Figure 3. Deletion and point mutation analyses of the X-domain residues responsible for its interaction with the ORF3 protein.

(a) Y2H analyses of the interaction potential of various deletion and point mutants of X with full length ORF3. Length of the deletion mutants is graphically represented as solid lines and amino acid position of point mutants are indicated in brackets. AD: Activation domain, BD: Binding domain, +++: Strong growth, ++: Moderate growth, +: Poor growth, −: No growth, L: Leucine, T: Tryptophan, H: Histidine, A: Adenine hemisulfate, Ar: Aureobasidin, “−”: Deficiency in the medium, “+”: Supplemented in the medium. (b) Quantitative α-galactosidase assay of Y2H gold transformants represented in Fig. 3a. Normalized α-galactosidase units are plotted as ± SEM of triplicate samples. (c) Alignment of X-domain sequence of different HEV isolates. Isoleucine 66^th, 67^th, Arginine 93^rd and Leucine 101^st, 102^nd are indicated in bold letters. (d) CoIP of Huh7 cell extract expressing empty vector or wild type X (X-HA) and ORF3 (ORF3-myc) or LL-EE mutant X (D11 X-HA) and ORF3 (ORF3-myc), immunoprecipitated using anti-HA antibody and immunoblotted using anti-myc (upper panel) or anti-HA (lower panel) antibodies.