These studies have addressed whether A3G uses predominately a deaminase-dependent or a deaminase-independent mechanism to inhibit the replication of a yeast retrotransposon, Ty1, a murine endogenous retrovirus, MusD, and a lentivirus, HIV-1. Minimal expression of a deaminase-defective mutant (A3G-E259Q) abolished A3G-mediated restriction of all three retroelements, while an equivalent mutation in the pseudoactive N-terminal zinc-binding domain (A3G-E67Q) did not. These results indicated that the antiretroelement activity of A3G is intrinsic to the protein, since restriction was dependent on E259 in three different systems. A3G-E259Q is virtually indistinguishable from wild-type A3G in terms of its cellular expression level, encapsidation ability, cellular localization, and dimerization capacity (these studies and unpublished data). Therefore, we strongly favor the conclusion that the deaminase activity of A3G is absolutely crucial for inhibiting the replication of Ty1, MusD, and HIV-1(ΔVif). Despite this requirement, the major uracil excision enzymes of yeast and humans did not appear to influence A3G-mediated retroelement restriction.
Our studies suggest that much of the previous data on A3G's deaminase-independent activity can be explained by expression differences. Consistent with previously published results, overexpression of the zinc-binding domain mutants A3G-E67Q and A3G-E259Q caused decreases in HIV-1(ΔVif) infectivity similar to those with wild-type A3G (23
). However, when A3G was expressed minimally, mutation of the catalytic glutamate (E259Q) eliminated A3G-mediated restriction. Furthermore, stable T-cell lines expressing A3G-E259Q at near-physiologic levels failed to inhibit spreading infections of HIV-1(ΔVif).
We previously demonstrated that A3G can inhibit Ty1 and mutate yeast genomic DNA (42
). The present study extends this work by showing that the mechanism of Ty1 restriction parallels those of MusD and HIV-1(ΔVif) restriction. This was particularly apparent at minimal expression levels. Together with the data revealing that lower levels of A3G expression actually triggered higher CanR
mutation frequencies, we hypothesize that higher expression levels (particularly in heterologous systems) may lead to A3G protein aggregation, to less functional protein (albeit more overall), and possibly to artifactual observations. For instance, one can easily envisage that the overexpression of A3G could lead to a higher-molecular-weight aggresome, which could immobilize the genomic RNA and/or Gag protein of assembling retroelements (see, e.g., references 7
, and 52
). Such an explanation readily accounts for Ty1 and HIV-1 data reported here and in previous publications (11
However, because A3G-E67Q does not inhibit Ty1, MusD, or HIV-1(ΔVif) as efficiently as wild-type A3G, we cannot completely eliminate the possibility that the N-terminal zinc-binding domain contributes some sort of deaminase-independent activity. Nevertheless, it should be noted that this possibility is unlikely, because the weak activity of A3G-E67Q can be attributed to lower expression levels and/or diminished encapsidation abilities. Our results are consistent with a report by Mbisa and colleagues, which showed that the catalytic activity of A3G is required for the inhibition of HIV-1 plus-strand transfer and integration (36
Since DNA cytosine deamination is critical for A3G-mediated restriction of Ty1, MusD, and HIV-1(ΔVif), the apparent lack of involvement of cellular UDGs was surprising. Unlike a recent report, which showed that UNG2 is necessary for efficient HIV-1 replication and A3G-mediated restriction of HIV-1(ΔVif), we showed that UNG2 had no effect on HIV-1 replication or on the ability of A3G to inhibit HIV-1(ΔVif) (53
). Furthermore, we showed that deletion of the only uracil excision enzyme in yeast (Ung1p) had no effect on A3G-mediated restriction of Ty1. The reasons for these discrepancies are not clear. However, our HIV-1 results are more consistent with two other recent reports, which showed that UNG2 has no effect on virus infectivity or on the accumulation of viral DNA in the presence of A3G (25
). One of these studies also showed that another cellular UDG (SMUG1), whose activity was not detected here in CEM-SS T cells, was not involved (25
). Moreover, our present data also addressed the UNG2 hypothesis without potential contributions from A3F (virus replication experiments using A3F- and A3G-expressing H9 cells [25
]) or complications from A3G overexpression (transient-transfection single-cycle infectivity experiments [36
]), thereby ruling out the possibility of the virus simply being too burdened with hypermutations to show infectivity recoveries in the absence of UNG2. Nevertheless, despite the strong requirement for the DNA deamination activity of A3G, the main uracil excision enzymes of yeast and humans appear dispensable.
Finally, it should be stressed that our data may not extend directly to other APOBEC3 proteins or other systems. Here we address only human A3G and Ty1, MusD, and HIV-1, but our data can certainly provide useful points for comparison. Similar experiments must be done with other systems (ideally with at least one reference element; such as HIV-1), because it is feasible that deaminase-independent mechanisms do indeed exist. For instance, data from several groups concur that the restriction of the endogenous retrotransposons L1 and Alu occurs by a deamination-independent mechanism (3
). We further recognize that future experiments must also endeavor to address each APOBEC3-retroelement interaction using the most physiologically relevant system possible. For instance, here we used CD4-positive human T cells to show that near-physiologic levels of an A3G catalytic mutant (E259Q) failed to inhibit the spreading replication of HIV-1(ΔVif).