Differential Evolution of Antiretroviral Restriction Factors in Pteropid Bats as Revealed by APOBEC3 Gene Complexity

Hayward, Joshua A; Tachedjian, Mary; Cui, Jie; Cheng, Adam Z; Johnson, Adam; Baker, Michelle L; Harris, Reuben S; Wang, Lin-Fa; Tachedjian, Gilda

We tested the activity of the bat A3 proteins for which expression was verified (supplementary table S1, Supplementary Material online) by assessing their ability to cause mutations in E. coli though a rifampicin mutagenesis assay, utilizing human A3B and A3G as positive controls because they are known to be expressed and enzymatically active in E. coli (Holden et al. 2008; Shi et al. 2015) . Mutations in the rpoB gene increase with mutagenic stress mediated by A3 (Harris et al. 2002; Shi et al. 2015) . Mutational frequencies above background levels were only observed for A3Z1 proteins, and we found that four bat A3Z1s showed a 10-fold or greater increase in mutation frequency ( fig. 3A ). In particular, A3Z1c isomer B showed over 1000-fold increase above empty vector and 100-fold above A3Z1c isomer A. To determine if the difference in mutation frequency correlated with differences in mutation signatures, we sequenced a PCR-amplified portion of the rpoB gene known to harbor hotspots for rifampicin resistance. Bat A3 mutation signatures were compared against human A3B and A3G ( fig. 3B ), which have dinucleotide sequence preferences for 5 0 -TC and 5 0 -CC, respectively. We found that bat A3Z1c isomer A was highly specific for causing mutation at a known 5 0 -TCG hotspot at nucleotide position 1585, which is also the preferred target site for A3B (Shi et al. 2015) . In contrast, A3Z1c isomer B targeted sites at position 1565, 1585, and 1592, which have 5 0 -TCT, 5 0 -TCG, and 5 0 -TCC contexts, respectively. Decreased specificity may account for the high mutation frequency observed in the rifampicin mutagenesis assays. These results show that bat A3 proteins are functionally active and can mutate cytosines in a diverse range of dinucleotide contexts.