In this study, we showed that AID from multiple species could inhibit the replication of the retrotransposon L1. We were surprised to find that many highly conserved residues of human AID were dispensable for L1 restriction, including the catalytic glutamate E58, the zinc-coordinating cysteines C87 and C90, the active site tryptophan W84 and the single-strand DNA binding arginines R24 and R112. These data combined to demonstrate a DNA deamination-independent mechanism. Similar DNA editing-independent L1 restriction mechanisms have also been documented for human A3B and A3G, but the replicative stage of the block has yet to be determined (22
). In contrast, restriction of L1 by human A3A clearly has a different set of genetic requirements dependent upon the analogous, conserved catalytic glutamate, zinc-coordinating cysteines and active site tryptophan [(25
) and this study]. Thus, at least two distinct mechanisms may serve to limit the transposition of L1 and similar non-LTR type retroelements in vertebrates. Additional studies will be required to pinpoint the step (or steps) at which AID/A3 proteins interfere with L1 replication.
Although the restriction of many retrotransposon and retrovirus substrates by multiple A3 proteins is clearly associated with G-to-A hypermutations in the coding DNA strand, a growing number of studies have indicated deamination-independent mechanisms (14
). Several prior studies showed that L1 restriction was not associated with A3 protein subcellular localization, as A3F (cytoplasmic) and A3B (nuclear) each inhibited L1 replication with similar efficiencies (22
). Our studies with AID (predominantly cytoplasmic) and AIDΔC (mostly nuclear) also indicated that sub-cellular compartmentalization is not a rate-limiting step in L1 restriction. Thus, we favor an L1 restriction model in which cytoplasmic AID (A3B, A3F or A3G) engages assembling L1 replication complexes co- or post-translationally (Supplementary Figure S7
). This cytoplasmic restriction model is supported indirectly by data showing that at least one family member, A3G, forms high-molecular mass cytoplasmic complexes, and that this complex is required for restriction of the L1-dependent retroelement Alu (24
). AID also appears capable of forming large cytoplasmic complexes (1
), and it is possible that at least some of the complex components are shared with A3G. A cytoplasmic restriction model is also appealing because it is the first to suggest a function for AID in the subcellular compartment in which it is predominantly located.
It is notable that G-to-A hypermutations have not been associated with L1 restriction by AID, A3B, A3F, A3G or even A3A, which requires key catalytic residues [(22
) and this study]. As mentioned above, a part of this apparent failure (at least for A3A) may be due to rapid degradation of edited L1's by cellular DNA repair enzymes. However, a reasonable alternative explanation is the possibility that editing may be co-factor or post-translational modification dependent. For instance, like the RNA editing family member APOBEC1, C-to-U deamination of its physiological substrate APOB mRNA is thought to require a co-factor called ACF. It is therefore plausible that AID (and also APOBEC3s) employ tissue-specific co-factors and/or post-translational modifications for maximal editing and/or restriction efficiency. AID may very well use distinct co-factors or modifications in germinal center B cells to edit antibody gene DNA than those that it may use in other tissues to inhibit L1 replication (such as the ovary). For instance, cofactors have recently been implicated for AID in antibody diversification and for A3G-dependent restriction of HIV-1 (87
We also confirmed a previous report that AID mRNA is expressed in ovarian tissue (82
), at least in mice, placing AID in a compartment where replication of L1 may have the greatest impact in vivo
). Although AID expression has also been detected in human testes (81
), we did not find it in mouse testes tissue; species-specific differences, sterile housing conditions and/or other factors may be responsible. Nevertheless, expression data [(80–82
) and this study], L1 restriction data (this study) and phylogenetic evidence that an AID
-like ancestor duplicated and diverged to give rise to the present day A3
) combine to indicate that AID may indeed also function in providing innate immunity to retroelements [as originally proposed in (45
); see also (90
)]. Future studies will be directed toward testing this hypothesis in vivo
, in mice or fish or an organism where both precise genetics and quantitative transposition assays are possible.
Endogenous retrotransposons make up a large portion of the mammalian genome, with L1 elements alone constituting around 20% of the human and mouse genomes (91
). With approximately 100 active copies in humans and around 3000 active copies in mice (93–95
), it is likely that cellular factors are required to maintain a balance between new insertions contributing to beneficial genetic diversity and causing deleterious gene disruptions. The APOBEC3 proteins are almost certainly one class of innate restriction factors. It is probable that A3
genes evolved from their more ancient family member, AID
, and fine-tuned the anti-retroelement activity during this process. In contrast to many of the APOBEC3s, which appear to have undergone a strong diversifying selection (41
appears to be under purifying selection (41
), which probably reflects the essential nature of AID
's function in antibody diversification. The A3
s may have therefore emerged to enable mammals to ‘keep up’ with the retroviruses and retrotransposons due to AID
's functional constraints.