Comment on "Inhibition of Hepatitis B Virus Replication by APOBEC3G"
The cytidine deaminase APOBEC3G (A3G) was recently identified
as a natural resistance gene that restricts efficient propagation
of human immunodeficiency virus and other retroviruses. The
enzyme induces massive cytidine to uridine (C
U) deamination
of single-stranded retroviral DNA, resulting in DNA degradation
or lethal guanine to adenine (G
A) hypermutation (
1). Hepadnaviruses,
including hepatitis B virus (HBV), replicate by reverse transcription
of a pregenomic RNA intermediate inside nucleocapsids, placing
them into the family of retroelements (
2). These observations,
along with an earlier report describing G
A hypermutations in
natural HBV variants (
3), raise the question of whether HBV
represents another potential target for A3G. Turelli
et al.
showed that this is indeed the case (
4). Surprisingly, however,
inhibition of viral pregenome packaging rather than induction
of G
A hypermutations was identified as the main antiviral mechanism.
No significant nucleotide changes were detected in a total of
40 polymerase chain reaction (PCR)–amplified HBV clones
derived from cotransfected Huh7 hepatoma cells. Turelli
et al.
discussed the possibility that A3G-mediated HBV editing may
occur in a different cellular context.
We investigated the potential antiviral effect of A3G in cotransfected Huh7 cells and another human hepatoma cell line, HepG2 (5). Our results confirm that A3G interferes with proper packaging of viral pregenomic RNA, resulting in a marked suppression of viral DNA synthesis (data not shown). To search for potential A3G-mediated editing of HBV DNA in nucleocapsids that may have escaped the block in RNA packaging, we PCR-amplified newly synthesized HBV DNA from supernatants or cell lysates of cotransfected cells and sequenced individual clones (6). Figure 1 summarizes the results obtained from three experiments in HepG2 cells and two experiments in Huh7 cells. In total, 430 individual clones were sequenced. In Huh7 cells, GA mutations were rare, irrespective of the presence or absence of A3G, thus confirming the finding of Turelli et al. (4). In A3G-expressing HepG2 cells, the majority of recovered sequences were wild-type as well. However, the number of clones bearing GA mutations and the overall number of GA mutations increased significantly (Fisher's exact test, P = 0.034), whereas other nucleotide substitutions were rare (Fig. 1). Further experiments revealed additional GA mutations in other regions of HBV DNA (Fig. 2), which suggests that they were caused by processive enzymatic activity rather than by global imbalances in the cellular nucleotide pool. Targeted sequence motifs matched well with the hallmarks of A3G action [(7), Fig. 2].
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Fig 1. GA mutations in newly synthesized HBV DNA produced in HepG2 or Huh7 hepatoma cells in the presence (+) or absence (–) of A3G. Nucleocapsid-associated HBV DNA was PCR-amplified, cloned, and sequenced with primer 5'-ACAGTAGCTCCAAATTCTTTA-3' (about 300 nucleotides per clone). Footnotes indicate the total number of sequenced clones and the number of clones displaying G A mutations. Boxes display the total number of the respective mutations.
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Fig. 2. Partial nucleotide sequence of four individual HBV genomes produced in HepG2 cells after transfection with a replication-competent HBV construct and an A3G expression vector. Nucleocapsid-associated HBV DNA was PCR-amplified, cloned, and sequenced with primer 5'-TTGCGGTGTTTGCTCTGAAGG-3' (clones nos. 1 and 2) or primer 5'-GATTTTTTGTATGATGTG-3' (clones nos. 3 and 4). Mutations are depicted with respect to the wild-type sequence above (wt). Asterisks represent nucleotide identity. Numbers indicate nucleotide positions relative to the start codon of the core protein. Underlined sequences represent preferred A3G targets (7).
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In conclusion, A3G displays a dual antiviral effect: (i) interference with pregenomic RNA packaging and (ii) induction of extensive GA mutations in a subset of HBV genomes. Interestingly, A3G-mediated editing of HBV DNA appears to be cell line–dependent. The cellular factor(s) accounting for differences in A3G deaminase activity remain to be defined. Conceivably, Huh7 cells either lack a cofactor that is important for deaminase activity or produce a suppressing factor. It is of note that HepG2 cells occasionally yielded some GA mutations even in the absence of transfected A3G. Because endogenous A3G expression in HepG2 cells was minute (8), this might reflect the activity of another deaminase. Nevertheless, the statistically significant overall increase in GA mutations and their distinctive distribution in individual clones clearly demonstrates that A3G can edit HBV DNA in cotransfected HepG2 cells.
Whether A3G plays a role in down-regulating HBV replication during natural infection remains an intriguing question. Although detectable by reverse transcription PCR, baseline expression levels of A3G are low in primary human hepatocytes (8). Furthermore, A3G mRNA is not induced by HBV infection or cytokines in livers of infected chimpanzees (9). On the other hand, hepadnaviruses have been detected in extrahepatic cells that express high levels of A3G, such as white blood cells (10). Thus, it is tempting to speculate that A3G-driven editing of HBV DNA may occur in extrahepatic cells and may contribute to the emergence of variants (3, 11). Clearly, further experiments are warranted to establish whether and how A3G can edit HBV DNA in nontransformed natural target cells.
Christine Rösler
Department of Medicine II
University of Freiburg
Hugstetter Strasse 55
D-79106 Freiburg, Germany
Josef Köck
Department of Medicine II
University of Freiburg
Hugstetter Strasse 55
D-79106 Freiburg, Germany
Michael H. Malim
Department of Infectious Diseases
Guy's, King's, and St. Thomas'
School of Medicine
King's College London
2nd Floor New Guy's House
GKT Guy's Hospital
London, SE1 9RT, UK
Hubert E. Blum
Department of Medicine II
University of Freiburg
Fritz von Weizsäcker*
Department of Medicine II
University of Freiburg
* To whom correspondence should be addressed. E-mail: fritz.weizsaecker{at}uniklinik-freiburg.de |
References and Notes
- 1. V. N. KewalRamani, J. M. Coffin, Science 301, 923 (2003).[Abstract/Free Full Text]
- 2. C. Seeger, W. S. Mason, Microbiol. Mol. Biol. Rev. 64, 51. (2000).[Abstract/Free Full Text]
- 3. S. Gunther et al., Virology 235, 104 (1997). [CrossRef] [ISI] [Medline]
- 4. P. Turelli, B. Mangeat, S. Jost, S. Vianin, D. Trono, Science 303, 1829 (2004).[Free Full Text]
- 5. American Type Culture Collection (ATCC), catalog no. HB-8065
- 6. Cells were transfected with a replication-competent HBV construct (12) and an expression vector encoding A3G (13). Nucleocapsid-associated viral DNA from culture supernatants was PCR-amplified with forward primer 2908 (5'-GCCCACCAAAGCTTGCCCAAGGTC-3') and reverse primer 1335 (5'-AATACAGGCCTCTCACTCTGG-3'). Purified PCR products were cloned into the EcoRI/Hind III sites of pUC19 (Invitrogen). Nucleocapsid-associated viral DNA from cytoplasmic lysate was amplified with forward wobble primer 2896 (5'-ACCACCRTRAACRCCCACC-3') and reverse primer 1305 (5'-GAGTTTGGTGGAAGGTTGTGG-3'). These PCR products were directly cloned with a TA Cloning Kit (Invitrogen). In additional experiments (Fig. 2, clones 3 and 4), nucleocapsid-associated viral DNA from cytoplasmic lysate was amplified with primers 2855 (5'-CCGGCAGATGAGAAGGCACAGACGG-3') and 556 (5'-TCCTTGGACTCATAAGGTGGG-3') and cloned into the EcoRI/SphI sites of pUC19 (Invitrogen). For sequencing of individual clones, primers 38 (5'-ACAGTAGCTCCAAATTCTTTA-3', Fig. 1), 1032 (5'-TTGCGGTGTTTGCTCTGAAGG-3'; Fig. 2, clones nos. 1 and 2) or 2218 (5'-GATTTTTTGTATGATGTG-3'; Fig. 2, clones nos. 3 and 4) were used.
- 7. Q. Yu et al., Nature Struct. Mol. Biol. 11, 435 (2004)
- 8. J. Köck, F. von Weizsäcker, unpublished data
- 9. S. Wieland, R. Thimme, R. H. Purcell, F. V. Chisari, Proc. Natl. Acad. Sci. U.S.A. 101, 6669 (2004).[Abstract/Free Full Text]
- 10. T. I. Michalak, Immunol. Rev. 174, 98 (2000). [CrossRef] [ISI] [Medline]
- 11. D. Milich, T. J. Liang, Hepatology 38, 1075 (2003). [CrossRef] [ISI] [Medline]
- 12. K. Reifenberg et al., J. Gen. Virol. 83, 991, 2002.[Abstract/Free Full Text]
- 13. A. M. Sheehy, Nature 418, 646, 2002. [CrossRef] [Medline]
- 14. Supported by grants from the Deutsche Forschungsgemeinschaft (We 1365/5-1), the Bundesministerium für Bildung und Forschung (01K19951; HepNet), and Gilead Sciences (DE-103-509).
Received for publication 18 May 2004. Accepted for publication 6 August 2004.
