Figure 1

Schematic diagram of expressed HEV ORF1 and multiple sequence alignment of the putative PCP domain in genotypes 1-4. ORF1 of HEV is shown schematically as a box containing a number of motifs identified by computer-assisted homology searching [2]. The motifs are: methyl/guanylyl transferase (M), Y domain (unknown function), papain-like cysteine protease (PCP?, the presence of which is tested in this study), proline rich region (P), X domain (poly ADP ribose phosphatase), helicase (H) and RNA-dependent RNA polymerase domain (R). For expression, ORF1 was amplified from pTM1HEV, a plasmid used to express ORF1 of the Burma strain of HEV (genotype 1) in our earlier study [1], and pSHEV3, an infectious cDNA clone of the swine US strain of HEV (genotype 3)[14](obtained from X.J. Meng), by PCR using primers that added a FLAG epitope at the N-terminus and an HA-epitope at the C-terminus of the ORF. The multiple sequence alignment consists of the putative PCP of representative members of the four HEV genotypes (Genotype 1, Burma strain M73218; Genotype 2, Mexico strain M74506; Genotype 3, swine US strain AF082843; Genotype 4, China T1 strain AJ272108). An alignment of 135 HEV ORF1 from HEV genomic sequences available on GenBank revealed that the putative cysteine catalytic residue (C483, boxed) is conserved while the putative catalytic histidine residue (H590, boxed) is present in genotype 1 sequences, but is not conserved in the other genotypes. It should be noted that the catalytic cysteine and histidine residues of the PCP in the NS-ORF of rubella virus are conserved in all eight rubella virus genotypes (Yumei Zhou, unpublished data).