The APOBEC3 family of cytidine deaminases has been shown to target a wide range of retroviruses, as well as the pararetrovirus human HBV. In the present study, the effects of the APOBEC3 proteins, in particular A3G, on HBV replication were examined. Our findings showed that A3G, A3F, A3C, and mA3 proteins were able to inhibit HBV DNA replication in two different hepatoma cell lines. Furthermore, the inhibitory effect of APOBEC3 proteins on HBV replication was mainly at the level of viral DNA, with only a minor effect on RNA packaging. The effect of A3G on HBV DNA was clearly independent of its DNA-editing function. Furthermore, the reduction of HBV DNA by A3G was due not to accelerated degradation but rather to very early blocks in viral reverse transcription. Finally, our results showed that the deamination-independent inhibition of HBV DNA synthesis by A3G could also target the DS DNA-RNA hybrid, in contrast to the strict SS DNA specificity of its deaminase activity.
An initial report on the effects of A3G on HBV replication suggested that A3G decreased viral DNA levels by inhibiting pgRNA packaging (52
). Subsequently, it was reported that A3G did not block viral RNA packaging per se; rather, the full-length pgRNA packaged in the presence of A3G was somehow rendered sensitive to exogenous nuclease whereas a packaged spliced pgRNA was not affected by A3G (41
). In agreement with the latter report, our results showed that A3G, as well as several other APOBEC3 proteins, had only a minor effect or none on pgRNA packaging, which is unlikely to explain its dramatic effect at the DNA level. In an attempt to test the nuclease sensitivity of the packaged viral RNA, we titrated the concentrations of the micrococcal nuclease (over a range of 20-fold) used to digest the cytoplasmic lysate before analysis of pgRNA packaging efficiency (D. H. Nguyen and J. Hu, unpublished results). However, we were unable to confirm the A3G-induced nuclease sensitivity of the packaged full-length pgRNA with any nuclease concentration tested. It remains possible that subtle differences in the exact conditions of nuclease digestion and nucleocapsid isolation might have been responsible for this apparent discrepancy.
Although some evidence has been reported that A3G may induce HBV DNA editing in a small fraction of viral DNA (37
), it is clear from our results, as well as those of others (41
), that the inhibitory effect of A3G on HBV DNA replication is largely independent of its deaminase activity. Although the DNA deamination function of the APOBEC proteins is well understood, the mechanism of this deamination-independent antiviral function, which also works on other retroviruses and retrotransposons (7
), is not yet clear. In principle, the decrease in HBV DNA could result from either an accelerated DNA degradation or a decreased synthesis. It has been suggested that the reduction in retroviral DNA levels by A3G results from the recognition and degradation of the edited (uracil-containing) reverse transcripts by host cellular repair enzymes, such as the uracil DNA glycosylase (UNG), which removes uracil from DNA to generate an abasic site that is subsequently cleaved by the host apurinic/apyrimidinic endonuclease. However, a recent study showed that the loss of UNG did not rescue the levels of viral reverse transcripts that were decreased by A3G (26
). We also obtained preliminary results suggesting that UNG activity is not required for the decrease in HBV DNA induced by A3G, since there was no rescue of HBV DNA levels by a bacteriophage UNG inhibitor (Ugi) (10
) (Nguyen and Hu, unpublished). Furthermore, attempts to look for enhanced DNA degradation by A3G, either in vivo or in vitro, did not reveal any such effect.
On the other hand, we were able to obtain evidence indicating that A3G may block early stages in viral reverse transcription. While our in vivo and in vitro DNA synthesis assays indicated that A3G did not block the synthesis of late DNA intermediates, as was also reported recently (41
), our studies using the RH mutant, which allowed the accumulation of early minus-strand DNA intermediates, demonstrated that A3G showed a stronger inhibitory effect on longer minus-strand DNA than on shorter DNA. This preferential inhibition of longer minus-strand DNA intermediates by A3G was also evident sometimes even with the WT HBV, but it could not be as readily demonstrated because these early intermediates failed to accumulate in the WT virus. An early block in viral DNA synthesis would explain how A3G could reduce HBV DNA without accelerating DNA degradation or its deaminase activity.
The exact mechanism of A3G inhibition of minus-strand DNA synthesis is not yet clear. A3G could block DNA synthesis by inhibiting the polymerase function, or alternatively, it could affect the template function of the pgRNA, e.g., by associating with the viral RNA (4
). The reported nuclease sensitivity of the packaged HBV pgRNA induced by A3G (41
) would be consistent with the notion that A3G may affect the structure of the pgRNA and, thus, its template function during reverse transcription. The apparent specificity for the synthesis of viral minus-strand, but not plus-strand, DNA by A3G also supports the view that A3G may interfere with the template function of the pgRNA, rather than the polymerase activity per se. The very early block in minus-strand DNA synthesis may suggest that A3G could inhibit the initiation step of minus-strand DNA synthesis, which is primed by the RT protein itself (protein priming) (29
), or block minus-strand primer translocation following protein priming (40
In contrast to the DNA-editing function of A3G, which acts strictly on SS DNA (4
), we have shown here that the editing-independent inhibition of viral reverse transcription by A3G could also act on a DS HBV RNA-DNA hybrid. Similar results were obtained with WT and an RH-defective DHBV mutant (S. H. Basagoudanavar and J. Hu, unpublished results). This reinforces the idea that the editing-dependent and -independent antiviral functions of the APOBEC proteins are fundamentally different mechanisms of cellular antiviral defense. Interestingly, A3G did not appear to significantly block HBV second-strand DNA synthesis from the small amounts of minus-strand DNA that escaped A3G inhibition. This suggests that A3G may not be able to function in the context of a DS DNA. In support of this notion, it has recently been shown that certain APOBEC proteins can target SS, but not DS, DNA viruses (5
In conclusion, our results provide further insights into the antiviral function of A3G. In particular, we have shown that the editing-independent inhibition of HBV replication was not due to viral DNA degradation. Rather, A3G appeared to inhibit very early steps of viral reverse transcription. Additionally, we have shown that A3G clearly inhibited the synthesis of DNA-RNA hybrids as well as SS DNA. The relative ease in detecting and analyzing various reverse transcription intermediates during HBV replication makes it a convenient model system to dissect further the detailed mechanism of A3G-mediated inhibition of reverse transcription.