Fig. 5.
PCBP1 promotes the binding of cGAS to DNA. a PCBP1 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 DNA and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblotting with anti-HA and anti-PCBP1 antibodies. b PCBP1 deficiency decreases the binding of cGAS to dsDNA in the MLFs. PCBP1-deficient and control MLFs were collected for in vitro pull-down assays as in a. c PCBP1 deficiency decreases the binding of cGAS to the HSV-1 DNA in the infected MLFs. PCBP1-deficient and control MLFs were infected with HSV-1, and 4 h later, the cell lysates were immunoprecipitated with anti-cGAS. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the HSV-1 genome. The graph shows the means ± SEM, n = 3. **P < 0.01 (unpaired t-test). d PCBP1 enhances the binding of cGAS to HIV-RT-DNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with biotinylated HIV-RT-DNA and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblotting with anti-HA and anti-PCBP1 antibodies. e PCBP1 deficiency decreases the binding of cGAS to HIV-RT-DNA in the MLFs. PCBP1-deficient and control MLFs were collected for in vitro pull-down assays as in d. f PCBP1 promotes cGAS binding to dsDNA in the ZCCHC3-deficient cells. ZCCHC3-deficient and control HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with biotinylated-HSV120 DNA and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblotting with anti-HA and anti-PCBP1 antibodies. g ZCCHC3 promotes cGAS binding to dsDNA in the PCBP1-deficient cells. PCBP1-deficient and control HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cells were collected for in vitro pull-down assays as in f. h Effects of PCBP1/ZCCHC3 double deficiency on the HSV-1-triggered transcription of downstream genes in the MLFs. Deficiency of PCBP1 was generated by the CRISPR-Cas9 method in the Zcchc3+/+ and Zcchc3−/− MLFs. The indicated MLFs (1 × 106) were untreated or infected with HSV-1 (MOI = 1) for 6 h before the qPCR analysis. The graph shows the means ± SEM, n = 3. **P < 0.01 (unpaired t-test)