Extended data Figure 5. ZAP mediates deleterious effects of CG-dinucleotides on HIV-1 replication.

a, Western blot analyses, using the indicated antibodies, following transfection of HeLa cells with the corresponding siRNAs, or control siRNAs, in the single-cycle replication assays described in Fig. 3a. Representative of 2 experiments. b, Western blot analysis of ZAP expression in control, CRISPR knockout MT4 cells and doxycycline-inducible ZAP-S reconstituted MT4 cells. Asterisks indicate protein species that appeared in some CRISPR knockout clones, reacted with an anti-ZAP antibody and arose after extended passage. These likely represent truncated forms of ZAP-L whose translation initiated at methionine codons 3′ to the CRISPR target site (that was near the ZAP N-terminus) Representative of 3 experiments. c, Western blot analysis (anti-Gag, anti-Env anti-GFP and anti-Tubulin) of viral, and cellular protein levels in cells and virions, 48h after a single-cycle WT and mutant HIV-1infection of ZAP^−/− MT4 cells that had been reconstituted with a doxycycline-inducible ZAP-S expression construct (ZAP_DI) and left untreated or treated with doxycycline. Representative of 3 experiments.