DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus.

Hepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into...

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Main Authors: Yonghe Qi, Zhenchao Gao, Guangwei Xu, Bo Peng, Chenxuan Liu, Huan Yan, Qiyan Yao, Guoliang Sun, Yang Liu, Dingbin Tang, Zilin Song, Wenhui He, Yinyan Sun, Ju-Tao Guo, Wenhui Li
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2016-10-01
Series:PLoS Pathogens
Online Access:https://doi.org/10.1371/journal.ppat.1005893
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spelling doaj-4a79b47722ce4d59b9fbecb8fa569e5b2021-04-21T17:37:21ZengPublic Library of Science (PLoS)PLoS Pathogens1553-73661553-73742016-10-011210e100589310.1371/journal.ppat.1005893DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus.Yonghe QiZhenchao GaoGuangwei XuBo PengChenxuan LiuHuan YanQiyan YaoGuoliang SunYang LiuDingbin TangZilin SongWenhui HeYinyan SunJu-Tao GuoWenhui LiHepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc) DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s) that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s) responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK), a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV.https://doi.org/10.1371/journal.ppat.1005893
collection DOAJ
language English
format Article
sources DOAJ
author Yonghe Qi
Zhenchao Gao
Guangwei Xu
Bo Peng
Chenxuan Liu
Huan Yan
Qiyan Yao
Guoliang Sun
Yang Liu
Dingbin Tang
Zilin Song
Wenhui He
Yinyan Sun
Ju-Tao Guo
Wenhui Li
spellingShingle Yonghe Qi
Zhenchao Gao
Guangwei Xu
Bo Peng
Chenxuan Liu
Huan Yan
Qiyan Yao
Guoliang Sun
Yang Liu
Dingbin Tang
Zilin Song
Wenhui He
Yinyan Sun
Ju-Tao Guo
Wenhui Li
DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus.
PLoS Pathogens
author_facet Yonghe Qi
Zhenchao Gao
Guangwei Xu
Bo Peng
Chenxuan Liu
Huan Yan
Qiyan Yao
Guoliang Sun
Yang Liu
Dingbin Tang
Zilin Song
Wenhui He
Yinyan Sun
Ju-Tao Guo
Wenhui Li
author_sort Yonghe Qi
title DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus.
title_short DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus.
title_full DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus.
title_fullStr DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus.
title_full_unstemmed DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus.
title_sort dna polymerase κ is a key cellular factor for the formation of covalently closed circular dna of hepatitis b virus.
publisher Public Library of Science (PLoS)
series PLoS Pathogens
issn 1553-7366
1553-7374
publishDate 2016-10-01
description Hepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc) DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s) that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s) responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK), a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV.
url https://doi.org/10.1371/journal.ppat.1005893
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