8A8N

Structure of self-assembling engineered protein nanocage (EPN) fused with hepatitis A pX protein

8A8N の概要

• エントリーDOI: 10.2210/pdb8a8n/pdb
• EMDBエントリー: 15234
• 分子名称: EPN-pX (1 entity in total)
• 機能のキーワード: hepatitis a, px, vp1-px, nanocage, icosahedral, protein nanoparticle, virus assembly, enveloped viruses, structural protein
• 由来する生物種: Human immunodeficiency virus type 1 BH10; 詳細
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 32934.23

構造登録者

Duyvesteyn, H.M.E.,Stuart, D.I. (登録日: 2022-06-23, 公開日: 2022-08-10, 最終更新日: 2022-08-31)

主引用文献

Shirasaki, T.,Feng, H.,Duyvesteyn, H.M.E.,Fusco, W.G.,McKnight, K.L.,Xie, L.,Boyce, M.,Kumar, S.,Barouch-Bentov, R.,Gonzalez-Lopez, O.,McNamara, R.,Wang, L.,Hertel-Wulff, A.,Chen, X.,Einav, S.,Duncan, J.A.,Kapustina, M.,Fry, E.E.,Stuart, D.I.,Lemon, S.M.
Nonlytic cellular release of hepatitis A virus requires dual capsid recruitment of the ESCRT-associated Bro1 domain proteins HD-PTP and ALIX.
Plos Pathog., 18:e1010543-e1010543, 2022

PubMed 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.

PubMed: 35969644
DOI: 10.1371/journal.ppat.1010543

実験手法

ELECTRON MICROSCOPY (6.7 Å)