Figure Supplementary 6: The effect of inhibitors, molecular deficiency, and anti-TLR3 mAb on HSV-1 infection.

(a) Progeny virus yields (PFU/ml) in supernatants from MEF-TLR3-Unc93B1 cells untreated (-) or treated for 2 h with Go 6976 (G) at 250 nM or Torin 1 (T) at 1 μM before the HSV-1 infection for 24 h at an MOI of 10. The progeny virus yields were determined with Vero cells. (b) WT and Rab7a^Δ/Δ MEF-TLR3-Unc93B1 cells were infected with the HSV-1 gB-RFP (red fluorescent protein) at an MOI of 10. RFP expression enables detection of HSV-1 in infected cells by confocal microscopy. After 6 h post-infection, the cells were immunostained with anti-TLR3 mAb, and the subcellular distribution of HSV-1 gB-RFP and TLR3 was analyzed. Statistical analyses of TLR3 colocalization with gB-RFP are shown. These experiments were repeated more than three times. (c) MEF-TLR3-Unc93B1 cells were treated with DMSO (Vehicle) or Torin 1 at 1 μM for 24 h and stained with Annexin V to determine cell viability. (d) WT mice (n = 10 per group) were intracranially administered with DMSO (Vehicle) or 6 μg of Torin1. Mice survival is shown. (e) WT mice were intracranially treated with 6 μg of Torin1. After 2 h, cell lysate was prepared, and immunoblotting of the indicated molecules was conducted. Grb2, a loading control. These experiments were repeated more than three times. (f) MEF-TLR3-Unc93B1 cells were left untreated or treated with control IgG or anti-TLR3 mAb (PaT3) at 4 or 20 μg/ml for 2 h. The treated cells were left unstimulated (U), stimulated (PIC) with poly(I:C) at 25 μg/ml for 24 h, or infected (HSV) with HSV-1 at an MOI of 10 for 24 h. The concentration of CCL5 and IFN-β in the supernatants was determined by ELISA. The results are represented by the mean value ± s.d. of triplicate wells. (g) WT mice (n = 8 per group) were intracranially infected with indicated doses of HSV-1 and mice survival was evaluated. (h, i) WT and Ifnar1^−/− mice were intracranially infected with 10³ PFU of HSV-1 (h) Mice survival is shown. n = 8 per group. (i) Virus yields in the brain at 2 days post infection. n > 3 per group. Statistical analysis was performed by the log-rank test (h) or the two-tailed Student’s t test (i). ***P < 0.001