Stable HepG2- and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus

Sun, Dongdong; Nassal, Michael

doi:10.1016/j.jhep.2006.05.019

Cited by 112 publications

(158 citation statements)

References 41 publications

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“…As a model of inducible, human cell-based conversion of RC-DNA to cccDNA, we exploited the fact that env − DHBV generates drastically more cccDNA (39) than does HBV even in human hepatoma cells and established a stable DHBVenv − Huh7 TetOFF cell line. Withdrawal of tetracycline (Tet)-initiated virus replication and RC-DNA formation, as expected and in contrast to analogous HBV lines (40), was accompanied by easily detectable accumulation of cccDNA from day 9 to day 18 postinduction (Fig. S6).…”

Section: Tdp2 Knockdown Impairs the Formation Of Hepadnaviral Cccdna Insupporting

confidence: 56%

“…HBV nucleocapsids obtained from induced cultures of TetOFF wild-type HBV HepG2.117 cells (40) were refractory to high-salt treatment, but freezing in the presence of 0.01% (wt/vol) ethidium bromide (EB) made the RC-DNA and doublestranded linear (dsL) DNA amenable to digestion by micrococcal nuclease (Fig. S3B), possibly via their increased space requirements upon EB intercalation; in line with this notion, ssDNA remained protected (Fig.…”

Section: Resultsmentioning

confidence: 66%

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Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses

Königer

Wingert

Marsmann

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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210

193

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Hepatitis B virus (HBV), the causative agent of chronic hepatitis B and prototypic hepadnavirus, is a small DNA virus that replicates by protein-primed reverse transcription. The product is a 3-kb relaxed circular DNA (RC-DNA) in which one strand is linked to the viral polymerase (P protein) through a tyrosyl-DNA phosphodiester bond. Upon infection, the incoming RC-DNA is converted into covalently closed circular (ccc) DNA, which serves as a viral persistence reservoir that is refractory to current anti-HBV treatments. The mechanism of cccDNA formation is unknown, but the release of P protein is one mandatory step. Structural similarities between RC-DNA and cellular topoisomerase-DNA adducts and their known repair by tyrosyl-DNA-phosphodiesterase (TDP) 1 or TDP2 suggested that HBV may usurp these enzymes for its own purpose. Here we demonstrate that human and chicken TDP2, but only the yeast ortholog of TDP1, can specifically cleave the Tyr-DNA bond in virus-adapted model substrates and release P protein from authentic HBV and duck HBV (DHBV) RC-DNA in vitro, without prior proteolysis of the large P proteins. Consistent with TPD2's having a physiological role in cccDNA formation, RNAi-mediated TDP2 depletion in human cells significantly slowed the conversion of RC-DNA to cccDNA. Ectopic TDP2 expression in the same cells restored faster conversion kinetics. These data strongly suggest that TDP2 is a first, although likely not the only, host DNA-repair factor involved in HBV cccDNA biogenesis. In addition to establishing a functional link between hepadnaviruses and DNA repair, our results open new prospects for directly targeting HBV persistence.hepatitis B virus persistence | TDP substrate specificity | virus-DNA repair interface M ore than 250 million people worldwide are chronically infected with hepatitis B virus (HBV) (1) and are at a highly increased risk for developing end-stage liver disease (2). Current treatments with IFN-α or nucleos(t)ide analogs (NAs) are only partially effective (3, 4). Importantly, they do not directly target the viral persistence reservoir, an episomal covalently closed circular (ccc) DNA form of the viral genome that serves as template for all viral transcripts; hence a few cccDNA molecules present in the liver can reactivate full viral replication. Eliminating infection thus will require the elimination of cccDNA. cccDNA is generated, upon infection, from the viral polymerase (P protein)-linked relaxed circular (RC) DNA present in incoming virions (Fig. 1A). The mechanism by which RC-DNA is converted to cccDNA is poorly understood, but it must involve multiple steps (see below). Because the ∼3-kb genomes of HBV and the other hepadnaviruses [e.g., from ducks (DHBV)] are too small to encode all the activities required for this conversion, these activities must be provided by the host cell.RC-DNA from all hepadnaviruses bears several molecular peculiarities that result from its generation by protein-primed reverse transcription (for reviews, see refs. 5 and 6). The pregenomic RNA (p...

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Section: Tdp2 Knockdown Impairs the Formation Of Hepadnaviral Cccdna Insupporting

confidence: 56%

Section: Resultsmentioning

confidence: 66%

Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses

Königer

Wingert

Marsmann

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

210

193

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“…Cells were treated with MG132 (Sigma-Aldrich, St. Louis, MO) as noted in the text. HepG2-1117 cells contain 1.05-fold HBV genome (subtype ayw) under inducible Tet-off control (33). Myc-tagged HBX and their mutants, Myc-tagged HBV X, S, S1, and C, were expressed by cloning the genes into the pcDNA3-based vector (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

The Hepatitis B Virus X Protein Disrupts Innate Immunity by Downregulating Mitochondrial Antiviral Signaling Protein

Wei¹,

Song

et al. 2010

The Journal of Immunology

210

185

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“…To date, many in vitro studies on lamivudine resistance have been reported [24][25][26][27] . In most of these, recombinant plasmids containing HBV full-length or fragment genome were constructed first, and then expressed in liver-derived cell lines.…”

Section: Discussionmentioning

confidence: 99%

Construction and expression of eukaryotic plasmids containing lamivudine-resistant or wild-type strains of Hepatitis B Virus genotype C

Fang²,

Li³

et al. 2008

WJG

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AIM:To construct eukaryotic expression plasmids of full-length Hepatitis B Virus (HBV) genotype C genome, which contain lamivudine-resistant mutants (YIDD, YVDD) or wild-type strain (YMDD), and to observe the expression of HBV DNA and antigens [hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg)] of the recombinant plasmids in HepG2 cells. METHODS: Three HBV full-length genomes were amplified from the plasmids pMD18T-HBV/YIDD, pMD18T-HBV/YVDD and pMD18T-HBV/YMDD, using PCR. Three recombinant plasmids were generated by inserting each of the PCR products into the eukaryotic expression vector pcDNA3.1 (+), between the Eco RI and Hin dⅢ sites. After being characterized by restriction endonuclease digestion, and DNA sequence analysis, the recombinant plasmids were transfected into HepG2 cells. At 48 and 72 h post-transfection, the levels of intracellular viral DNA replication were detected by real-time PCR, and the expression of HBsAg and HBeAg in the cell culture supernatant was determined by ELISA. RESULTS: Restriction endonuclease digestion and DNA sequence analysis confirmed that the three recombinant plasmids were correctly constructed. After transfecting the plasmids into HepG2 cells, high levels of intracellular viral DNA replication were observed, and HBsAg and HBeAg were secreted into the cell culture supernatant. CONCLUSION: Eukaryotic expression plasmids pcDNA3.1 (+)-HBV/YIDD, pcDNA3.1 (+)-HBV/YVDD or pcDNA3.1 (+)-HBV/YMDD, which contained HBV genotype C full-length genome, were successfully constructed. After transfection into HepG2 cells, the recombinant plasmids efficiently expressed HBV DNA, HBsAg and HBeAg. Our results provide an experimental basis for the further study of HBV lamivudine-resistant mutants.

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Stable HepG2- and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus

Cited by 112 publications

References 41 publications

Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses

Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses

The Hepatitis B Virus X Protein Disrupts Innate Immunity by Downregulating Mitochondrial Antiviral Signaling Protein

Construction and expression of eukaryotic plasmids containing lamivudine-resistant or wild-type strains of Hepatitis B Virus genotype C

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