Fig 2: SLC3A2 is required for the entry step of the HCV life cycle. (A) Huh7.5 cells were transfected with the indicated siRNAs. At 48 h after siRNA transfection, cells infected with HCVpp derived from either genotype 1b (H77) or genotype 2a (JFH1) for 6 h. VSVpp was used as a control. Cell medium was replaced with fresh DMEM containing antibiotics. At 48 h postinfection, cells were harvested and then luciferase activity was determined. (B) Huh7.5 cells were transfected with either negative or SLC3A2-specific siRNA for 24 h, followed by transfection with Flag-tagged siRNA-resistant SLC3A2 mutant plasmid. At 24 h after plasmid transfection, cells were infected with HCVpp derived from either H77 (left) or JFH1 (right). At 48 h postinfection, total cells were harvested and luciferase activities were determined. (C) (Left panel) Schematic diagram of the plasmids used for the production of HCV-LP. (Right panel) Huh7.5 cells were transfected with either negative or SLC3A2-specific siRNAs. At 48 h after siRNA transfection, cells were infected with HCV-LP. At 48 h postinfection, total cells were harvested and luciferase activities were determined for single cycle infection. Data represent averages from at least three independent experiments. The asterisks indicate significant differences (***p < 0.001) from the activity for the negative control. (D) (Left panel) Huh7.5 cells were transfected with the indicated siRNAs for 48 h. The cells were incubated with Jc1 at 4 °C for 2 h for binding. The cells were washed with PBS and then bound virions were determined by analyzing HCV RNA levels by qRT-PCR. (Right panel) Huh7.5 cells were transfected with the indicated siRNAs. At 48 h after transfection, cells were incubated with Jc1 at 4 °C for 2 h. The cells were washed with PBS and then temperature was shifted to 37 °C for 4 h. The cells were trypsinized and washed twice with PBS to remove free virions. Internalized HCV virions were indirectly determined by analyzing HCV RNA levels by qRT-PCR. (E) HEK293T cells were cotransfected with Flag-tagged SLC3A2 and either vector or V5-tagged E2 expression plasmid. At 36 h after transfection, total cell lysates were immunoprecipitated with an anti-Flag antibody and bound proteins were analyzed by immunoblot analysis using an anti-E2 antibody and secondary antibodies. To verify E2 protein, we used another kind of secondary antibody, E2(s), which did not recognize heavy chain. (F) Total cell lysates prepared as described in (E) were immunoprecipitated with anti-V5 antibody and bound proteins were detected by immunoblot analysis using an anti-Flag antibody. (G) HEK293T cells were cotransfected with Flag-tagged LAT1 and either vector or V5-tagged E2 plasmid. At 30 h after transfection, total cell lysates were immunoprecipitated with an anti-V5 antibody and bound proteins were analyzed by immunoblot analysis using an anti-Flag antibody.

Fig 3: SLC3A2/LAT1 complex is upregulated by HCV infection. (A) Huh7.5 cells were either mock-infected or infected with Jc1 for the indicated time intervals. Both mRNA (top panel) and protein (bottom panel) levels of LAT1 were analyzed either by qRT-PCR or immunoblot analysis, respectively. (B) Huh7.5 cells were treated with either reducing or non-reducing buffer as described in Methods. Total cell lysates were subjected to native gel electrophoresis and immunoblotted with either SLC3A2 or LAT1 antibodies, respectively. (C) Huh7.5 cells were either mock-infected or infected with Jc1 for 4 and 8 days, respectively. Total cell lysates prepared by native lysis buffer were immunoblotted with the indicated antibodies.

Fig 4: Characterization of m-reMAIT cells.(A) Flow cytometric profiles of m-reMAIT cells. mMR1-tet staining and expression of T-cell receptor ß (TCRß), CD4, CD8, CD25, and CD44 and the transcription factors PLZF and ROR?t in m-reMAIT cells on differentiation day 18. (B) 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) dose-dependent activation of m-reMAIT cells. The percentages of CD69+ cells among m-reMAIT cells challenged with the indicated concentration of 5-OP-RU in the presence of WT3 (?), WT3/mMR1 (?), CH27 (?), and CH27/mMR1(¦). (C) MR1-dependent activation of m-reMAIT cells. The percentage of CD69+ cells among m-reMAIT cells cultured with CH27/mMR1, challenged as in (B) in the presence of the anti-MR1 antibody (¦) or the isotype control antibody (?). (D) 5-OP-RU dose-dependent activation. The percentage of m-reMAIT cells expressing CD69 upon a challenge with various concentrations of 5-OP-RU. Representative data from two independent experiments are shown. (E) mMR1-tet dose-dependent activation. The percentage of m-reMAIT cells expressing CD69 upon a challenge with the indicated amounts of mMR1-tet. Representative data from two independent experiments are shown. (F) 5-OP-RU- and mMR1-tet-induced cytokines and chemokines. m-reMAIT cells were stimulated with various concentrations of 5-OP-RU (?) or mMR1-tet (¦) and the resultant cytokines and chemokines were quantified with LegendPlex. The concentrations at which each reagent induced a similar degree of activation (% CD69) are shown as relative concentrations (0.1–100 nM for 5-OP-RU and 0.01–10 µg/ml for mMR1-tet). The number on the X-axis corresponds to that in (D) and (E). (G) Tyrosine phosphorylation elicited with 5-OP-RU. A Western blot analysis with PY99 (anti-phosphotyrosine). Upon a challenge with different concentrations of 5-OP-RU for 30 min, the cell lysate from m-reMAIT cells was separated on SDS-PAGE (5 × 105/lane), and subjected to Western blotting. Lane 1, 0; lane 2, 0.1; lane 3, 1.0; lane 4, 10; lane 5, 100; lane 6, 1000; and lane 7, 10,000 (nM). Phosphorylated proteins are indicated with arrows. (H) Time course of tyrosine phosphorylation. A Western blot analysis with PY99. The cell lysate from m-reMAIT cells challenged with 100 nM of 5-OP-RU for the indicated time was separated on SDS-PAGE (5 × 105/lane) and subjected to Western blotting. Lane 1, 0; lane 2, 15; lane 3, 30; lane 4, 60; lane 5, 150; lane 6, 300 (min) . Arrows indicate phosphorylated proteins. (I) Linker for the activation of T cells (LAT) as a phosphorylated 37-kD protein. A Western blot analysis with PY99, anti-LAT, and anti-ß-actin. The cell lysate prepared as described in (H) for 60 min was subjected to Western blotting. A blot with PY99 (upper panel), anti-LAT (middle panel), and anti-ß-actin (lower panel). Phosphorylated LAT and LAT as well as ß-actin are indicated (arrow). Lane 1, 0; lane 2, 0.1; lane 3, 1.0; lane 4, 10; lane 5, 100; lane 6, 1000; lane 7, 10,000 (nM). (J) Tyrosine phosphorylation induced by mMR1-tet. A Western blot analysis with PY99. The cell lysate from m-reMAIT cells challenged with the indicated amounts of unlabeled mMR1-tet for 60 min was subjected to Western blotting (5 × 105/lane). Lane 1, 0; lane 2, 0.43; lane 3, 1.3; lane 4, 4.3; lane 5, 13 (µg/ml). Arrows indicate phosphorylated proteins. MAIT: mucosal-associated invariant T cell. Figure 1—source data 1.Characterization of m-reMAIT cells.The type of antigen-presenting cell (APC), reMAIT cells, and the concentration of 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) (nM) used for activation assays. Activation is shown as the percentage of CD69+ cells among reMAIT cells (B). The percentage of CD69+ MAIT cells challenged with 5-OP-RU in the presence of isotype control or anti-MR1 antibody (in the presence of CH27m as APC) (C). The percentage of CD69+ MAIT cells challenged with 5-OP-RU or mMR1-tet (D, E). Production of the cytokines and chemokines from m-reMAIT cells challenged with 5-OP-RU or mMR1-tet (F). MAIT: mucosal-associated invariant T cell. Figure 1—source data 2.Original gel electrophoresis panels for Figure 1G–J.