The primary intent of this structure-function study was to better understand molecular determinants of the interaction between A3G and the antagonistic HIV-1 Vif protein. A detailed knowledge of the interaction between these proteins is of particular interest for the rational design of future therapeutic approaches that aim to harness natural protection against HIV-1 infection by preventing the Vif-mediated degradation of A3G. We chose to mutate residues 119 to 146 of A3G because the region contains residue 128, which has previously been shown to be pivotal in facilitating the formation of A3G-Vif complexes (4
In addition to confirming the role of residue 128 in the interaction with Vif, we identified two additional amino acids, P129 and D130, whose alteration created proteins that displayed Vif-resistant phenotypes (Fig. ). More specifically, we found that insensitivity to Vif was conferred by mutating the aspartic acid residue at position 128 or 130 to the positively charged residue lysine. However, we did observe that residue 128 is more sensitive to mutation than 130, which can accommodate several amino acid changes and still behave like wild-type A3G, implying that D128 may play a more prominent role in the Vif interaction (Fig. ). Nevertheless, simultaneous mutation of residues D128 and D130 to alanine yielded a protein that was markedly more resistant to Vif than either singly mutated protein (Fig. ), suggesting a substantial degree of redundancy between these positions. In sum, these data indicate that the Vif-A3G interaction is dependent on electrostatic interactions, which is fully consistent with recent data demonstrating that mutation of positively charged residues in HIV-1 Vif can compensate for the D128K mutation in A3G (45
). In addition to these electrostatic determinants, there also appears to be a rather specific structural requirement for the interaction, as evidenced by the strong Vif-resistant phenotypes of mutant A3G proteins carrying either alanine or glycine in place of proline at position 129 (Fig. ). Though it is most likely that Vif contacts A3G directly in this region, we emphasize that direct binding awaits formal demonstration. For this reason, we use the term “interaction” to encompass both direct and indirect contacts.
While our current study has provided novel insight into the interaction between HIV-1 Vif and A3G, the 3-amino-acid motif D128-P129-D130 is unlikely to represent a complete description of motifs within APOBEC proteins that are capable of interacting with HIV-1 Vif. More specifically, (i) one study reported that residues 54 to 124 of A3G are sufficient for coimmunoprecipitation with Vif, which would suggest the presence of an additional point (or points) of contact (9
); (ii) the Vif proteins of HIV-2 and SIVmac
are able to mediate the degradation of both wild-type A3G and the D128K mutant, indicating that these Vif proteins have different requirements for functional interactions with A3G (17
); and (iii) the interaction between HIV-1 Vif and human A3F is different from the interaction with A3G, as illustrated by the demonstration that amino acid differences in HIV-1 Vif can differentially affect the ability to neutralize A3F versus A3G (51
). Indeed, the human A3F protein contains an ERD motif at the position equivalent to the DPD motif of A3G (defined here), yet mutation of the glutamic acid in A3F does not affect sensitivity to Vif (10
); moreover, the naturally Vif-resistant human A3B protein also has this ERD motif at the equivalent position, again implying that an interaction with Vif requires more than just acidic amino acids at this position. Lastly, like A3G, A3F contains two CDA domains, and we noted that the C-terminal domain contains a DTD motif within the predicted β4-α3 loop. However, mutation of the first aspartic acid in this motif to lysine (D310K) did not affect downregulation by Vif (data not shown). Taken together, these assorted findings indicate that the DPD motif at residues 128 to 130 in human A3G represents a Vif interaction domain that is unique to this protein. A full understanding of the molecular determinants of this interaction will clearly depend on future structural analyses of Vif-A3G complexes. In this regard, it is interesting that the recently resolved structure of APOBEC2 (41
) reveals that the corresponding glutamic acid-proline-glutamic acid motif (residues 159 to 161) also straddles the N terminus of helix α3 (Fig. ) and that the acidic side chains protrude outward from the helix, rendering them accessible for interactions with protein ligands.
In addition to defining this Vif interaction motif, our analyses revealed the existence of an adjacent motif that plays a central role in the packaging of A3G into virus particles (Fig. ). Alanine substitution mutations in this 4-amino-acid cluster spanning residues 124 to 127 generated proteins displaying reduced levels of antiviral activity against HIV-1/Δvif (Fig. ) that correlated with losses in virion incorporation (Fig. and ). Specifically, mutants Y124A and W127A were almost entirely excluded from virions and had minimal effects on infectivity, whereas the Y125A and F126A proteins showed reduced levels of packaging and intermediate infectivity phenotypes. Further mutation analysis of residue 127 indicated that the presence of tryptophan at this position is particularly important, as all substitutions tested resulted in substantial diminutions of antiviral activity (Fig. ). A principal role for residues 124 and 127 in A3G function is also reflected in the strong conservation of these residues within A3G proteins from different species, whereas more variation was observed at the less important positions 125 and 126 (not shown).
It has been reported by several groups that incorporation of A3G into virus particles depends on a combination of its interaction with the NC domain of Gag and nonspecific RNA binding (6
). Although the losses of antiviral activity for mutants in the 124-to-127 region correlated with defects in packaging, we found, using a previously described pull-down assay (12
) and lysates from transfected 293T cells, that proteins with mutations at positions 124, 125, and 126 interacted with HIV-1 Gag in an RNA-dependent manner but that the W127A mutant protein did not (data not shown). Thus, packaging capabilities cannot simply be ascribed to Gag-A3G-RNA interactions as monitored by this assay system. The reasons for these discrepancies are not yet evident and will require further investigation; however, one potential contributing factor to A3G packaging may be the requirement for spatial proximity between A3G and the cellular sites of viral particle assembly, and it is conceivable that this motif may participate in determining correct subcellular localization. A further possibility is that this motif may bind to an as-yet-unidentified molecular partner that may specify virus incorporation. In sum, further work is required to define the role of residues 124 to 127 in HIV-1 packaging and to determine how this region combines with other conserved elements of the N-terminal CDA of A3G that also contribute to encapsidation (18
In this study, we demonstrated that crucial determinants for A3G interaction with Vif and its incorporation into HIV-1 particles are localized to a 7-amino-acid element spanning residues 124 to 130. Consistent with the notion that this region interacts with viral and cellular factors, structure predictions for A3G and comparisons with APOBEC2 place it in a loop region that connects β4 to α3 (Fig. ). On the basis of our alanine scan, the residues that confer these two attributes appear to be distinct from each other. The W127L protein provides the only data that may dispute this: it is no longer packaged (Fig. ) yet has lost susceptibility to Vif-induced degradation (Fig. ). Since other mutations at position 127 remain Vif sensitive, it seems most likely that the W127L mutant is misfolded and consequently broadly nonfunctional (though it remains formally possible that this mutation genuinely influences both attributes). As discussed above, pharmacologic inhibition of the Vif-A3G interaction represents a potential therapeutic approach for suppressing HIV-1 replication. Should such an inhibitory agent interact with A3G at the DPD motif, it will be important to preserve the biological activities of the neighboring YYFW motif: failure to do so could lead to reduced packaging and antiviral efficacy. Further work defining the precise functions and structures of these motifs will inform rational drug design in this area.