Fig 2: HSV-1 restriction by OPTN occurs via a TBK1-independent pathway. HeLa cells were infected with 0.1 multiplicity of infection (MOI) HSV-1 McKrae strain for 24 h. (A) Representative confocal imaging of WT, OPTN KO, and TBK1 KO HeLa cells. (B) Plaque assay data for HeLa 24 hpi. (C) Immunoblot for HeLa cells against HSV-1 proteins gB and VP-16. (D and E) Quantification of expression of gB and VP-16 relative to GAPDH from Immunoblot data using ImageJ. n = 3 independent replicates per group. A one-way analysis of variance test was used to determine statistical significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns not significant.

Fig 3: HSV-1 proteins UL-46 and γ134.5 can interact with OPTN via TBK1. (A) Immunoblot for WT cells exposed to 0.1 MOI HSV-1 Mckrae strain (WT) and HSV-1 Mckrae Δ γ134.5 strain(Δ γ134.5) for 24 h. Quantification of expression of OPTN relative to GAPDH from Immunoblot data using ImageJ. (B) Immunoblot for WT cells was exposed to 0.1 MOI HSV-1 KOS strain (WT) and HSV-1 KOS Δ UL46 strain(ΔUL46) for 24 h. Quantification of expression of OPTN relative to GAPDH from Immunoblot data using ImageJ. (C) Immunoblot for TBK1 KO cells was exposed to 0.1 MOI HSV-1 KOS strain (WT) and HSV-1 KOS Δ UL46 strain (ΔUL46) for 24 h. Quantification of expression of OPTN relative to GAPDH from Immunoblot data using ImageJ; n = 3 independent replicates per group. A one-way analysis of variance test was used to determine statistical significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. (D and E) Plaque assay data after 24 hpi. n = 3 independent replicates per group. A two-tailed Student’s t test was used to determine statistical significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. (F) Representative confocal imaging of WT and TBK1 KO cells infected with HSV-1 UL46gfp.

Fig 4: Transient knockdown of OPTN can restrict HSV-1 infection in HCE without aid from TBK1. (A) Representative confocal imaging of WT, siOPTN, and siTBK1 cells infected with 0.1 MOI HSV-1 McKrae for 24 h. (B) Plaque assay data after 24 hpi. (C) Immunoblot for HCE cells against HSV-1 proteins gB and VP-16. (D and E) Quantification of expression of gB and VP-16 relative to GAPDH from Immunoblot data using ImageJ. n = 3 independent replicates per group. A one-way analysis of variance test was used to determine statistical significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns not significant.