The α2 domain of HLA-DR is crucial for H18-mediated entry.

(A) Surface representation of a crystal structure (PDB code: 1DLH) [41] of HLA-DR bound to an antigenic peptide. The α1, α2, β1, and β2 domains are colored in different shades of gray (light to dark in the order indicated), and the peptide is colored in brown. (B) Surface representation of HLA-DR with residues predicted by model 1 to form the H18-binding interface in purple. The bar graph depicts the number of HLA-DR residues that contribute to H18 binding within the indicated domains and in total. Numbers above the bars show contribution of the domains to the predicted interacting surface area. (C) Illustration of the chimeric HLA-DR used in (E) and (F) with the indicated motifs of the α2 domain replaced by their HLA-DM-derived counterparts. HLA-DR-derived subdomains are colored in beige; HLA-DM-derived subdomains are colored in dark blue. (D) Amino acid alignment of the α2 domain of HLA-DR (black) and HLA-DM (blue), highlighting the amino acid sequences of loops and β-sheets shown in (C). (E) Flow cytometric analysis of the surface expression of the indicated MHC-II complexes transiently expressed in HEK293T cells. The bar graph depicts the median fluorescence intensity of the β-chain of cells that are double positive for the α- and β-chain (see S1C Fig), normalized to HLA-DR (dashed line). Underlying data: S2 Data. (F) Flow cytometric quantification of VSV-H18-infected, GFP-positive HEK293T cells transiently expressing the respective chimeric MHC-II complexes. The percentage of GFP-positive cells is normalized to that of infected, HLA-DR-expressing cells (dashed line). Error bars represent SD. Underlying data: S2 Data. (G) Ribbon (left) and surface representations (right) of HLA-DR showing the H18 binding surfaces S1 and S2. β-sheet 3, loop 4, and β-sheet 4 (S1) are in violet, and β-sheet 6, loop 7, and β-sheet 7 (S2) in pink. (H) Surface representation of the HLA-DR structure highlighting S1 and S2 among the residues that constitute the H18 binding sites predicted by model 1. The bar graph highlights the percentage of these residues (black coloring) among the predicted H18 interacting residues. For statistical analysis, one-way ANOVA followed by Dunnett test was performed. * P P P P < 0.0001, ns, not significant. HLA-DR, human leukocyte antigen DR; MHC-II, major histocompatibility complex class II.