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Discussion
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.
Establishment and regulation of cccDNA pool in virally infected hepatocytes
We demonstrated in this study that cccDNA is formed from incoming virion DNA in HepG2-NTCP cells at as early as 24 h post infection and establishes the pool size of approximately 3 copies of cccDNA per infected cell within a few days of infection. The kinetics of cccDNA accumulation as well as two lines of independent evidence obtained from HBV-ΔHBc infection of HepG2-NTCP cells and viral DNA polymerase inhibitor treatment of wild-type HBV-infected cells strongly support the notion that intracellular amplification does not play a significant role in the establishment of cccDNA pool in the HBV-infected hepatoma cells. This observation is consistent with the findings from HBV infection of primary human hepatocytes and HepaRG cells [38, 39], but distinct from DHBV infection of primary duck hepatocytes where significant intracellular cccDNA amplification occurs in a manner regulated by the level of its large envelope protein expression [19, 20]. However, intracellular amplification of cccDNA has been observed in HepG2-derived cell lines supporting constitutive or inducible HBV replication [40–42]. Developing therapeutics against chronic HBV infection requires better understanding the contribution of intracellular cccDNA amplification in the maintenance of persistent infection, and further investigation of the activity and regulation of this pathway in HBV-infected hepatocytes in vivo is thus warranted.
Role of HBV core protein in cccDNA function
An elegant study by Chisari and colleagues demonstrated that although DHBV deficient for capsid protein expression (DHBVΔcp) infected primary duck hepatocytes and produced similar amounts of cccDNA from the incoming virions as did wild-type DHBV, the cccDNA in DHBVΔcp-infected hepatocytes was significantly less efficiently transcribed into viral RNAs, suggesting an important role of capsid protein in DHBV cccDNA transcription [43]. However, our results showed that HBV-ΔHBc infected HepG2-NTCP cells and expressed viral genes at a similar efficiency as wild-type HBV did. The results therefore suggest that the synthesis of HBV capsid protein may not significantly modify HBV cccDNA transcription activity. Interestingly, DHBV capsid protein is structurally distinct from the capsid proteins of mammalian hepadnaviruses [44] and may have HBx-like function in regulation of DHBV cccDNA transcription. However, the possibility that HBV capsid proteins from in-coming virions interact with cccDNA and promote its transcriptional activity cannot be completely ruled out. It has been shown that HBV capsid protein is a structural component of viral cccDNA minichromosome and its binding reduces the nucleosomal spacing of the minichromosome [45]. In addition, it has also been suggested that capsid protein promotes an epigenetic permissive state of HBV cccDNA by binding on CpG islands of cccDNA [46]. Of note, some non-nucleoside analogue compounds targeting capsid protein can dysregulate functional HBV capsid assembly [47–52]. Those capsid assembly effectors may alter the amounts and/or structure of cccDNA-bound capsid protein and consequentially interfere with cccDNA metabolism and function [53]. Intriguingly, interferon-stimulated gene (ISG) APOBEC3A seems to have a role in the destruction of cccDNA by direct interaction with HBV core protein [54]. Further investigation on the differential roles of capsid proteins in regulation of cccDNA function should shed light on this aspect of hepadnaviral pathobiology.
Role of viral DNA polymerase in cccDNA formation
We showed herein that, similarly to DHBV and WHV, completion of plus strand DNA synthesis during de novo HBV infection of HepG2-NTCP cells is not sensitive to viral DNA polymerase inhibitors, suggesting the reaction is most likely catalyzed by a host DNA polymerase. In support of this hypothesis, by following the fate of viral DNA sequence during conversion of rcDNA into cccDNA, Seeger and colleagues demonstrated that independent of a viral enzymatic activity, a cellular DNA polymerase may fill in the 3’ end of both DNA strands [55]. The observed slight reduction of cccDNA amounts in ADV or ETV treated cells in this study could indicate either a minor contribution of viral DNA polymerase to cccDNA formation or an off-target inhibition of cellular functions required for cccDNA formation. Interestingly, studies of HBV cccDNA biosynthesis via intracellular amplification pathway in HepG2-derived stable cell lines, such as HepAD38 or HepDES19 cells, suggested that deproteinization and uncoating of progeny rcDNA require the completion of plus strand DNA synthesis, which requires viral DNA polymerase activity [41, 42]. Hence, different from rcDNA in virion particles with various length of incompletely synthesized plus-stranded DNA, the precursor rcDNA for cccDNA synthesis from the intracellular amplification pathway may have a very short gap in plus strand DNA and thus distinct DNA repair enzymes may be recruited to convert the rcDNA to cccDNA. Moreover, it had been shown that a small fraction of cccDNA can be formed from dslDNA via NHEJ DNA repair pathway. The cellular DNA polymerases required for cccDNA synthesis through intracellular amplification pathway and from dslDNA remain to be determined in future studies.
Role of host cellular DNA polymerase in cccDNA biosynthesis and implications
While our results demonstrated that POLK plays a critical role in cccDNA formation during de novo HBV infection of cultured HepG2-NTCP, HepaRG and PTHs, we also showed that POLL and POLH play a role in cccDNA formation, albeit at a lesser extent. It is currently not clear whether each of the cellular DNA polymerases plays a redundant or distinct role in de novo cccDNA synthesis. As mentioned above, plus-strand DNA in rcDNA in virions has gaps of heterogeneous lengths. It is possible that depending on the length of gaps, distinct DNA repair complexes containing different repairing DNA polymerases are recruited to fill the gaps with different length. If this is the case, the three DNA polymerases may play non-redundant roles and be involved in conversion of distinct rcDNA precursors into cccDNA. Alternatively, each of the three cellular DNA polymerases may participate in a different DNA repair complex to fill the plus strand gaps, irrespective of their length, but in a different efficiency. These two possibilities will need to be further investigated.
POLK plays a functional role in nucleotide excision repair (NER) pathway by filling the gap produced upon excision of damaged nucleotides [56, 57]. The activity of POLK is partially dependent on the growth state of the cells, and reaches maximum activity under conditions of low deoxynucleotide concentration such as in non-dividing cells [56]. A previous study showed treatment of HBV-transfected HepG2 cells with aphidicolin arrested cells in the G1 phase could result in enhancement of cccDNA synthesis [58]. Consistent with this observation, we found that the efficiency of HBV infection closely correlates with the number of G0/G1 phase cells in HepG2-NTCP cultures. Therefore, HBV cccDNA formation may preferentially occur at G0/G1 phase of cell cycle, supporting the notion that HBV infects non-dividing cells, so that cccDNA is formed and stably exists in quiescent hepatocytes [59]. It is thus conceivable that cell cycle-dependent factor(s) or protein post translational modification affecting the physiologic state of hepatocytes may regulate the formation of HBV cccDNA.
Moreover, because cellular DNA polymerases must work in concert with other DNA repair proteins to restore the structure of damaged DNA, other DNA repair proteins in NER pathway may also play a role in HBV cccDNA formation. For example, it is possible that HBV hijacks cellular endonuclease (e.g. XPG) or exonuclease (e.g. Exo1) to cleave the capped RNA primer to leave a free 5’ end of plus strand DNA of rcDNA, and followed by POLK or other cellular DNA polymerase, such as POLL and POLH, to fill the gap using minus strand DNA as a template. Additionally, POLK has been shown to work together with POLD to fill in single stranded DNA gaps [56, 60] and XRCC1-Lig3 is required for ligation of NER-induced breaks in quiescent cells [61]; hence it will be interesting to test whether those host enzymes are involved in cccDNA formation.
In conclusion, taking advantage of highly efficient genetic manipulation of HepG2-NTCP HBV infection system, and in combination with studies using recombinant HBV virus and chemical inhibitors, we rigorously demonstrated that cellular DNA polymerase κ substantially contributes to HBV cccDNA formation in HepG2-NTCP cells. Our findings shed new light on the molecular mechanism of cccDNA formation and may facilitate the development of novel therapeutics to cure chronic hepatitis B. |
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发表于 2016-10-27 17:06
