We performed here a structure/function study of the determinants dictating A3A functions, and we identified 12 residues at the core of A3A-editing and restriction activities. Mutations at these residues affect the blockade of LINE-1 retrotransposition, the inhibition of AAV replication, and the decrease in gene expression from foreign plasmid DNA without affecting the expression levels or subcellular localization of the protein. Structural modeling and molecular docking guided by experimental observations predicted that these residues form a groove that can accommodate a single-stranded DNA molecule. The restriction and editing defects of the mutants coincided with their inability to prevent the accumulation of LINE-1 reverse transcription products, using experimental conditions where no effect of A3A on LINE-1 RNA transcript levels or G418 resistance induced by a neomycin phosphotransferase-expressing plasmid could be detected (Fig. ). However, some effect on transfected LINE-1 DNA could be noted, which translated in a moderate, albeit significant, decrease in the recovery of hygromycin-resistant colonies. Why this was observed in the absence of an impact on the amounts of plasmid-generated LINE1 RNA is unclear. However, it is worth noting that the bulk of reverse transcription-competent LINE-1 RNA likely is produced shortly after transfection from unintegrated circular plasmid DNA. In contrast, the induction of hygromycin-resistant colonies requires that the plasmid DNA not only migrates to the nucleus but also becomes linearized and integrated, and hence it follows a path where it may be more exposed to A3A-induced degradation. It is clear that investigating thoroughly the molecular mechanism of A3A-induced LINE-1 inhibition would greatly benefit from a system allowing for the production of retrotransposition-competent LINE-1 from an integrated locus rather than from a transfected plasmid. Remarkably, mutants found here to alter the ability of A3A to block LINE-1 were equally defective for AAV-2 inhibition and the perturbation of gene expression from plasmid DNA. It was demonstrated that A3A acts on AAV by decreasing levels of viral replicative intermediates (12
). Our data support a model whereby all three effects of A3A are linked to interference with the synthesis or stability of some DNA intermediate. The absence of the editing activity of A3A on double-stranded DNA in vitro
) and the restricted size of the putative DNA-binding groove revealed by our structural model suggest that this DNA intermediate is a single-stranded molecule.
We recently reported that A3A mutants with no detectable levels of deaminase activity in vitro
still can block AAV-2 replication (42
). These data suggest that A3A acts through distinct or simply additive effects. Supporting evidence for multiple yet overlapping functions in APOBEC3 proteins is the segregation of the DNA editing from the RNA-binding activities of the single-domain cytidine deaminase A3C (51
). Similarly, it could be that the editing-independent blockade of HIV replication observed with high levels of A3G (5
) stems from the sequestration of single-stranded viral DNA products by the cytidine deaminase (26
). Accordingly, A3A could interfere with LINE-1/AAV genome replication by interfering with DNA replication before inducing its editing and degradation. In the absence of a high-resolution structure of an APOBEC3 protein bound to its substrate, the modalities of the interaction of these molecules with nucleic acids remain speculative. Distinct DNA-binding models have been proposed for the A3G C terminus based on mutagenesis and/or chemical shift perturbations. One of these models (24
) is in agreement with the putative DNA-binding groove predicted by our study (Fig. and ) and with results obtained from the constrained molecular docking of the 5′-TTC
A target sequence on A3A, while another model suggests that residues on the A3G C-terminal loop 4 participate in DNA binding (17
). Since several highly conserved residues on A3A loop 4 do not contribute to A3A restriction activities on LINE/AAV (data not shown), this loop probably is not relevant to the restriction function of this protein. Moreover, the two helices linking the active site to loop 4 (helix 2 and 3) do not harbor residues with protruding positive charges or aromatic groups that could interact with single-stranded polynucleotides. Although the aromatic residue F75 found to be essential for A3A deaminase activity but not AAV-2 restriction (42
) resides on helix 2, it points inside the protein and likely participates in the stability of secondary-structure elements, as predicted by in silico
alanine scanning (data not shown). Since catalytic-site residues also map on helix 2, mutation at position F75 may affect the local structure of the enzyme and reduce the editing activity while leaving intact other putative DNA-binding functions. In agreement, we found here that the mutation of R69, which is adjacent to the zinc-coordinating residue H70, had a more significant impact on editing than on LINE-1 or AAV restriction. Alternatively, F75L may alter the ability of A3A to access its target by preventing its interaction with an unknown partner.
Despite the absence of the intrinsic restriction activity of the C-terminal domain of human A3G, the comparison of A3A and A3G C terminus sequences from different primates reveals that residues that may contribute to the restriction activities of A3A are highly conserved between primate A3A and the A3G C-terminal homologous sequences. Two remarkable exceptions are the residues K30 and K60 of human A3A, which generally are conserved among A3A primate sequences yet are absent from A3G C-terminal domain orthologues. Remarkably, introducing simultaneously the two residues into the C-terminal half of human A3G conferred on this molecule the ability to block LINE-1 replication (Fig. ). Only one of the two mutations (P247K) seemed to be sufficient for the gain of activity against AAV-2, a result in line with a previous experiment that showed that the transfer of a patch of residues on A3A loop 3 (including K60) to the A3G C terminus was sufficient to confer antiviral activity against AAV-2 (42
). Since we failed to recapitulate the complete restriction activity of wild-type A3A, additional residues are likely to be of functional importance. It is expected that less conserved residues or residues present in secondary structure elements play an additional role in the specific activity of A3A. In agreement, a recent study found that a series of residues under positive selection influences murine APOBEC3 restriction activity against Friend virus (49
). In addition, extra residues, such as the three-amino-acid patch found specifically on human A3G C terminus loop 1 but not on A3A or more ancient A3G homologues, could limit the restriction activity of the enzyme regardless of the presence of other beneficial amino acids. Interestingly, residues homologous to human A3A K30 and K60 are absent from human A3C (51
), which is far less active than A3A against LINE-1 (7
) or AAV-2 (12
One potential consequence of these structural variations is a difference in affinity for DNA binding. Although this affinity still needs to be determined for A3A, the surprisingly high Kd
(dissociation constant) (>10 μM) of the A3G C terminus for single-stranded DNA (11
) suggests that this domain needs to be present at a high concentration close to the target DNA for efficient editing and antiviral activity, as is the case with full-length A3G in HIV particles (58
). The purification of wild-type and mutant A3A and the evaluation of their respective Kd
s for DNA binding will be required to validate this hypothesis. Residues on loop 6 influence A3G C terminus sequence preference at positions −1 and −2 relative to the edited cytidine (10
). It has been shown that swapping the loop between different APOBEC homologues can modify their editing sequence preference accordingly (29
). Surprisingly, swapping functionally important residues of loop 6 (D133 and Y136) from A3A into the A3G C-terminal domain failed to confer any gain-of-function activity against LINE-1 (not illustrated). Another study found that replacing the variable region of A3A loop 6 with the one from the A3G N terminus is sufficient to allow the targeting of A3A to HIV virions (19
). On A3G, these residues are critical for binding to specific small cellular RNAs and for virion incorporation (9
). Although it is tempting to speculate that the identity of the target sequence plays less of a role for the editing-dependent restriction of LINE-1 elements than for specific binding to certain small cellular RNAs, this hypothesis will need to be tested by a thorough analysis of the influence of loop 6 residues on cytidine deamination target site preference. Since human A3A and A3G C-terminal domains are predicted to have a common phylogenetic origin (32
), these data suggest that the specific restriction activities of A3A evolved after the divergence from the common ancestor gene but before the emergence of New World monkeys and was maintained throughout primate evolution. In agreement, preliminary data suggest that at least one New World monkey orthologue (marmoset A3A) is endowed with restriction activity against LINE-1 (data not shown). This hypothesis needs to be challenged by more in-depth vertical studies of APOBEC3 molecules.
The ability of A3G and A3C to bind to small cellular RNAs is one major determinant for packaging into retroviral particles and subsequent antiviral activity (9
). Future studies will indicate whether the loss of editing and restriction activities of the A3A mutants identified here stem from decreased binding affinity for single-stranded DNA. This finding would be consistent with a model in which critical positions in APOBEC3 proteins influence the modalities of their interactions with polynucleotidic sequences, thereby determining their spectrum of action against mobile genetic elements.