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

Date Published: October 26, 2016

Publisher: Public Library of Science

Author(s): 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, Jianming Hu.

http://doi.org/10.1371/journal.ppat.1005893

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.

Partial Text

Despite the availability of effective vaccines for more than three decades, hepatitis B virus (HBV) still persistently infects 240 million people worldwide [1, 2]. Antiviral therapies with nucleos(t)ide analog inhibitors of HBV reverse transcriptase dramatically reduce the viral load, significantly improve the liver function and lower the incidence of liver failure and hepatocellular carcinoma, but fail to cure the viral infection [3, 4], due to the persistence of covalently closed circular (ccc) DNA in the nuclei of infected hepatocytes [5–8]. Hence, better understanding the molecular mechanisms underlying the formation and maintenance of cccDNA is critical for development of novel therapeutics to cure chronic HBV infection.

Synthesis of cccDNA is a critical, but not well-understood step in the life cycle of hepadnaviruses. Our current study characterized the kinetics of cccDNA formation in HBV infected cells and obtained strong evidence suggesting that cellular POLK plays a crucial role in cccDNA synthesis in de novo HBV infection. In addition, our findings reported herein also provide important clues for further investigation of viral and cellular factors in cccDNA biosynthesis and regulation.

 

Source:

http://doi.org/10.1371/journal.ppat.1005893

 

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