Figure 7. Reconstitution of WT ATG4A and LC3B2 in P1 and P2 primary fibroblasts, respectively, rescues autophagy and the antiviral response following HSV2 infection.

(a) Flow cytometric analysis (CytoID) of HSV2-activated autophagy P1 primary fibroblasts electroporated with L90I ATG4 or wt ATG4A with quantification of ATG4 expression. (b) Flow cytometric analysis (CytoID) of HSV2-activated autophagy in P2 primary fibroblasts electroporated with L109M LC3B2 or wt LCB3B with quantification by immunoblotting of LC3B2 protein expression. (c, d) Plaque assay for HSV2 replication in supernatants from P1 (c) and P2 (d) fibroblasts electroporated with the relevant mutant and wt alleles of ATG4A and LC3B2. (e, f) Cytotoxicity measured as LDH release from untreated and HSV2-infected (MOI 1, 24h) primary fibroblasts from P1 (e) and P2 (f) electroporated with the relevant mutant and wt alleles of ATG4A and LC3B2. (g,h) Wt fibroblasts were depleted for endogenous LCB32 by CRISPR/Cas9 technology or electroporated with the guide AAVS1 as control, followed by expression of either WT LC3B2 or L109M LC3B2 by electroporation, and (g) measurement of LC3-I and LCB3-II levels in cells after xh and h) quantification of HSV2-induced autophagy in the modified cells by CytoID. (i) Plaque assay showing HSV2 replication in the supernatants of cells from (g,h). (j-m) Expression of increasing amounts of L90I ATG4A and L109M LC3B2 mutants in WT fibroblasts by electroporation and (j.k) measurement of protein levels and (l,m) HSV2-induced autophagy in the cells from (j,k) by CytoID. The experiments shown were performed at least twice. For the histograms in panel a, b,c,d,h and i, data are shown as mean values +/- st.dev. 1Way ANOVA was used for the data presented in panel a,b,c,d,h, whereas Mann-Whitney was used for panel i * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns, not significant.