ABSTRACTAPOBEC3 deaminases (A3s) provide mammals with an anti-retroviral barrier by catalyzing dC-to-dU deamination on viral ssDNA. Within primates, A3s have evolved diversely via gene duplications and fusions. Human APOBEC3C (hA3C) efficiently restricts the replication of viral infectivity factor (vif)-deficient Simian immunodeficiency virus (SIVΔvif), but for unknown reasons, it inhibits HIV-1Δvif weakly. In catarrhines (Old World monkeys and apes), the A3C loop 1 displays the conserved amino acid pair WE, while the corresponding consensus sequence in A3F and A3D is the largely divergent pair RK, which is also the inferred ancestral sequence for the last common ancestor of A3C|D|F in primates. Here, we report that modifying the WE residues in hA3C loop 1 to RK leads to stronger interactions with ssDNA substrate, facilitating catalytic function, which resulted in a drastic increase in both deamination activity and the ability to restrict HIV-1 and LINE-1 replication. Conversely, the modification hA3F_WE resulted only in a marginal decrease in HIV-1Δvif inhibition. The two series of ancestral gene duplications that generated A3C, A3D-CTD and A3F-CTD allowed neo/subfunctionalization: A3F-CTD maintained the ancestral RK residues in loop 1, while strong evolutionary pressure selected for the RK→WE modification in catarrhines A3C, possibly allowing for novel substrate specificity and function.AUTHOR SUMMARYThe restriction factors of the APOBEC3 (A3) family of cytidine deaminases inhibit the replication of Vif-deficient retroviruses mainly by mutating their viral genomes. While there are seven A3 proteins (A3A-A3H) found in humans only A3G and A3F potently inhibit HIV-1 replication. A3C in general and its retroviral restriction capacity have not been widely studied probably due to its weak anti-HIV-1 activity, however, it displays a strong antiviral effect against SIV. Understanding the role of A3C is important because it is highly expressed in CD4+ T cells, is upregulated upon HIV-1 infection, and is distributed cell-wide. In this study, we report that replacing two residues in loop 1 of A3C protein with conserved positively-charged amino acids enhance the substrate DNA binding, which markedly facilitates its deamination-dependent antiviral activity against HIV-1 as well as increasing the restriction of LINE-1 retroelements. Furthermore, our evolutionary analysis demonstrates that the pressure that caused the loss of potential loop 1 residues occurred only in A3C but not in primate homologues. Overall, our study highlights the possibility of A3C acting as a super restriction factor, however, this was likely evolutionarily selected against to achieve a balance between anti-viral/anti-LINE-1 activity and genotoxicity.
Evolutionary Analysis of HIV-1 Pol Proteins Reveals Representative Residues for Viral Subtype Differentiation