Fig. 6

ZCCHC3 binds to dsDNA and facilitates the binding of cGAS to dsDNA. a ZCCHC3 enhances the binding of cGAS to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA and anti-ZCCHC3. b ZCCHC3-deficiency decreases the binding of cGAS to dsDNA in HEK293 cells. ZCCHC3-KO HEK293 clones were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HEK293 cells were transfected with HA-cGAS. Twenty hours after transfection, cells were collected for in vitro pull-down assays similarly as in a. c ZCCHC3-deficiency decreases the binding of endogenous cGAS to dsDNA in MLFs or MEFs. Zcchc3^+/+ and Zcchc3^−/− MLFs or MEFs were collected for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-cGAS and anti-ZCCHC3. d MST measurement of binding affinities. Binding affinities between the indicated recombinant proteins as well as between the indicated recombinant proteins and synthetic dsDNA HSV60 were measured by MST. The purified recombinant proteins were stained with Coomassie blue (right gel). e Effects of reconstitution of ZCCHC3 or ZCCHC3 (1-340) on HSV-1-induced transcription of downstream genes in Zcchc3^−/−MLFs. Zcchc3^−/− MLFs were reconstituted with murine ZCCHC3 or ZCCHC3(1-340) by lentiviral-mediated gene transfer. The reconstituted MLFs were left un-infected or infected with HSV-1 for 6 h before qPCR analysis. f Effects of ZCCHC3 on transcription of downstream genes induced by HSV120 in cGas^+/+ and cGas^−/− L929 cells. cGas^+/+ and cGas^−/− L929 cells stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. *P < 0.05, **P < 0.01 (unpaired t test). Data are representative of at least two experiments with similar results (mean ± SD, n = 3 independent samples in e, f)