository / Type: Initial release

Assembly

Deposited unit

A: ORF3a protein

B: ORF3a protein

C: Apolipoprotein A-I

D: Apolipoprotein A-I

hetero molecules

Summary:

• 123 kDa, 4 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	123,434	6
Polymers	121,946	4
Non-polymers	1,488	2
Water	0

1

Summary:

• Idetical with deposited unit
• defined by author
• Evidence: cross-linking

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555		1

Components

#1: Protein

A B ORF3a protein / Accessory protein 3a / Protein 3a / Protein U274 / Protein X1

Details:

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

C D Apolipoprotein A-I / Apolipoprotein AI / Apo-AI / ApoA-I / Apolipoprotein A1 / MSP1D1 scaffold protein

Details:

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

A-401 B-401 ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE / Discrete optimized protein energy

Details:

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

• Has ligand of interest: N

Experimental details

Experiment

• Experiment: Method: ELECTRON MICROSCOPY
• EM experiment: Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

Sample preparation

• Component: Name: Structure of SARS-CoV-1 Orf3a in late endosome/lysosome-like environment, MSP1D1 nanodisc; Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
• Molecular weight: Experimental value: NO
• Source (natural): Organism: Homo sapiens (human)
• Source (recombinant): Organism: Escherichia coli (E. coli)
• Buffer solution: pH: 7.5
• Specimen: Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
• Vitrification: Cryogen name: ETHANE

Electron microscopy imaging

• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company
• Microscopy: Model: FEI TITAN KRIOS
• Electron gun: Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
• Electron lens: Mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
• Image recording: Electron dose: 50 e/Ų / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13970
• EM imaging optics: Energyfilter name: GIF Bioquantum

Processing

EM software

ID	Name	Version	Category
1	Topaz	0.9	particle selection
4	CTFFIND	4	CTF correction
9	cryoSPARC	3	initial Euler assignment
10	RELION	3.1	final Euler assignment
12	RELION	3.1	3D reconstruction

• CTF correction: Type: NONE
• Symmetry: Point symmetry: C₂ (2 fold cyclic)
• 3D reconstruction: Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 162607 / Num. of class averages: 1 / Symmetry type: POINT
• Atomic model building: Protocol: AB INITIO MODEL