Research Article: Uracil DNA Glycosylase Counteracts APOBEC3G-Induced Hypermutation of Hepatitis B Viral Genomes: Excision Repair of Covalently Closed Circular DNA

Date Published: May 16, 2013

Publisher: Public Library of Science

Author(s): Kouichi Kitamura, Zhe Wang, Sajeda Chowdhury, Miyuki Simadu, Miki Koura, Masamichi Muramatsu, Aleem Siddiqui.


The covalently closed circular DNA (cccDNA) of the hepatitis B virus (HBV) plays an essential role in chronic hepatitis. The cellular repair system is proposed to convert cytoplasmic nucleocapsid (NC) DNA (partially double-stranded DNA) into cccDNA in the nucleus. Recently, antiviral cytidine deaminases, AID/APOBEC proteins, were shown to generate uracil residues in the NC-DNA through deamination, resulting in cytidine-to-uracil (C-to-U) hypermutation of the viral genome. We investigated whether uracil residues in hepadnavirus DNA were excised by uracil-DNA glycosylase (UNG), a host factor for base excision repair (BER). When UNG activity was inhibited by the expression of the UNG inhibitory protein (UGI), hypermutation of NC-DNA induced by either APOBEC3G or interferon treatment was enhanced in a human hepatocyte cell line. To assess the effect of UNG on the cccDNA viral intermediate, we used the duck HBV (DHBV) replication model. Sequence analyses of DHBV DNAs showed that cccDNA accumulated G-to-A or C-to-T mutations in APOBEC3G-expressing cells, and this was extensively enhanced by UNG inhibition. The cccDNA hypermutation generated many premature stop codons in the P gene. UNG inhibition also enhanced the APOBEC3G-mediated suppression of viral replication, including reduction of NC-DNA, pre-C mRNA, and secreted viral particle-associated DNA in prolonged culture. Enhancement of APOBEC3G-mediated suppression by UNG inhibition was not observed when the catalytic site of APOBEC3G was mutated. Transfection experiments of recloned cccDNAs revealed that the combination of UNG inhibition and APOBEC3G expression reduced the replication ability of cccDNA. Taken together, these data indicate that UNG excises uracil residues from the viral genome during or after cccDNA formation in the nucleus and imply that BER pathway activities decrease the antiviral effect of APOBEC3-mediated hypermutation.

Partial Text

The hepatitis B virus (HBV) is one of the major causative factors of liver cirrhosis and hepatocellular carcinoma. Chronic inflammation due to persistent HBV infection plays a major causative role in these severe liver diseases. However, it is still unknown how HBV establishes persistent infection and how this infection results in these diseases [1], [2]. The HBV genome in virions forms a relaxed circular DNA (rcDNA) that is converted into covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes. The cccDNA transcribes all viral RNAs including pregenomic (pg) RNA as a replicative RNA intermediate. In the cytoplasm, pgRNA, viral core, and polymerase proteins are assembled into the nucleocapsid (NC), after which the pgRNA is converted into an rcDNA by viral polymerase activity. The mature NCs are transferred to either the endoplasmic reticulum to be secreted after combining with envelope proteins or the nucleus to form cccDNA again for the next replication cycle. Although the host repair system is thought to play a major role in conversion of rcDNA into cccDNA, the molecules responsible for the conversion have not been determined experimentally [3], [4], [5].

To avoid the mutagenic impact of dUTP misincorporation or cytosine deamination, organisms have dUTPase and uracil DNA glycosylases, including UNG. Some viruses such as poxviruses (vaccinia viruses) or herpesviruses (HSV-1 and cytomegaloviruses) also encode UNG homologs, and primate lentiviruses incorporate host UNG into the virion through interaction with the viral Vpr protein [29], [32], [59]. However, the involvement of uracil excision activity during replication and infection of these viruses has not been fully investigated. The effect of Vpr-bound UNG on the deaminated HIV-1 DNA is apparently controversial, although it is thought to be involved in DNA repair [31], [32] or DNA degradation [33] or to have no role [34], [35], [36]. Thus, it seems that there is no unified view whether UNG plays a protective or suppressive role in viral replication. In this study, using in vitro models of HBV and DHBV, we investigated the role of UNG activity in hypermutation and viral replication in the presence of A3G. We found that UNG inhibition resulted in the enhancement of A3G-induced NC-DNA hypermutation. This study for the first time also found that the A3G protein induced cccDNA hypermutation (Figures 5 and 7). The cccDNA hypermutation was enhanced on UNG inhibition and subsequently resulted in significant decrease in viral production (Figure 7), suggesting a protective role of UNG for viral replication against cccDNA hypermutation.




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