Fig 5. CRISPR targeting of TRIM25 leads to increased virus replication and both the RING and CCD domains of TRIM25 are required for ZAP activation.
(A) Wild type (clone E) and TRIM25lo ZC3HAV1-knockout 293T cells (clones D and F) were transfected with empty vector or vector expressing ZAPS or ZAPL and infected with Toto1101/Luc (MOI = 0.01) 2 days post-transfection. (B) TRIM25lo ZC3HAV1-knockout 293T cells (clones D and F) were reconstituted with expression of FL or truncated TRIM25 (ΔRING, ΔCCD) and/or ZAPS or ZAPL, and infected with Toto1101/Luc (MOI = 10) 2 days post-transfection. (A and B) The data is representative of 2 independent experiments performed on both clones D and F. Cell lysates were harvested for measurement of luciferase activity at 24 h p.i. Relative luciferase units represent the level of SINV replication. Asterisks indicate statistically significant differences (Student’s t-test, *, p<0.05; **, p<0.005; ***, p<0.0005).