An HLA-DR α2 pocket formed by β-sheet 6, loop 7, and β-sheet 7 is crucial for H18-mediated entry.

(A, B) Surface representation of HLA-DM (A) and HLA-DR (B, left panel) (PDB codes: 2BC4 and 1DLH, respectively) [41,42] highlighting the H18 binding surfaces S1 and S2. β-sheet 3, loop 4, and β-sheet 4 (S1) are highlighted in violet, while β-sheet 6, loop 7, and β-sheet 7 (S2) are highlighted in pink. Stick representation of the amino acid make-up of the narrow pocket formed by β-sheet 6, loop 6, and β-sheet 7 on HLA-DR (B, right panel), highlighting positions V190 and R189. (C) Cross-species conservation of the respective amino acids of HLA-DR β-sheet 6, loop 7, and β-sheet 7 across 300 homologs calculated with ConSurf-DB [21,22]. The bar graph depicts the conservation score for each amino acid ranging from 1 (lowest conservation) to 9 (highest conservation). The corresponded HLA-DM sequence is depicted in blue. Underlying data: S4 Data. (D) Flow cytometric analysis of the surface expression of HLA-DR harboring the indicated amino acid substitutions in HEK293T. The bar graph depicts the median fluorescent intensity of the β-chain determined from cells that fall into the HLA-D α-chain and HLA-D β-chain double positive gate (S1B Fig). Values were normalized to wild-type HLA-DR (dashed line). (E) T cell activation by BHK21 cells expressing HLA-DR1 with the V190A and R189A mutation relative to wild-type HLA-DR1. Underlying data: S4 Data. (F, G) Susceptibility of HEK293T cells expressing the indicated HLA-DR variants to VSV-H18 infection determined by flow cytometry (F) or by fluorescent microscopy (scale bar, 100 μm) (G). Underlying data: S4 Data. (H) pH-induced polykaryon formation of HEK293T cells expressing GFP and H18 or EV with BHK21 cells expressing the indicated HLA-DR variants. Images are representatives of 3 independent experiments. (I, J) Infection rate of HEK293T cells expressing HLA-DR harboring the indicated mutation with authentic H18N11 virus (I) or VSV-H17 (J). Percentage of entry positive cells are normalized to that of infected, HLA-DR-expressing cells. Underlying data: S4 Data. (K, L) Flow cytometric analysis of the surface expression of Artibeus jamaicensis MHC-II (Aj-DR) harboring the indicated amino acid substitution in transfected HEK293T cells (K) and their susceptibility to VSV-H18 infection (L). Surface levels are normalized to the wild-type Aj-DR as described in (D). Underlying data: S4 Data. Error bars represent SD. For statistical analysis, one-way ANOVA followed by Dunnett test was performed for panels D, F, I, J, K, and L, and unpaired Student t test was used for panel E. * P P P P < 0.0001, ns, not significant. EV, empty vector; HEK293T, human embryonic kidney 293T; HLA-DR, human leukocyte antigen DR.