Concerted action of activation-induced cytidine deaminase and uracil-DNA glycosylase reduces covalently closed circular DNA of duck hepatitis B virus

Covalently closed circular DNA (cccDNA) forms a template for the replication of hepatitis B virus (HBV) and duck HBV (DHBV). Recent studies suggest that activation-induced cytidine deaminase (AID) functions in innate immunity, although its molecular mechanism of action remains unclear, particularly regarding HBV restriction. Here we demonstrated that overexpression of chicken AID caused hypermutation and reduction of DHBV cccDNA levels. Inhibition of uracil-DNA glycosylase (UNG) by UNG inhibitor protein (UGI) abolished AID-induced cccDNA reduction, suggesting that the AID/UNG pathway triggers the degradation of cccDNA via cytosine deamination and uracil excision. © 2013 Federation of European Biochemical Societies

RNA editing of hepatitis B virus transcripts by activation-induced cytidine deaminase.

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. The mechanism by which AID triggers SHM and CSR has been explained by two distinct models. In the DNA deamination model, AID converts cytidine bases in DNA into uridine. The uridine is recognized by the DNA repair system, which produces DNA strand breakages and point mutations. In the alternative model, RNA edited by AID is responsible for triggering CSR and SHM. However, RNA deamination by AID has not been demonstrated. Here we found that C-to-T and G-to-A mutations accumulated in hepatitis B virus (HBV) nucleocapsid DNA when AID was expressed in HBV-replicating hepatic cell lines. AID expression caused C-to-T mutations in the nucleocapsid DNA of RNase H-defective HBV, which does not produce plus-strand viral DNA. Furthermore, the RT-PCR products of nucleocapsid viral RNA from AID-expressing cells exhibited significant C-to-T mutations, whereas viral RNAs outside the nucleocapsid did not accumulate C-to-U mutations. Moreover, AID was packaged within the nucleocapsid by forming a ribonucleoprotein complex with HBV RNA and the HBV polymerase protein. The encapsidation of the AID protein with viral RNA and DNA provides an efficient environment for evaluating AID's RNA and DNA deamination activities. A bona fide RNA-editing enzyme, apolipoprotein B mRNA editing catalytic polypeptide 1, induced a similar level of C-to-U mutations in nucleocapsid RNA as AID. Taken together, the results indicate that AID can deaminate the nucleocapsid RNA of HBV

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

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. © 2013 Kitamura et al

APOBEC3 deaminases induce hypermutation in human papillomavirus 16 DNA upon beta interferon stimulation

Apolipoprotein B mRNA-editing catalytic polypeptide 3 (APOBEC3) proteins are interferon (IFN)-inducible antiviral factors that counteract various viruses such as hepatitis B virus (HBV) and human immunodeficiency virus type 1 (HIV-1) by inducing cytidine (C)-to-uracil (U) mutations in viral DNA and inhibiting reverse transcription. However, whether APOBEC3 proteins (A3s) can hypermutate human papillomavirus (HPV) viral DNA and exhibit antiviral activity in human keratinocyte remains unknown. Here we examined the involvement of A3s in the HPV life cycle using cervical keratinocyte W12 cells, which are derived from low-grade lesions and retain episomal HPV16 genomes in their nuclei. We focused on the viral E2 gene as a potential target for A3-mediated hypermutation because this gene is frequently found as a boundary sequence in integrated viral DNA. Treatment of W12 cells with beta interferon (IFN-ß) increased expression levels of A3s such as A3A, A3F, and A3G and induced C-to-U conversions in the E2 gene in a manner depending on inhibition of uracil DNA glycosylase. Exogenous expression of A3A and A3G also induced E2 hypermutation in W12 cells. IFN-ß-induced hypermutation was blocked by transfection of small interfering RNAs against A3G (and modestly by those against A3A). However, the HPV16 episome level was not affected by overexpression of A3A and A3G in W12 cells. This study demonstrates that endogenous A3s upregulated by IFN-ß induce E2 hypermutation of HPV16 in cervical keratinocytes, and a pathogenic consequence of E2 hypermutation is discussed. © 2014, American Society for Microbiology

Group IIA secreted phospholipase A2 controls skin carcinogenesis and psoriasis by shaping the gut microbiota

Besides promoting inflammation by mobilizing lipid mediators, group IIA secreted phospholipase A2 (sPLA2-IIA) prevents bacterial infection by degrading bacterial membranes. Here, we show that, despite the restricted intestinal expression of sPLA2-IIA in BALB/c mice, its genetic deletion leads to amelioration of cancer and exacerbation of psoriasis in distal skin. Intestinal expression of sPLA2-IIA is reduced after treatment with antibiotics or under germ-free conditions, suggesting its upregulation by gut microbiota. Metagenome, transcriptome, and metabolome analyses have revealed that sPLA2-IIA deficiency alters the gut microbiota, accompanied by notable changes in the intestinal expression of genes related to immunity and metabolism, as well as in the levels of various blood metabolites and fecal bacterial lipids, suggesting that sPLA2-IIA contributes to shaping of the gut microbiota. The skin phenotypes in Pla2g2a–/– mice are lost (a) when they are cohoused with littermate WT mice, resulting in the mixing of the microbiota between the genotypes, or (b) when they are housed in a more stringent pathogen-free facility, where Pla2g2a expression in WT mice is low and the gut microbial compositions in both genotypes are nearly identical. Thus, our results highlight a potentially new aspect of sPLA2-IIA as a modulator of gut microbiota, perturbation of which affects distal skin responses

RNA editing of hepatitis B virus transcripts by activation-induced cytidine deaminase

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. The mechanismby which AID triggers SHMand CSR has been explained by two distinct models. In the DNA deamination model, AID converts cytidine bases in DNA into uridine. The uridine is recognized by the DNA repair system, which produces DNA strand breakages and point mutations. In the alternative model, RNA edited by AID is responsible for triggering CSR and SHM. However, RNA deamination by AID has not been demonstrated. Here we found that C-to-T and G-to-A mutations accumulated in hepatitis B virus (HBV) nucleocapsid DNA when AID was expressed in HBV replicating hepatic cell lines. AID expression caused C-to-T mutations in the nucleocapsid DNA of RNase H-defective HBV, which does not produce plus-strand viral DNA. Furthermore, the RT-PCR products of nucleocapsid viral RNA from AID-expressing cells exhibited significant C-to-T mutations, whereas viral RNAs outside the nucleocapsid did not accumulate C-to-U mutations. Moreover, AID was packaged within the nucleocapsid by forming a ribonucleoprotein complex with HBV RNA and the HBV polymerase protein. The encapsidation of the AID protein with viral RNA and DNA provides an efficient environment for evaluating AID\u27s RNA and DNA deamination activities. A bona fide RNA-editing enzyme, apolipoprotein B mRNA editing catalytic polypeptide 1, induced a similar level of C-to-U mutations in nucleocapsid RNA as AID. Taken together, the results indicate that AID can deaminate the nucleocapsid RNA of HBV

TGF-β Suppression of HBV RNA through AID-Dependent Recruitment of an RNA Exosome Complex

Transforming growth factor (TGF)-β inhibits hepatitis B virus (HBV) replication although the intracellular effectors involved are not determined. Here, we report that reduction of HBV transcripts by TGF-β is dependent on AID expression, which significantly decreases both HBV transcripts and viral DNA, resulting in inhibition of viral replication. Immunoprecipitation reveals that AID physically associates with viral P protein that binds to specific virus RNA sequence called epsilon. AID also binds to an RNA degradation complex (RNA exosome proteins), indicating that AID, RNA exosome, and P protein form an RNP complex. Suppression of HBV transcripts by TGF-β was abrogated by depletion of either AID or RNA exosome components, suggesting that AID and the RNA exosome involve in TGF-β mediated suppression of HBV RNA. Moreover, AID-mediated HBV reduction does not occur when P protein is disrupted or when viral transcription is inhibited. These results suggest that induced expression of AID by TGF-β causes recruitment of the RNA exosome to viral RNP complex and the RNA exosome degrades HBV RNA in a transcription-coupled manner. © 2015 Liang et al

Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

金沢大学医薬保健研究域医学系Hepatitis B virus (HBV) is one of the major etiological pathogens for liver cirrhosis and hepatocellular carcinoma. Chronic HBV infection is a key factor in these severe liver diseases. During infection, HBV forms a nuclear viral episome in the form of covalently closed circular DNA (cccDNA). Current therapies are not able to efficiently eliminate cccDNA from infected hepatocytes. cccDNA is a master template for viral replication that is formed by the conversion of its precursor, relaxed circular DNA (rcDNA). However, the host factors critical for cccDNA formation remain to be determined. Here, we assessed whether one potential host factor, flap structure-specific endonuclease 1 (FEN1), is involved in cleavage of the flap-like structure in rcDNA. In a cell culture HBV model (Hep38.7-Tet), expression and activity of FEN1 were reduced by siRNA, shRNA, CRISPR/Cas9-mediated genome editing, and a FEN1 inhibitor. These reductions in FEN1 expression and activity did not affect nucleocapsid DNA (NC-DNA) production, but did reduce cccDNA levels in Hep38.7-Tet cells. Exogenous overexpression of wild-type FEN1 rescued the reduced cccDNA production in FEN1-depleted Hep38.7-Tet cells. Anti-FEN1 immunoprecipitation revealed the binding of FEN1 to HBV DNA. An in vitro FEN activity assay demonstrated cleavage of 5′-flap from a synthesized HBV DNA substrate. Furthermore, cccDNA was generated in vitro when purified rcDNA was incubated with recombinant FEN1, DNA polymerase, and DNA ligase. Importantly, FEN1 was required for the in vitro cccDNA formation assay. These results demonstrate that FEN1 is involved in HBV cccDNA formation in cell culture system, and that FEN1, DNA polymerase, and ligase activities are sufficient to convert rcDNA into cccDNA in vitro

Associations between the orexin (hypocretin) receptor 2 gene polymorphism Val308Ile and nicotine dependence in genome-wide and subsequent association studies

Impact of the HCRTR2 gene risk variant on schizotypal personality traits (mean ± SD). (DOC 54 kb