Amino‐terminal epitopes are exposed when full‐length open reading frame 2 of hepatitis E virus is expressed in Escherichia coli , but carboxy‐terminal epitopes are masked

We constructed a panel of overlapping and non‐overlapping fragments of cDNA derived from open reading frame 2 (ORF2) of hepatitis E virus (HEV) and fused to the gene encoding glutathione S‐transferase (GST), from which proteins were expressed in Eschericia coli. IgG‐specific immunoreactivity against each protein was measured by Western immunoblotting using sera from experimentally infected Rhesus macaques ( Macaca mulatta ) or from HEV‐infected patients. Under these conditions, full‐length ORF2 protein (GST‐ORF2) was strongly reactive with acute‐phase sera from either macaques or patients, but was poorly reactive with convalescent sera. Recombinant protein GST‐ORF2.3, representing amino acids 1–110 of the 660 encoded by ORF2, demonstrated a pattern of reactivity largely indistinguishable from the full‐length protein. Conversely, GST‐ORF2.1, representing amino acids 394–660 of the ORF2 protein was strongly reactive with both acute‐ and convalescent‐phase sera. Extension of GST‐ORF2.1 towards the N‐terminus led to a progressive loss of convalescent‐phase reactivity, apparent with as few as 20 additional HEV‐specific amino acids. Deletion of 40 or more amino acids from the N‐terminus of ORF2.1 also led to reduced convalescent‐phase reactivity, however a protein representing this “reactive” region, containing amino acids 394–473, was poorly reactive, suggesting that the convalescent‐reactive epitopes are conformational. Expression of full‐length ORF2 protein in E. coli therefore masks the convalescent‐reactive epitopes within the C‐terminal part of the protein, without affecting N‐terminal, acute‐reactive epitopes. J. Med. Virol. 52:289–300, 1997. © 1997 Wiley‐Liss, Inc.