Nonlytic cellular release of hepatitis A virus requires dual capsid recruitment of the ESCRT-associated Bro1 domain proteins HD-PTP and ALIX

DOI: 10.1371/journal.ppat.1010543

Authors: Takayoshi Shirasaki (The University of North Carolina at Chapel Hill) , Hui Feng (The University of North Carolina at Chapel Hill) , Helen M. E. Duyvesteyn (The Wellcome Centre for Human Genetics, University of Oxford; Diamond Light Source) , William G. Fusco (The University of North Carolina at Chapel Hill) , Kevin L. Mcknight (The University of North Carolina at Chapel Hill) , Ling Xie (The University of North Carolina at Chapel Hill) , Mark Boyce (The Wellcome Trust Centre for Human Genetics, University of Oxford) , Sathish Kumar (Stanford University School of Medicine) , Rina Barouch-Bentov (Stanford University School of Medicine) , Olga González-López (The University of North Carolina at Chapel Hill) , Ryan Mcnamara (The University of North Carolina at Chapel Hill) , Li Wang (The University of North Carolina at Chapel Hill) , Adriana Hertel-Wulff (The University of North Carolina at Chapel Hill) , Xian Chen (The University of North Carolina at Chapel Hill) , Shirit Einav (Stanford University School of Medicine; Chan-Zuckerberg BioHub) , Joseph A. Duncan (The University of North Carolina at Chapel Hill) , Maryna Kapustina (The University of North Carolina at Chapel Hill) , Elizabeth E. Fry (The Wellcome Trust Centre for Human Genetics, University of Oxford) , David I. Stuart (The Wellcome Trust Centre for Human Genetics, University of Oxford; Diamond Light Source) , Stanley M. Lemon (The University of North Carolina at Chapel Hill)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Plos Pathogens , VOL 18

State: Published (Approved)
Published: August 2022

Abstract: 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.

Journal Keywords: 293T cells; Transfection; Viral packaging; Co-immunoprecipitation; Cell membranes; Fluorescence imaging; Hepatitis A virus; Protein domains

Diamond Keywords: Hepatitis A Virus; Viruses

Subject Areas: Biology and Bio-materials


Technical Areas:

Added On: 22/08/2022 11:47

Discipline Tags:

Pathogens Infectious Diseases Health & Wellbeing Structural biology Life Sciences & Biotech

Technical Tags: