科学杂志：治疗乙肝

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Inhibition of Hepatitis B Virus Replication by APOBEC3G

Science, Vol 303, Issue 5665, 1829 , 19 March 2004


Priscilla Turelli,* Bastien Mangeat,* Stephanie Jost,* Sandrine Vianin, Didier Trono

Department of Microbiology and Molecular Medicine and "Frontiers in Genetics" Research Program, University of Geneva, Switzerland.




* These authors contributed equally to this work.

To whom correspondence should be addressed. E-mail: [email protected]



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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).



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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 Vif 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. [View Larger Version of this Image (61K GIF file)]  


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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.


References and Notes

1. V. N. KewalRamani, J. M. Coffin, Science 301, 923 (2003).[Abstract/Free Full Text]
2. X. Yu et al., Science 302, 1056 (2003).[Abstract/Free Full Text]
3. B. Mangeat et al., Nature 424, 99 (2003).[CrossRef][ISI][Medline]
4. Materials and Methods are available as supporting online material on Science Online.
5. M. Biermer, R. Puro, R. J. Schneider, J. Virol. 77, 4033 (2003).[Abstract/Free Full Text]
6. L. G. Guidotti, F. V. Chisari, Annu. Rev. Immunol. 19, 65 (2001).[CrossRef][ISI][Medline]
7. S. L. Ngui, R. Hallet, C. G. Teo, Rev. Med. Virol. 9, 183 (1999).[CrossRef][ISI][Medline]
8. C. J. Oon et al., Vaccine 13, 699 (1995).[CrossRef][ISI][Medline]
9. We thank L. Perrin, M. Strubin, S. Bontron, O. Leupin, and S. Liao for help and comments. Supported by Swiss National Science Foundation and Roche Research Foundation.

[此贴子已经被作者于2004-4-11 9:19:40编辑过]

金错刀

研究人员提出，对治疗慢性乙肝来说，一种基因方法也许是可行的。全世界约有2.5亿人患有慢性乙肝，它是导致肝癌的首要原因。Priscilla Turelli和同事指出，酶APOBEC3G能保护细胞免受逆转录酶病毒的感染，因为这个酶能使细胞的基因组产生很多的变异使病毒不可能再复制。作者报告说，酶APOBEC3G也能抑止乙肝病毒的复制，乙肝病毒也是一种DNA病毒。他们认为，APOBEC3G使用了一种与对付其他病毒不同的机理来阻止乙肝病毒。