Mutational comparison of the single-domained APOBEC3C and double-domained APOBEC3F/G anti-retroviral cytidine deaminases provides insight into their DNA target site specificities

Human APOBEC3F and APOBEC3G are double-domained deaminases that can catalyze dC→dU deamination in HIV-1 and MLV retroviral DNA replication intermediates, targeting T–C or C–C dinucleotides, respectively. HIV-1 antagonizes their action through its vif gene product, which has been shown (at least in the case of APOBEC3G) to interact with the N-terminal domain of the deaminase, triggering its degradation. Here, we compare APOBEC3F and APOBEC3G to APOBEC3C, a single-domained deaminase that can also act on both HIV-1 and MLV. We find that whereas APOBEC3C contains all the information necessary for both Vif-binding and cytidine deaminase activity in a single domain, it is the C-terminal domain of APOBEC3F and APOBEC3G that confer their target site specificity for cytidine deamination. We have exploited the fact that APOBEC3C, whilst highly homologous to the C-terminal domain of APOBEC3F, exhibits a distinct target site specificity (preferring Y–C dinucleotides) in order to identify residues in APOBEC3F that might affect its target site specificity. We find that this specificity can be altered by single amino acid substitutions at several distinct positions, suggesting that the strong dependence of APOBEC3-mediated deoxycytidine deamination on the 5′-flanking nucleotide is sensitive to relatively subtle changes in the APOBEC3 structure. The approach has allowed the isolation of APOBEC3 DNA mutators that exhibit novel target site preferences

The AID/APOBEC family of nucleic acid mutators

The AID/APOBECs, a group of cytidine deaminases, represent a somewhat unusual protein family that can insert mutations in DNA and RNA as a result of their ability to deaminate cytidine to uridine. The ancestral AID/APOBECs originated from a branch of the zinc-dependent deaminase superfamily at the beginning of the vertebrate radiation. Other members of the family have arisen in mammals and present a history of complex gene duplications and positive selection. All AID/APOBECs have a characteristic zinc-coordination motif, which forms the core of the catalytic site. The crystal structure of human APOBEC2 shows remarkable similarities to that of the bacterial tRNA-editing enzyme TadA, which suggests a conserved mechanism by which polynucleotides are recognized and deaminated. The AID/APOBECs seem to have diverse roles. AID and the APOBEC3s are DNA mutators, acting in antigen-driven antibody diversification processes and in an innate defense system against retroviruses, respectively. APOBEC1 edits the mRNA for apolipoprotein B, a protein involved in lipid transport. A detailed understanding of the biological roles of the family is still some way off, however, and the functions of some members of the family are completely unknown. Given their ability to mutate DNA, a role for the AID/APOBECs in the onset of cancer has been proposed

Flow-cytometric visualization of C>U mRNA editing reveals the dynamics of the process in live cells

<div><p>APOBEC1 is the catalytic subunit of the complex that edits ApolipoproteinB (ApoB) mRNA, which specifically deaminates cytidine 6666 to uracil in the human transcript. The editing leads to the generation of a stop codon, resulting in the synthesis of a truncated form of ApoB. We have developed a method to quantitatively assay ApoB RNA editing in live cells by using a double fluorescent mCherry-EGFP chimera containing a ∼300bp fragment encompassing the region of ApoB subject to RNA editing. Coexpression of APOBEC1 together with this chimera causes specific RNA editing of the ApoB fragment. The insertion of a stop codon between the mCherry and EGFP thus induces the loss of EGFP fluorescence. Using this method we analyze the dynamics of APOBEC1-dependent RNA editing under various conditions. Namely we show the interplay of APOBEC1 with known interactors (ACF, hnRNP-C1, GRY-RBP) in cells that are RNA editing-proficient (HuH-7) or -deficient (HEK-293T), and the effects of restricted cellular localization of APOBEC1 on the efficiency of the editing. Furthermore, our approach is effective in assaying the induction of RNA editing in Caco-2, a cellular model physiologically capable of ApoB RNA editing.</p></div

The Vif Protein of HIV Triggers Degradation of the Human Antiretroviral DNA Deaminase APOBEC3G

AbstractAPOBEC3G is a human cellular enzyme that is incorporated into retroviral particles and acts to restrict retroviral replication in infected cells by deaminating dC to dU in the first (minus)-strand cDNA replication intermediate [1–5]. HIV, however, encodes a protein (virion infectivity factor, Vif [6, 7]), which overcomes APOBEC3G-mediated restriction but by an unknown mechanism. Here, we show that Vif triggers APOBEC3G degradation by a proteasome-dependent pathway and that an 80 amino acid region of APOBEC3G surrounding its first zinc coordination motif is sufficient to confer the ability to partake in an interaction involving Vif. Inhibitors of this interaction might therefore prove therapeutically useful in blocking Vif-mediated APOBEC3G destruction

Splice Variants of Activation Induced Deaminase (AID) Do Not Affect the Efficiency of Class Switch Recombination in Murine CH12F3 Cells

<div><p>Activation Induced Deaminase (AID) triggers the antigen-driven antibody diversification processes through its ability to edit DNA. AID dependent DNA damage is also the cause of genetic alterations often found in mature B cell tumors. A number of splice variants of AID have been identified, for which a role in the modulation of its activity has been hypothesized. We have thus tested two of these splice variants, which we find catalytically inactive, for their ability to modulate the activity of endogenous AID in CH12F3 cells, a murine lymphoma cell line in which Class Switch Recombination (CSR) can be induced. In contrast to full-length AID, neither these splice variants or a catalytically impaired AID mutant affect the efficiency of Class Switch Recombination. Thus, while a role for these splice variants at the RNA level remains possible, it is unlikely that they exert any regulatory effect on the function of AID.</p></div

Schematic representation of the splice variants of AID and their activity in bacteria.

<p>(A) The exonic structure of the splice isoforms is shown. The position of functional features of the full-length AID (AID-FL) is indicated: the catalytic domain, the cytoplasmic retention signal (CRS; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121719#pone.0121719.ref020" target="_blank">20</a>]) and the nuclear export signal (NES; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121719#pone.0121719.ref016" target="_blank">16</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121719#pone.0121719.ref019" target="_blank">19</a>]). The coding sequence appears in grey and the retained intron 3 is indicated (AID-ivs3) by the angle-striped pattern. The asterisks indicate the AID isoforms tested in the study. (B) Western blot analysis showing the expression levels of AID-FL, AID-ΔE4, AID-ivs3, or an empty plasmid in KL16 bacteria after induction with IPTG. Equal amounts of protein lysates (10 μg) were loaded on SDS-PAGE. The apparent molecular weights from prestained protein ladder are shown on left. The arrows indicate the expected molecular weight of the AID isoforms. The asterisk indicates an unspecific band. The asterisk indicates an unspecific band. (C) Rifampicin assay using the various AID isoforms. Only the revertants resistant to rifampicin can grow. While AID induces a mutator phenotype (<i>P</i><10⁻³ by Dunn’s multiple comparison test), the tested splice variants display levels of revertants similar to the negative control (empty plasmid). The mutation rate is calculated after normalization with Ampicillin resistant viable colonies. The median value is indicated.</p