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.
Highlights
Despite the availability of effective vaccines for more than three decades, hepatitis B virus (HBV) still persistently infects 240 million people worldwide [1, 2]
In HepG2-NTCP cells infected with HBV that is deficient in core protein production, we unambiguously demonstrated that viral DNA polymerase activity is not required for cccDNA formation in a de novo infection
To investigate the molecular mechanism of HBV cccDNA formation, we first determined the kinetics of cccDNA synthesis in wild-type HBV infected HepG2-NTCP cells
Summary
Despite the availability of effective vaccines for more than three decades, hepatitis B virus (HBV) still persistently infects 240 million people worldwide [1, 2]. HBV is the prototype member of Hepadnaviridae family and contains a relaxed circular (rc) partially double-stranded DNA genome with its DNA polymerase covalently linked to the 5’ terminus of minus strand [9]. The formation and intracellular amplification of cccDNA plays a central role in the establishment and maintenance of persistent infection. Tdp gene knockout only slows down the formation of duck hepatitis B virus (DHBV) cccDNA from intracellular amplification pathway, but does not inhibit HBV cccDNA formation in HBV infection of HepG2-NTCP cells [25, 26]. Ku80, a component of non-homologous end joining DNA repair pathway, has been reported to play an essential role in the synthesis of DHBV cccDNA from dslDNA, but not rcDNA [29]
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