Inhibition of Hepatitis B Virus Replication by APOBEC3G

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Science  19 Mar 2004:
Vol. 303, Issue 5665, pp. 1829
DOI: 10.1126/science.1092066

To replicate efficiently, viruses must overcome innate defense mechanisms. Human APOBEC3G is a cytidine deaminase that represents one such barrier, conferring broad intracellular antiretroviral protection. The enzyme is packaged in virions and, during reverse transcription, deaminates deoxycytidine residues to deoxyuridine (dU) in the growing minus-strand of viral DNA. These dU-rich reverse transcripts are either degraded or yield proviruses that are largely nonfunctional due to G-to-A hypermutation (1). Most lentiviruses escape APOBEC3G inhibition via expression of a protein called Vif, which prevents the virion incorporation of the deaminase and triggers its proteasomal degradation (2). APOBEC3G is otherwise able to block a wide spectrum of distantly related retroviruses (3). To ask whether it can interfere with other retroelements, we examined its effect on hepatitis B virus (HBV).

HBV chronically infects more than 250 million individuals worldwide and is a leading cause of liver insufficiency and hepatocellular carcinoma. Retroviruses synthesize the proviral DNA mostly after they enter target cells. In contrast, HBV packages a partially double-stranded DNA genome produced by reverse transcription of pregenomic RNA within subviral core particles found in virus producer cells.

Co-transfection of the Huh7 hepatoma cell line with an HBV-producing plasmid and APOBEC3G-expressing or control vectors led to comparable levels of viral transcription, translation, and extracellular production of HBsAg viral surface antigen (fig. S1, A to C). However, cells expressing the deaminase released at least 50 times less HBV DNA than control cells (fig. S1C). Correspondingly, there was a dose-dependent decrease in the levels of intracellular core-associated viral DNA in cells transfected with APOBEC3G, an effect that was completely abrogated by HIV-1 Vif (Fig. 1A; fig. S1D). APOBEC3G could be co-immunoprecipitated with HBcAg in the cytoplasm of HBV-transfected cells (Fig. 1B), reminiscent of its association with the retroviral reverse transcription complex (3).

Fig. 1.

(A) Southern blot analysis of core-associated HBV DNA purified from HBV-transfected Huh7 cells in the presence or absence of APOBEC3G-HA, with or without HIV-1 Vif or the reverse transcriptase inhibitor 3TC. A3G, APOBEC3G; RC, relaxed circular DNA; DL, double-stranded linear DNA; SS, single-stranded DNA. (B) Western blot analysis of cytoplasmic extracts from Huh7 cells transfected with the indicated plasmids (HBV and A3G), without (Lysate) or with (IP) prior immunoprecipitation with an HBcAg-specific antibody. (C) Real-time PCR quantification of intracellular core-associated HBV DNA (open bars) and single-round scoring of ΔVif HIV infectivity in the supernatant (solid bars) of Huh7 cells with WT APOBEC3G or the indicated APOBEC3G mutants [Cys291 → Ser291 (C291S) and C288S]. Antiviral activity of WT was set at 100% in each case. (D) Real-time PCR quantification of core-associated HBV RNA in Huh7 cells transfected with the indicated plasmids. (Inset) Western blot analysis showing that the levels of core protein are unaffected by APOBEC3G. NT, nontransfected.

In view of this result and of the known mechanism of APOBEC3G antiretroviral action, we thought that its most likely HBV target would be the nascent viral DNA. However, we detected no significant nucleotide change in sequences obtained by polymerase chain reaction (PCR) amplification of the low levels of core-associated HBV DNA present in APOBEC3G-expressing cells (4). This suggested that APOBEC3G might act on HBV and retroviruses through different mechanisms. Confirming this hypothesis, catalytically inactive APOBEC3G derivatives no longer inhibited vif-defective HIV-1 but did conserve wild-type levels of activity on HBV (Fig. 1C). Core-associated HBV RNA was reduced in the presence of APOBEC3G (Fig. 1D). The block of HBV DNA accumulation by APOBEC3G thus seems to result primarily from an inhibition of viral pregenomic RNA packaging.

During the course of an acute infection, HBV DNA clearance apparently occurs through noncytopathic mechanisms (5, 6). APOBEC3G, or a related protein, might participate in this type of antiviral response. Although APOBEC3G is not normally expressed in the liver, it is possible that it is induced in the course of HBV infection, for instance under the influence of cytokines.

G-to-A hypermutation can influence HBV pathogenesis. Specific G-to-A changes yield HBeAg-negative HBV variants, often isolated from patients with acute fulminant hepatitis, as well as HBV vaccine escape mutants (7, 8). Our experimental system did not reveal APOBEC3G-mediated HBV editing, but this might occur in particular cellular contexts or with specific viral strains.

Our work broadens the spectrum of known viruses inhibited by APOBEC3G. Whether the sole role of this protein is to provide antiviral protection or whether it first evolved to fulfill other functions within the cell is an interesting question.

Supporting Online Material

Materials and Methods

Fig. S1


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