The secreted form of ORF2 (ORF2S) is glycosylated and dimerized.

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The secreted form of ORF2 (ORF2S) is glycosylated and dimerized. The secreted form of ORF2 (ORF2S) is glycosylated and dimerized. (A) Effects of PNGase F, O-glycosidase (O-Gly-ase), and neuraminidase (NA) treatment on ORF2 in virions and in supernatants of Huh-7 cells transfected with a WT HEV RNA or a variant carrying mutations in three putative N-linked glycosylation sites (N137/310/562Q). ORF2 was detected by a chimpanzee convalescent serum (Ch1313). (B) HEV persistently infected Huh-7 cells were treated with brefeldin A (BFA) for 24 h or left untreated (Ctrl). Cells and supernatants were collected and analyzed by immunoblotting with indicated antibodies. (C) Effects of β-mecaptomethanol (β-ME) and heat treatment (95 °C, 10 min) on the size of ORF2S in a native protein gel. (D) Determination of the size of ORF2S by size-exclusion chromatography. The majority of ORF2S was eluted as peak 1 with an estimated size of 180 kDa. The column contaminants were eluted in the second peak (peak 2). The presence of ORF2S in peak fractions was confirmed by Western blot analysis using Ch1313. Xin Yin et al. PNAS 2018;115:18:4773-4778 ©2018 by National Academy of Sciences