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Structure of self-assembling engineered protein nanocage (EPN) fused with hepatitis A pX proteinStructure of self-assembling engineered protein nanocage (EPN) fused with hepatitis A pX protein
Structural highlights
FunctionA0A0F7Q1W1_9PICO Acts as a primer for viral RNA replication and remains covalently bound to viral genomic RNA. VPg is uridylylated prior to priming replication into VPg-pUpU. The VPg-pUpU is then used as primer on the genomic RNA poly(A) by the RNA-dependent RNA polymerase to replicate the viral genome.[ARBA:ARBA00002133] Affects membrane integrity and causes an increase in membrane permeability.[ARBA:ARBA00004017] Plays a role in the assembly of the 12 pentamers into an icosahedral structure. Has not been detected in mature virions, supposedly owing to its small size.[ARBA:ARBA00002016] VP0 precursor is a component of the immature procapsids.[ARBA:ARBA00003724]Q9WXS1_THEMA Publication Abstract from PubMedAlthough 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. Nonlytic cellular release of hepatitis A virus requires dual capsid recruitment of the ESCRT-associated Bro1 domain proteins HD-PTP and ALIX.,Shirasaki T, Feng H, Duyvesteyn HME, Fusco WG, McKnight KL, Xie L, Boyce M, Kumar S, Barouch-Bentov R, Gonzalez-Lopez O, McNamara R, Wang L, Hertel-Wulff A, Chen X, Einav S, Duncan JA, Kapustina M, Fry EE, Stuart DI, Lemon SM PLoS Pathog. 2022 Aug 15;18(8):e1010543. doi: 10.1371/journal.ppat.1010543. , eCollection 2022 Aug. PMID:35969644[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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