Figure 7. SIDT2 is required for RLR-induced MAVS activation in bystander cells during HSV-1 infection.

(A) PECs from Sidt2^+/+ and Sidt2^−/− mice infected with 1×10⁷ PFU at 16 h p.i. were stained with J2 dsRNA antibody (red) and DAPI (blue) and imaged by confocal microscopy. Scale bar = 10 μm. (B) The proportion of each cell occupied by punctate dsRNA staining was quantified using FIJI software. (C) Sidt2^+/+ and Sidt2^−/− MEFs stably expressing MAVS-YFP were infected with 1 MOI HSV-1-mCherry for 48 h, and imaged via confocal microscopy to assess for MAVS aggregation (arrows) in uninfected, bystander cells. Scale bar = 80 μm. (D) Individual cells were segmented and HSV-1-mCherry infected cells were excluded using FIJI software. Uninfected bystander cells (250 cells per condition) were scored for the appearance of MAVS aggregates. (E) Sidt2^+/+ (Cas9 only), Sidt2^−/− (Cas9 only), MAVS^−/− and Sidt2^−/− MAVS^−/− MEFs were infected with 1 MOI mCherry-tagged HSV-1, and IFNβ was measured in cell culture supernatant at 96 h p.i. via ELISA. (F) IL-6 and (G) TNF-α were measured in cell culture supernatant from shTLR3 and Sidt2^−/− shTLR3 MEFs infected with 1 MOI mCherry-tagged HSV-1 96h p.i. Sidt2^+/+ and Sidt2^−/− MEFs transduced with retroviral vector lacking shRNA were used as controls. IFN-β, IL-6 and TNF-α in non-infected cells were below the limit of assay detection. Data is representative of results from 2–3 independent experiments and expressed as the mean ± SEM of triplicate wells. ^* P < 0.05, ^** P < 0.01, n.s. = not significant. See also Figure S6.