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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
Christian Königer a,b,
Ida Wingert a,
Moritz Marsmann a,
Christine Rösler a,
Jürgen Beck a, 1, and
Michael Nassa la,1
Author Affiliations
aDepartment of Internal Medicine 2/Molecular Biology, University Hospital Freiburg, D-79106 Freiburg, Germany; and
bFaculty of Biology, University of Freiburg, D-79104 Freiburg, Germany
Edited by Francis V. Chisari, The Scripps Research Institute, La Jolla, CA, and approved July 28, 2014 (received for review May 29, 2014)
Significance
Chronic hepatitis B virus (HBV) infection puts >250 million humans at risk for developing liver cirrhosis and liver cancer. Current therapies are not curative because they do not target HBV´s persistence reservoir, the plasmid-like covalently closed circular DNA (cccDNA). RNA production from cccDNA initiates the generation of progeny virus via protein-primed reverse transcription, yielding viral polymerase-linked relaxed-circular DNA (RC-DNA). Its conversion, upon infection, into cccDNA requires multiple poorly understood steps, including polymerase removal. We found that the host enzyme tyrosyl-DNA-phosphodiesterase 2 (TDP2), important for repair of cellular protein–DNA adducts, performed this step in vitro and that TDP2 depletion impaired the conversion of RC-DNA to cccDNA in cells. These data establish a functional link between HBV and cellular DNA repair and pave the way for targeting HBV persistence.
Abstract
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.
1To whom correspondence may be addressed. Email: [email protected] or [email protected].
Author contributions: C.K., J.B., and M.N. designed research; C.K., I.W., M.M., C.R., and J.B. performed research; C.K., M.M., J.B., and M.N. analyzed data; and C.K., J.B., and M.N. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10 ... 1/-/DCSupplemental.
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发表于 2014-10-8 18:52
