Figure 3.
BLAST uncovers reactivity to a distinct region of the HHV6 U27 protein with sequence similarity to DIP2A. (a) BLAST search results demonstrating alignment of DIP2A peptide hits to HHV6 U27 protein, 7/9 amino acids were identical, and among nonidentical amino acids from U27 (red), only T (blue in DIP2A) → D (red in U27) is a nonconservative substitution. (b) HelixFold rendering of HHV6 U27 protein, with the BepiPred-predicted linear epitope highlighted in blue. (c) HHV6 alanine scanning schematic (left) and resulting impact on HHV6 reactivity (right); reactivity is normalized to percentage of wild-type reactivity (red bar). Alanine mutants with <20% of wild-type reactivity (gray bars) indicate U27 amino acids 4–30 as the critical region for antibody reactivity, whereas mutations outside this region (blue bars) had a lesser impact on reactivity. Locations of the BepiPred-predicted linear epitope (yellow), DIP2A similarity (blue), and immunoproteasome processing (orange) all converge on this region of reactivity. (d) Point mutations of the nonidentical residues in HHV6 (red) and DIP2A (blue); when reacted with AIH patient serum (n = 2), the wild-type HHV6 sequence leads to 8× enrichment over input library, more than twice that of DIP2A wild-type sequence. Mutating the D found in the HHV6 wild-type sequence (red) to the T found in DIP2A (blue; a nonconservative change) abolished this effect. (e) Luminex-based orthogonal validation assay confirms significant reactivity to the HHV6 U27 and DIP2A peptides in AIH patients positive for the DIP2A antibody by PhIP-seq (red bar/circles) compared with AIH patients negative for DIP2A by PhIP-seq (blue circle) or healthy controls (blue triangle); gray dashed line indicates mean + 2 SD of DIP2A-negative patients and healthy controls. Normalized reactivity (peptide–BSA/BSA alone) is plotted on the y axis. Preblocking with the putative cross-reactive peptide (lightly shaded bars) decreased reactivity and, in the case of HHV6 preincubation, led to loss of significant DIP2A reactivity (NS, far right). (f) HHV6 IgG index in AIH patients positive for DIP2A (red circles) compared with AIH patients negative for DIP2A (blue circles, middle), and healthy controls (blue circles, right); the assay cutoff for positivity is denoted by the dashed gray line; AIH patients positive for DIP2A by PhIP-seq had significantly higher HHV6 IgG titers than AIH patients negative for DIP2A (P < 0.001) or healthy controls (P < 0.005).

BLAST uncovers reactivity to a distinct region of the HHV6 U27 protein with sequence similarity to DIP2A. (a) BLAST search results demonstrating alignment of DIP2A peptide hits to HHV6 U27 protein, 7/9 amino acids were identical, and among nonidentical amino acids from U27 (red), only T (blue in DIP2A) → D (red in U27) is a nonconservative substitution. (b) HelixFold rendering of HHV6 U27 protein, with the BepiPred-predicted linear epitope highlighted in blue. (c) HHV6 alanine scanning schematic (left) and resulting impact on HHV6 reactivity (right); reactivity is normalized to percentage of wild-type reactivity (red bar). Alanine mutants with <20% of wild-type reactivity (gray bars) indicate U27 amino acids 4–30 as the critical region for antibody reactivity, whereas mutations outside this region (blue bars) had a lesser impact on reactivity. Locations of the BepiPred-predicted linear epitope (yellow), DIP2A similarity (blue), and immunoproteasome processing (orange) all converge on this region of reactivity. (d) Point mutations of the nonidentical residues in HHV6 (red) and DIP2A (blue); when reacted with AIH patient serum (n = 2), the wild-type HHV6 sequence leads to 8× enrichment over input library, more than twice that of DIP2A wild-type sequence. Mutating the D found in the HHV6 wild-type sequence (red) to the T found in DIP2A (blue; a nonconservative change) abolished this effect. (e) Luminex-based orthogonal validation assay confirms significant reactivity to the HHV6 U27 and DIP2A peptides in AIH patients positive for the DIP2A antibody by PhIP-seq (red bar/circles) compared with AIH patients negative for DIP2A by PhIP-seq (blue circle) or healthy controls (blue triangle); gray dashed line indicates mean + 2 SD of DIP2A-negative patients and healthy controls. Normalized reactivity (peptide–BSA/BSA alone) is plotted on the y axis. Preblocking with the putative cross-reactive peptide (lightly shaded bars) decreased reactivity and, in the case of HHV6 preincubation, led to loss of significant DIP2A reactivity (NS, far right). (f) HHV6 IgG index in AIH patients positive for DIP2A (red circles) compared with AIH patients negative for DIP2A (blue circles, middle), and healthy controls (blue circles, right); the assay cutoff for positivity is denoted by the dashed gray line; AIH patients positive for DIP2A by PhIP-seq had significantly higher HHV6 IgG titers than AIH patients negative for DIP2A (P < 0.001) or healthy controls (P < 0.005).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal