Fig. 1.
HSV-1 UL56 is an inhibitor of viral DNA-triggered innate antiviral response. (A) UL56 inhibits cGAS-MITA-mediated activation of the IFNβ promoter, ISRE and NF-κB. HEK293-MITA cells (1×105) were transfected with the IFNβ promoter (50 ng), ISRE (50 ng) or NF-κB (2 ng) reporter plasmids, and expression plasmids for cGAS (100 ng), or an empty vector as well as the UL56 plasmids for 24 h before luciferase assays were performed. The immunoblot analysis was performed with the indicated antibodies. (B) UL56 inhibits HSV-1- and HCMV-, but not SeV-triggered transcription of antiviral genes in HFFs. HFFs stably-expressing UL56 or control cells were left uninfected or infected with HSV-1 (MOI=1), HCMV (MOI=1) or SeV (MOI=1) for the indicated times before qPCR analysis. (C) UL56 inhibits HSV-1-triggered transcription of antiviral genes. MLFs stably-expressing UL56 or control cells were left uninfected or infected with HSV-1 (MOI=1) for the indicated times before qPCR analysis. (D) Both the N-terminal PPxY motifs and C-terminal transmembrane domain of UL56 are essential for its inhibition of HSV-1-triggered transcription of antiviral genes. MLFs stably-expressing UL56, transmembrane domain-deletion (ΔTM) or PY motif-mutated (P/A) mutant were left uninfected or infected with HSV-1 (MOI=1) for 6 h before qPCR analysis. (E) UL56 inhibits UV-inactivated HSV-1-triggered transcription of antiviral genes. HFFs stably-expressing UL56 were left uninfected or infected with untreated or UV-inactivated HSV-1 (MOI=1) for the indicated times before qPCR analysis. (F) UL56 inhibits dsDNA-triggered transcription of antiviral genes. HFFs and MLFs stably-expressing UL56 were mock-transfected or transfected with HSV120 for the indicated times, or with HT-DNA or DNA90 for 4 h before qPCR analysis. (G) UL56 inhibits dsDNA-triggered phosphorylation of MITA, TBK1, and IRF3. MLFs stably-expressing UL56 were mock-transfected or transfected with HSV120 for the indicated times before immunoblot analysis was performed with the indicated antibodies.
The data shown are means ± SD (A–F) from one representative experiment performed in triplicates (3 technical repeats). Similar data were obtained from at least two independent experiments. ∗, p < 0.05; ∗∗, p < 0.01 (Student's unpaired t-test).