Fig. 5.

UL56 impairs cGAS binding to viral DNA. (A) UL56 impairs the binding of cGAS to dsDNA. HEK293T cells were transfected with the indicated plasmids. Twenty-four hours later, the cell extracts were incubated with biotinylated-HSV120 and streptavidin agarose for 3 h. The bound proteins were analyzed by immunoblots with the indicated antibodies. (B) MST measurement of binding affinities. Binding affinities between GST-cGAS and the synthetic dsDNA HSV60 in the presence of UL56 or BSA were measured by MST. (C) UL56 impairs the binding of cGAS to HSV-1 DNA. HEK293T cells were transfected with HA-cGAS and Flag-UL56 or an empty vector for 20 h. After transfection, cells were infected with HSV-1 (MOI=1) for 3 h. The cell lysate was then immunoprecipitated with control mouse IgG or anti-HA. The protein-bound DNA was extracted and analyzed by qPCR analysis. Specific cGAS-bound DNA amount in each sample was calculated by subtraction of anti-HA-precipitated DNA minus control mouse IgG-precipitated DNA. The specific cGAS-bound DNA amount from the empty vector-transfected sample was treated as 1.0, and the relative specific cGAS-bound DNA amount from UL56-transfected sample was calculated by dividing the specific raw DNA amount with the specific cGAS-bound DNA amount from the empty vector-transfected sample. (D) Deficiency of UL56 increases binding of endogenous cGAS to HSV-1 DNA. MLFs were infected with HSV-1 (MOI=1) or HSV-1ΔUL56 (MOI=1) for 12 h. The cell lysate was then immunoprecipitated with control mouse IgG or anti-cGAS (mouse). The protein-bound DNA was extracted and analyzed by qPCR analysis. Specific cGAS-bound DNA amount in each sample was calculated by subtraction of anti-cGAS-precipitated DNA minus control IgG-precipitated DNA. The specific cGAS-bound DNA amount from HSV-1-infected sample was treated as 1.0, and the relative specific cGAS-bound DNA amount from HSV1ΔUL56-infected sample was calculated by dividing the specific raw DNA amount with the specific cGAS-bound DNA amount from the HSV-1-infected sample. (E) UL56 inhibits HT-DNA- or HSV-1-induced synthesis of cGAMP. THP-1 cells stably-expressing UL56 or control cells were transfected with HT-DNA for 4 h or infected with HSV-1 (MOI=1) for the indicated times. cGAMP in the cell extracts was measured by ELISA. (F) UL56 inhibits cGAS enzymatic activity in vitro. Purified His-cGAS and His-UL56 or BSA were mixed with ATP, GTP, and HT-DNA in the reaction buffer. After incubation, samples were collected for Mono Q analysis of cGAMP production. (G) Effects of UL56 on cGAMP-induced transcription of antiviral genes. Digitonin-permeabilized MLFs stably-expressing UL56 or control cells (1×10⁶) were transfected with cGAMP (0.1 μg) for 3 h before qPCR analysis.

The data shown are means ​± ​SD (C-E, G) 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).