National Institutes of Health/National Cancer Institute (NIH/NCI)
R01-AI103083
United States
National Institutes of Health/National Cancer Institute (NIH/NCI)
R01-AI131685
United States
National Institutes of Health/National Cancer Institute (NIH/NCI)
R01-AI088255
United States
National Institutes of Health/National Cancer Institute (NIH/NCI)
R01-AI150095
United States
Wellcome Trust
ALR00750
United Kingdom
Medical Research Council (MRC, United Kingdom)
MR/N00065X/1
United Kingdom
Citation
Journal: PLoS Pathog / Year: 2022 Title: Nonlytic cellular release of hepatitis A virus requires dual capsid recruitment of the ESCRT-associated Bro1 domain proteins HD-PTP and ALIX. Authors: Takayoshi Shirasaki / Hui Feng / Helen M E Duyvesteyn / William G Fusco / Kevin L McKnight / Ling Xie / Mark Boyce / Sathish Kumar / Rina Barouch-Bentov / Olga González-López / Ryan ...Authors: Takayoshi Shirasaki / Hui Feng / Helen M E Duyvesteyn / William G Fusco / Kevin L McKnight / Ling Xie / Mark Boyce / Sathish Kumar / Rina Barouch-Bentov / Olga González-López / Ryan McNamara / Li Wang / Adriana Hertel-Wulff / Xian Chen / Shirit Einav / Joseph A Duncan / Maryna Kapustina / Elizabeth E Fry / David I Stuart / Stanley M Lemon / Abstract: Although picornaviruses are conventionally considered 'nonenveloped', members of multiple picornaviral genera are released nonlytically from infected cells in extracellular vesicles. The mechanisms ...Although picornaviruses are conventionally considered 'nonenveloped', members of multiple picornaviral genera are released nonlytically from infected cells in extracellular vesicles. The mechanisms underlying this process are poorly understood. Here, we describe interactions of the hepatitis A virus (HAV) capsid with components of host endosomal sorting complexes required for transport (ESCRT) that play an essential role in release. We show release of quasi-enveloped virus (eHAV) in exosome-like vesicles requires a conserved export signal located within the 8 kDa C-terminal VP1 pX extension that functions in a manner analogous to late domains of canonical enveloped viruses. Fusing pX to a self-assembling engineered protein nanocage (EPN-pX) resulted in its ESCRT-dependent release in extracellular vesicles. Mutational analysis identified a 24 amino acid peptide sequence located within the center of pX that was both necessary and sufficient for nanocage release. Deleting a YxxL motif within this sequence ablated eHAV release, resulting in virus accumulating intracellularly. The pX export signal is conserved in non-human hepatoviruses from a wide range of mammalian species, and functional in pX sequences from bat hepatoviruses when fused to the nanocage protein, suggesting these viruses are released as quasi-enveloped virions. Quantitative proteomics identified multiple ESCRT-related proteins associating with EPN-pX, including ALG2-interacting protein X (ALIX), and its paralog, tyrosine-protein phosphatase non-receptor type 23 (HD-PTP), a second Bro1 domain protein linked to sorting of ubiquitylated cargo into multivesicular endosomes. RNAi-mediated depletion of either Bro1 domain protein impeded eHAV release. Super-resolution fluorescence microscopy demonstrated colocalization of viral capsids with endogenous ALIX and HD-PTP. Co-immunoprecipitation assays using biotin-tagged peptides and recombinant proteins revealed pX interacts directly through the export signal with N-terminal Bro1 domains of both HD-PTP and ALIX. Our study identifies an exceptionally potent viral export signal mediating extracellular release of virus-sized protein assemblies and shows release requires non-redundant activities of both HD-PTP and ALIX.
Name: EPN-pX / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all Details: Associated model just contains EPN-01 component since associated tag and pX could not be confidentally resolved.
Source (natural)
Organism: Homo sapiens (human)
Recombinant expression
Organism: Homo sapiens (human) / Recombinant cell: HEK293T
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Macromolecule #1: EPN-pX
Macromolecule
Name: EPN-pX / type: protein_or_peptide / ID: 1 Details: Model just contains EPN-01 component since associated tag and pX could not be confidentally resolved.,Model just contains EPN-01 component since associated tag and pX could not be confidentally resolved. Number of copies: 1 / Enantiomer: LEVO
Source (natural)
Organism: Human immunodeficiency virus type 1 BH10
Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 293 K / Instrument: GATAN CRYOPLUNGE 3 / Details: Blot time 4-5 s..
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Electron microscopy
Microscope
TFS GLACIOS
Image recording
Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 1803 / Average electron dose: 46.0 e/Å2
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Rigid body fit monomer in chimera, generated biological unit then checked in coot and residues to be altered done so in coot followed by one cycle of rigid body refinement of entire icosahedral assembly in phenix, defining each monomeric unit as a separate body and considering ncs constraints to check for clashes.
Refinement
Space: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient
Output model
PDB-8a8n: Structure of self-assembling engineered protein nanocage (EPN) fused with hepatitis A pX protein
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