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

FIG. 2. Mammalian APOBEC3 phylogeny. The phylogenetic relationships of mammalian A3 proteins were inferred using the maximum likelihood method and JTT þ G model with 1,000 bootstrap replicates. A3 subgroups Z1, Z2, and Z3 are paralogs, which have expanded independently. Bootstrap values for nodes represented in >40% of trees are shown. A3Z1, A3Z2/Z2A, A3Z2B, and A3Z3 clades are shown in green, orange, pink, and blue, respectively. The scale represents the number of amino acid substitutions per site. Differential Evolution of Antiretroviral Restriction Factors in Pteropid Bats . doi:10.1093/molbev/msy048 MBE Ancient Bat Retroviruses Were Hypermutated by APOBEC3 A3 activity results in C to U lesions in the negative (cDNA) strand, which lead to positive (genomic) strand G to A mutations in endogenous retroviruses (ERVs) within host genomes (Perez-Caballero et al. 2008) . A3 proteins also exhibit local sequence-context bias in the nucleobases they deaminate. The presence of strand-biased, context-specific A3 mutagenic signatures in ERVs constitutes unambiguous evidence of a past encounter with one or more cellular A3 enzymes (Lee et al. 2008) . To assess hypermutation of pteropid ERVs, we identified a group of closely related ERVs from each of the Betaretrovirus and Gammaretrovirus genera present in the genome of P. vampyrus. We interrogated the P. vampyrus genome since the P. alecto genome assembly does not contain sufficient contiguous endogenous retroviral sequences to conduct this analysis. A previously reported group of bat endogenous betaretroviruses, sub-classified as Group 7 betaretroviruses, was selected and a group of bat endogenous gammaretroviruses (herein described as Group 1 gammaretroviruses) was identified in the manner described previously (Hayward et al. 2013 ). Phylogenies were generated using the maximum likelihood method and an ancestral reconstruction was performed by extrapolation of the common ancestor of each group (see supplementary fig. S4 , Supplementary Material online). Mutations in each ERV were identified by comparison against the group's common ancestor. Strand-biased G to A hypermutation and contextspecific GG and GA dinucleotide preferences were identified in both groups ( fig. 5 ). These results are consistent with the hypothesis that ancient bat retroviruses were subjected to A3-mediated hypermutation, supporting the expected role of bat A3 proteins as antiviral restriction factors. The divergence times of the bat A3Z1 genes were estimated using a molecular clock dating analysis that indicates these genes arose through duplication events beginning approximately 27 Ma (supplementary fig. S5 , Supplementary Material online).