N149 and G150 within the HLA-DR α2 loop 4 are critical for H18-mediated entry.

(A) Cross-species conservation of the respective amino acids of HLA-DR loop 4 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 corresponding HLA-DM sequence is depicted in blue. Underlying data: S3 Data. (B) Surface expression of the variants of the chimera 6c harboring the indicated HLA-DM-specific point mutations in loop 4 on transfected HEK293T cells. 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). Underlying data: S3 Data. (C) VSV-H18 infection rate of cells expressing the indicated chimera number 6c variants. The percentage of GFP-positive cells is normalized to that of infected HLA-DR-expressing cells. Underlying data: S3 Data. (D, E) Surface expression of HLA-DR harboring the indicated HLA-DM-specific amino acid substitutions in loop 4 in transfected HEK293T cells (D) and VSV-H18 infection rate (E). Underlying data: S3 Data. (F) GFP-positive cells observed by fluorescent microscopy in HEK293T cells expressing the indicated HLA-DR variants (scale bar, 100 μm). (G) pH-induced polykaryon formation of HEK293T cells expressing GFP and H18 or an EV with BHK21 cells expressing the indicated HLA-DR variants. Images are representatives of 3 independent experiments. (H) Principle of the T cell activation assay. BHK21 cells transiently expressing HLA-DR1 (HLA-DRB1*01:01) were loaded with the HA307-319 peptide and cocultured with CH7C17Jurkat T cells transgenic for the HA1.7 TCR. Binding of the HA1.7 TCR to HLA-DR1–HA307-319 peptide complex results in the surface expression of CD69, an early marker of T cell activation. Scheme was created with BioRender.com. (I) T cell activation by BHK21 cells transiently expressing HLA-DR1 with the indicated HLA-DM-specific point mutations in the loop 4 of the α2 domain relative to wild-type HLA-DR1. Underlying data: S3 Data. Error bars represent SD. For statistical analysis, one-way ANOVA followed by Dunnett test was performed for panels B-E, and unpaired Student t test was used for panel I. * P P P < 0.0001, ns: not significant. EV, empty vector; HLA-DR, human leukocyte antigen DR; TCR, T cell receptor.