Figure 3. DNA transfection and DNA virus infection induce IFNβ through cGAMP.

(A) Chemically synthesized cGAMP (100 nM) was delivered to digitonin-permeabilized L929 cells for indicated times, then IFNβ RNA and secreted protein were measured by q-RT-PCR (inset) and ELISA, respectively. Unless noted otherwise, the error bars in this and all other panels represent standard errors of the mean (n=3). (B) Similar to (A), except that different concentrations of cGAMP were delivered into L929 cells for 8 hr followed by q-RT-PCR analyses of IFNβ RNA. (C) Similar to (B), except that different concentrations of cGAMP and cdi-GMP were delivered into L929 cells followed by ELISA assays for IFNβ. (D) L929 cells were infected with HSV-1Δ34.5 or VSV-ΔM51-GFP, transfected with HT-DNA, or mock treated. An aliquot of the cell extracts was directly analyzed for IRF3 dimerization (top), whereas another aliquot was heated to denature proteins and the heat-resistant supernatant was assayed for its ability to stimulate IRF3 dimerization in permeabilized Raw264.7 cells (bottom). (E) The heat-resistant supernatant from (D) was fractionated by HPLC using a C18 column, and the presence of cGAMP in the fractions was measured by mass spectrometry using SRM. (F) L929 cells were transfected with 4 μg/ml HT-DNA for the indicated time, then IFNβ RNA was measured by q-RT-PCR and IRF3 dimerization was analyzed by native PAGE. Aliquots of the cell extracts were tested for the presence of cGAMP based on its ability to induce IRF3 dimerization after delivery into Raw264.7 cells. (G) THP1 cells were infected with HSV-1Δ34.5 and Vaccinia virus (VACV) for 6 hr, then the activation of endogenous IRF3 and generation of cGAMP activity were measured as described in (F).