title: Hepatitis E virus capsid protein assembles in 4M urea in the presence of salts.
abstract: The hepatitis E virus (HEV) capsid protein has been demonstrated to be able to assemble into particles in vitro. However, this process and the mechanism of protein-protein interactions during particle assembly remain unclear. In this study, we investigated the assembly mechanism of HEV structural protein subunits, the capsid protein p239 (aa368-606), using analytical ultracentrifugation. It was the first to observe that the p239 can form particles in 4M urea as a result of supplementation with salt, including ammonium sulfate [(NH₄)₂SO₄], sodium sulfate (Na₂SO₄), sodium chloride (NaCl), and ammonium chloride (NH₄Cl). Interestingly, it is the ionic strength that determines the efficiency of promoting particle assembly. The assembly rate was affected by temperature and salt concentration. When (NH₄)₂SO₄ was used, assembling intermediates of p239 with sedimentation coefficient values of approximately 5 S, which were mostly dodecamers, were identified for the first time. A highly conserved 28-aa region (aa368-395) of p239 was found to be critical for particle assembly, and the hydrophobic residues Leu³⁷², Leu³⁷⁵, and Leu³⁹⁵ of p239 was found to be critical for particle assembly, which was revealed by site-directed mutagenesis. This study provides new insights into the assembly mechanism of native HEV, and contributes a valuable basis for further investigations of protein assembly by hydrophobic interactions under denaturing conditions.
authors.affiliation: National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Life Sciences, Xiamen University, Xiamen 361005, People's Republic of China.
authors.forename: Chunyan
authors.initials: C
authors.lastname: Yang
authors.forename: Huirong
authors.initials: H
authors.lastname: Pan
authors.forename: Minxi
authors.initials: M
authors.lastname: Wei
authors.forename: Xiao
authors.initials: X
authors.lastname: Zhang
authors.forename: Nan
authors.initials: N
authors.lastname: Wang
authors.forename: Ying
authors.initials: Y
authors.lastname: Gu
authors.forename: Hailian
authors.initials: H
authors.lastname: Du
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Shaowei
authors.initials: S
authors.lastname: Li
authors.forename: Ningshao
authors.initials: N
authors.lastname: Xia
mesh_terms: D000643:Ammonium Chloride / Q000737:chemistry; D000645:Ammonium Sulfate / Q000737:chemistry; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016752:Hepatitis E virus / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D007202:Indicators and Reagents / Q000737:chemistry*; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009994:Osmolar Concentration; D010316:Particle Size; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D058849:Protein Refolding / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D058767:Protein Unfolding / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry; D013431:Sulfates / Q000737:chemistry; D013696:Temperature; D014508:Urea / Q000737:chemistry*
chemical_list: D036022:Capsid Proteins; D007202:Indicators and Reagents; D050505:Mutant Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013431:Sulfates; D000643:Ammonium Chloride; C012036:sodium sulfate; D012965:Sodium Chloride; D014508:Urea; D000645:Ammonium Sulfate