title: DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus.
abstract: 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.
authors: National Institute of Biological Sciences, Beijing, China.
authors: Yonghe
authors: Y
authors: Qi
authors: National Institute of Biological Sciences, Beijing, China.|Graduate program in School of Life Sciences, Peking University, Beijing, China.
authors: Zhenchao
authors: Z
authors: Gao
authors: National Institute of Biological Sciences, Beijing, China.
authors: Guangwei
authors: http://orcid.org/0000-0003-3350-5147
authors: G
authors: Xu
authors: National Institute of Biological Sciences, Beijing, China.|Graduate program in School of Life Sciences, Peking University, Beijing, China.
authors: Bo
authors: B
authors: Peng
authors: National Institute of Biological Sciences, Beijing, China.|College of Life Sciences Beijing Normal University, Beijing, China.
authors: Chenxuan
authors: http://orcid.org/0000-0002-9960-1302
authors: C
authors: Liu
authors: National Institute of Biological Sciences, Beijing, China.
authors: Huan
authors: H
authors: Yan
authors: National Institute of Biological Sciences, Beijing, China.
authors: Qiyan
authors: http://orcid.org/0000-0001-7253-0420
authors: Q
authors: Yao
authors: National Institute of Biological Sciences, Beijing, China.
authors: Guoliang
authors: http://orcid.org/0000-0001-5403-5741
authors: G
authors: Sun
authors: National Institute of Biological Sciences, Beijing, China.|School of Life Science, Tsinghua University, Beijing, China.
authors: Yang
authors: http://orcid.org/0000-0002-4952-7714
authors: Y
authors: Liu
authors: National Institute of Biological Sciences, Beijing, China.|Graduate program in School of Life Sciences, Peking University, Beijing, China.
authors: Dingbin
authors: D
authors: Tang
authors: National Institute of Biological Sciences, Beijing, China.
authors: Zilin
authors: Z
authors: Song
authors: National Institute of Biological Sciences, Beijing, China.
authors: Wenhui
authors: W
authors: He
authors: National Institute of Biological Sciences, Beijing, China.
authors: Yinyan
authors: Y
authors: Sun
authors: Baruch S. Blumberg Institute, Doylestown, Pennsylvania, United States of America.
authors: Ju-Tao
authors: JT
authors: Guo
authors: National Institute of Biological Sciences, Beijing, China.
authors: Wenhui
authors: W
authors: Li
mesh_terms: D015139:Blotting, Southern; D002460:Cell Line; D004270:DNA, Circular / Q000235:genetics* / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D055786:Gene Knockout Techniques; D006509:Hepatitis B / Q000235:genetics* / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics / Q000378:metabolism; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering; D014162:Transfection; D014779:Virus Replication / Q000235:genetics; D000099270:Y-Family DNA Polymerases
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase; D034741:RNA, Small Interfering; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases