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

Assessment of the functionality of bat A3 proteins through a rifampicin mutagenesis assay revealed several A3Z1 proteins as potent mediators of cytosine deamination with A3Z1 isomer B demonstrating a mutational frequency greater than human A3B and A3G. Although some A3Z1 proteins, and A3 proteins of other subtypes were not found to be catalytically active in this assay, numerous factors could account for inactivity within E. coli, such as protein misfolding or missing cellular cofactors and localization requirements. The capacity of bat A3 proteins to restrict retroviral infection was observed through the use of HIV-1 as a model target for A3 functionality. Four bat A3 proteins representing single and double Z-domain proteins of the Z2A, Z2B, and Z3 subtypes were observed to be capable of inhibiting HIV-1 infectivity in a dose-dependent manner. The bat A3Z1 proteins found to be mutagenic were not found to be capable of restricting HIV-1 infectivity. A possible explanation for this is that not all bat A3 proteins are capable of restricting this specific virus, as is the case for human A3 proteins such as A3B and A3C which inhibit SIV but not HIV, whereas A3F and A3G are potent inhibitors of HIV (Sheehy et al. 2003; Yu et al. 2004; Zheng et al. 2004) . Although many endogenous retroviruses are present in bat genomes (Cui et al. 2012; Hayward et al. 2013) , no exogenous bat retroviruses have yet been reported. To address our expectation that bat retroviruses could be subjected to restriction by bat A3 proteins we performed a hypermutation analysis of the endogenized retroviruses present in the genome of P. vampyrus and found evidence of strand-biased, context-dependent, cytosine deamination of both beta-and gammaretroviruses. These results implicate bat A3 proteins as the mediators of G to A hypermutation in bat retroviruses.