Extended Data Fig. 8. TMEFF1 restricts the early translocation of HSV-1 to the cell nucleus by interfering with gD-NECTIN-1-mediated viral entry.
a, Representative gating strategy for HEK293T cells stably expressing NECTIN-1, not treated or treated with recombinant His-tagged HSV-1 gD for 150 min. b, Mean fluorescence intensity (MFI) of surface gD binding in NECTIN-1-positive or NECTIN-1-negative fractions of HEK293T cells stably expressing NECTIN-1, following incubation with different concentrations of His-tagged gD (gD-His-Tag) for 150 min. c, Histogram of surface NECTIN-1 expression in WT and TMEFF1 KO HEK293T cells incubated with recombinant His-tagged HSV-1 gD (5 µg/ml) for 150 min. The data shown are representative of three independent experiments. d, Histogram of surface NECTIN-1 expression in WT and TMEFF1 KO HEK293T cells infected with HSV-1 (MOI 10) for 45 min. The data shown are representative of three independent experiments. e, NECTIN1 mRNA levels (left panel) as determined by RT-qPCR in total RNA, and protein expression in cell total lysates as assessed by immunoblotting (right panel), in TMEFF1 KO or parental WT HEK293T cells upon gD treatment for 150 min or without treatment. f, NECTIN1 mRNA levels (left panel) as determined by RT-qPCR in total RNA, and protein expression in cell total lysates as assessed by immunoblotting (right panel), in TMEFF1 KO or parental WT HEK293T cells after infection with HSV-1 for 45 min, or without infection. g, Representative contour plots of the RFP signal in WT, TMEFF1 KO, NECTIN-1 KO, and TMEFF1 and NECTIN-1 double KO HEK293T cells infected with HSV-1-RFP (MOI 10) at 8 hpi. h, Basal mRNA levels for NECTIN-1, HVEM, and PILRa, as assessed by RT-qPCR, in WT parental or TMEFF1 KO hPSC-derived cortical neurons and HEK293T cells (left panel), and WT HeLa cells (right panel). The data shown are the mean ± SEM from three independent experiments. i, MFI of N-ter YFP-tagged HVEM on the cell surface in NECTIN-1 KO and TMEFF1 and NECTIN-1 double KO HEK293T cells transfected with an empty vector (EV), or N-ter YFP-tagged HVEM-expressing plasmid. The data shown are the mean ± SEM from four independent experiments. j, Percentage of HSV-1-positive cells, as assessed in an assay of HSV-1 translocation to the cell nucleus 10 h after infection with an RFP-reporter HSV-1, in NECTIN-1 KO or TMEFF1 and NECTIN-1 double KO HEK293T cells transfected with an empty vector (EV) or HVEM-expressing plasmid. The data are presented as the mean ± SEM from four independent experiments. Statistical analysis was conducted with Kruskal-Wallis tests with Dunn’s test for multiple comparisons. ns: not significant.