A3G and A3F do not inhibit L1 retrotransposition. HeLa-JVM cells were co-transfected with an L1 construct tagged with the retrotransposition indicator cassette and an APOBEC expression plasmid or control (pK/β-arr). Two different L1 constructs were used, a wildtype L1 (pDK101) and an L1 that contains the ColE1 origin of replication 3’ of the neomycin resistance cassette (SP101/L1.3K7). Three days post transfection cells were subjected to G418 for 12 days to select for retrotransposition events. (A) Representative results for the effect of A3G and A3F on L1 retrotransposition. Colonies were stained with crystal violet to visualize retrotransposition events. (B) For each experiment, colonies were counted from six wells and retrotransposition efficiency was determined relative to the control (pK/β-arr), which was set at 100%. Data are the average of at least two independent experiments, with standard deviation indicated. For A3G and A3F, similar results were observed in HeLa cell lines that stably expressed the A3G and A3F proteins (data not shown).

A3G, A3Gm, and A3F do not induce detectable hypermutation of retrotransposed Alu elements. HeLa-HA cells were transfected as described in Figure 2. As an additional control, cells were co-transfected with only an Alu construct and an L1 construct (No APOBEC). After four days of hygromycin selection, DNA was harvested from two wells to yield independent DNA samples. PCR was then performed to amplify the spliced neo gene from integrated Alu elements. PCR products were purified, cloned, and sequenced. For each reaction, a total of 12–14 sequences were examined for base changes from two independent genomic DNA samples. As seen previously, we sometimes detected the simultaneous mutation of three nucleotides (two C-to-T changes and one A-to-C change) surrounding the splice site present in the neo indicator cassette in controls or in cells transfected with an APOBEC3 protein (Bogerd et al., 2006b). These mutations have been removed from the data presented.

A3G inhibits Alu retrotransposition. HeLa-HA cells were co-transfected with an Alu construct tagged with the retrotransposition indicator cassette, an L1 construct, and an APOBEC protein or control (pK/β-arr). We used two different L1 constructs that lack the retrotransposition indicator cassette, a wildtype L1 (JM101/L1.3Δneo) and a L1 that lacks ORF1 coding sequence (ORF2Δneo). Three days post-transfection, cells were subjected to G418 for 12 days to select for retrotransposition events. (A) Representative results for the effect of A3G and A3F on Alu retrotransposition. (B) For each experiment, colonies were counted from two flasks and retrotransposition efficiency was determined relative to the control (pK/β-arr), which was set at 100%. Data are the average of at least two independent experiments, with standard deviation 20 indicated. For A3G, the retrotransposition frequency range was 5–23% (JM101/L1.3Δneo) in 5 independent experiments and 11–34% (ORF2Δneo) in 4 independent experiments as one experiment was removed due to low transfection efficiency. For A3Gm, the retrotransposition frequency range was 11–31% (JM101/L1.3Δneo) and 13–35% (ORF2Δneo) in 4 independent experiments.

A3G and A3F do not inhibit ORF1mneoI retrotransposition. HeLa-JVM cells were cotransfected with an L1 construct tagged with the retrotransposition indicator cassette that lacks the ORF2 coding sequence (ORF1mneoI), an L1 construct (pCep5’UTRORF2Δneo), and an APOBEC protein or control (pK/β-arr). Three days post-transfection, cells were subjected to G418 to select for retrotransposition events. (A) Representative results for the effect of A3G and A3F on ORF1mneoI retrotransposition. (B) For each experiment, colonies were counted from two flasks and retrotransposition efficiency was determined relative to the control (pK/β-arr), which was set at 100%. Data are the average of two independent experiments, with standard deviation indicated.