Fig 2: Ligand Binding Blocks HSV-1-Mediated, but Not HCMV-Mediated, Inhibition of Endogenous MR1 Surface Expression(A and B) HFFs were mock or HSV-1 infected in parallel. Cells were either treated with Ac-6-FP (5 µM) for (A) 24 h prior to infection (pre) or (B) at 14 h p.i. (post) before staining for surface MR1, MHC I at 18 h p.i., and analysis by flow cytometry. Fold change relative to mock infected cells is graphed. Statistical significance was calculated by paired Student’s t test; **p < 0.005; ***p < 0.0005; ****p < 0.0001 (n = 3).(C and D) HFFs were mock or HCMV infected in parallel. Cells were either treated with Ac-6-FP (5 µM) for (C) 24 h prior to infection (pre) or (D) at 8 h p.i. (post) before staining for surface MR1, MHC I at 24 h p.i., and analysis by flow cytometry. Fold change relative to mock infected cells is graphed. Statistical significance was calculated by paired Student’s t test; **p < 0.005; ***p < 0.0005 (n = 3).

Fig 3: HSV-1 Us3 Expression Modulates Surface MR1(A) 293T cells transfected with plasmid expressing HSV-1 Us3 (pSY10-Us3).(B) HFFs transduced with lentivirus expressing HSV-1 Us3 were treated with Ac-6-FP (5 µM) for 6 h prior to staining for surface MR1, analysis by flow cytometry, with gating on GFP expression to identify Us3-expressing GFP+ cells. Fold change relative to GFP- cells is graphed. Statistical significance was calculated by paired Student’s t test (n = 3).(C and D) ARPE-19 MR1 (C) or HFFs (D) were mock, HSV-1 KOS, or ?Us3 infected in parallel. Cells were treated with Ac-6-FP (5 µM) at 24 h prior to infection (pre) or at 14 h p.i. (post) before staining for surface MR1 at 18 h p.i. and analysis by flow cytometry. Fold change relative to mock infected cells is graphed. Statistical significance was calculated by ANOVA; *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001 (ARPE-19 MR1 n = 4; HFF n = 5).(E) Cell lysates from mock, HSV-1 KOS-, or ?Us3-infected ARPE-19 MR1 cells harvested at 2, 4, 6, or 18 h p.i. and immunoblotted for MR1, viral gD, and GAPDH. Data are representative of 2 independent experiments.

Fig 4: Inhibition of MAIT cell activation by folic acid, but not 5–methylfolate, photometabolites. (A–C) Docked conformation of 6–FP (A), (R)–5m–6–FP (B) and (S)–5m–6–FP (C) to the MR1 binding site. Cdock affinity scores are highlighted in bold. The carbonyl of 6–FP interacts with the positively charged residues ARG9 via hydrogen bonding and ARG94 via water mediated hydrogen bonding. A π–stacking interaction with TYR7 is also observed. The (R) and (S) 5–m–6–FP enantiomers show similar positioning within the pocket but have no π–stacking interaction with TYR7. Ligand carbons are marked in grey while protein carbons are marked in orange. Hydrogen bonding and hydrophobic interactions are depicted as yellow and blue dashed lines, respectively. (D–F) Folic acid (FA) and 5–methyltetrahydrofolate (5–MTHF) solutions (80 μM in PBS) were exposed to UVA radiation in the presence or absence of equimolar amounts (80 μM) of the natural photosensitizer riboflavin. The extent of their respective photodegradation was determined by UV–Vis spectrophotometry (D). The release of inhibitory MR1 ligands was assessed by the staining intensity (as mean fluorescence intensity, MFI) of cell surface MR1 on antigen presenting (C1R.MR1) cells (E) and the associated inhibition of 5–OP–RU–mediated activation of a MAIT cell line (Jurkat.MAIT), as determined by the expression of the activation marker CD69 (F). 6–FP (5 μM and 10μM) was used as a control. Each symbol represents the mean value of an independent experiment (2–4), each run in triplicates. Statistics were calculated using one–way ANOVA with Tukey post–test, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns = non–significant.

Fig 5: HSV-1 Infection Inhibits Ligand and Bacterially Induced MAIT TCR-Driven Activation(A) Representative histograms of CD69 expression on Jurkat MAIT (JM) cells incubated with fixed mock or HSV-1-infected HFFs left untreated or treated with 5-OP-RU (10 µM) or E. coli (200 colony-forming units [CFUs]/cell) as indicated.(B and C) The fold change in CD69 median fluorescence intensity (MFI) compared to mock cells incubated with JM cells for (B) 5-OP-RU, n = 6, and (C) E. coli, n = 4.(D) Mock or HSV-1-fixed infected fibroblasts in co-culture with JM cells were incubated with 5-OP-RU (10 µM) overnight before staining for surface CD69 expression. The fold change in CD69 MFI compared to mock cells is graphed (n = 6).(E) Representative histograms of CD69 expression of JM cells incubated with fixed mock or HSV-1-infected ARPE-19 MR1 treated with 5-OP-RU (10 µM) or E. coli (200 CFUs/cell) as indicated.(F) The fold change in CD69 MFI compared to mock untreated ARPE-19 MR1 co-cultured with JM cells is graphed (n = 4). Statistical significance was calculated by two-way ANOVA; ***p < 0.0005; ****p < 0.0001.