DNA落后链合成机器的核心组件对于乙型肝炎病毒cccDNA的形成

StephenW

Core components of DNA lagging strand synthesis machinery are essential for hepatitis B virus cccDNA formation

    Lei Wei & Alexander Ploss

Nature Microbiology volume 5, pages715–726(2020)Cite this article

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Abstract

Chronic hepatitis B virus (HBV) infections result in 887,000 deaths annually. The central challenge in curing HBV is eradication of the stable covalently closed circular DNA (cccDNA) form of the viral genome, which is formed by the repair of lesion-bearing HBV relaxed circular DNA delivered by the virions to hepatocytes. The complete and minimal set of host factors involved in cccDNA formation is unknown, largely due to the lack of a biochemical system that fully reconstitutes cccDNA formation. Here, we have developed experimental systems where various HBV relaxed-circular-DNA substrates are repaired to form cccDNA by both cell extracts and purified human proteins. Using yeast- and human-extract screenings, we identified five core components of lagging-strand synthesis as essential for cccDNA formation: proliferating cell nuclear antigen, the replication factor C complex, DNA polymerase δ, flap endonuclease 1 and DNA ligase 1. We reconstituted cccDNA formation with purified human homologues, establishing these as a minimal set of factors for cccDNA formation. We further demonstrated that treatment with the DNA-polymerase inhibitor aphidicolin diminishes cccDNA formation both in biochemical assays and in HBV-infected human cells. Together, our findings define key components in HBV cccDNA formation.

StephenW

DNA落后链合成机器的核心组件对于乙型肝炎病毒cccDNA的形成至关重要

    雷威和亚历山大·普拉斯

Nature Microbiology第5卷，第715-726页（2020年）引用本文

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抽象

慢性乙型肝炎病毒（HBV）感染每年导致887,000人死亡。治愈HBV的主要挑战是根除病毒基因组的稳定的共价闭合环状DNA（cccDNA）形式，这种形式是由病毒体递送到肝细胞的带损伤的HBV松弛环状DNA的修复形成的。 cccDNA形成所涉及的宿主因子的完整和最少集合是未知的，主要是由于缺乏能够完全重构cccDNA形成的生化系统。在这里，我们开发了实验系统，其中各种HBV松弛的环状DNA底物都可以通过细胞提取物和纯化的人类蛋白质进行修复，从而形成cccDNA。通过酵母和人提取物筛选，我们确定了滞链合成的五个核心成分是cccDNA形成所必需的：增殖细胞核抗原，复制因子C复合体，DNA聚合酶δ，襟翼内切核酸酶1和DNA连接酶1。 cccDNA的形成与纯化的人类同源物有关，将其确定为cccDNA形成的最小因素。我们进一步证明了用DNA聚合酶抑制剂Aphidicolin的治疗在生化测定和HBV感染的人类细胞中均减少了cccDNA的形成。总之，我们的发现定义了HBV cccDNA形成的关键成分。

StephenW

The persistence of covalently closed circular (ccc) DNA after a long‐term therapy of viral DNA polymerase inhibitors attributes to their failure of curing chronic hepatitis B virus (HBV) infection. However, lack of knowledge on the molecular mechanism of cccDNA biosynthesis, metabolic stability and transcriptional regulation impedes the discovery of therapeutics for selective eradication or silencing of cccDNA minichromosomes.

The persistence of covalently closed circular (ccc) DNA after a long-term therapy of viral DNA
polymerase inhibitors attributes to their failure of curing chronic hepatitis B virus (HBV) infection.
However, lack of knowledge on the molecular mechanism of cccDNA biosynthesis, metabolic
stability and transcriptional regulation impedes the discovery of therapeutics for selective eradication
or silencing of cccDNA minichromosomes. Recently, Wei and Ploss reported a biochemical system
that can efficiently convert recombinant rcDNA into cccDNA in yeast and human hepatoma cell
extracts and found that the five core components of DNA lagging strand synthesis are sufficient for
cccDNA synthesis in vitro (Wei L and Ploss A. Core components of DNA lagging strand
synthesis machinery are essential for hepatitis B virus cccDNA formation. Nature Microbiology,
2020, 5:715–726). This is the first robust cell-free cccDNA synthesis system suitable for the
identification and biochemical analysis of cellular components involving in cccDNA biosynthesis.
Synthesis of cccDNA from its precursor, the relaxed circular double stranded DNA (rcDNA)
derived from virions during a de novo infection or progeny nucleocapsids in the cytoplasm of infected
hepatocytes, is catalyzed by the host cellular DNA repair machinery. However, because most of DNA
repair proteins are essential for cell survival and cccDNA synthesis is catalyzed by functionally
redundant DNA repair pathways, it is difficult to identify the cellular components involving in
cccDNA synthesis by conventional cell-based genetic approaches. By far, several cellular DNA repair
proteins, including DNA polymerases (Pol) α, δ, κ, and λ, tyrosyl-DNA phosphodiesterase 2 (TDP2),
flap endonuclease 1 (FEN1), DNA topoisomerases and DNA ligases (LIG) 1 and 3, as well as
ATR-CHK1 pathway have been identified to involve in cccDNA synthesis in hepatocytes (Fig. 1)
However, the biochemical mechanism of these cellular proteins in cccDNA synthesis remains to be
determined. An in vitro biochemical system capable of converting rcDNA to cccDNA is thus highly
desired.

Instead of using deproteinized rcDNA (DP-rcDNA) prepared from hepatoma cells supporting
HBV replication as the substrate for in vitro cccDNA synthesis, Wei and Ploss utilized recombinant
rcDNA molecules (RrcDNA) mimicking the rcDNA with or without DNA polymerase attached at the
5’ terminus of minus strand DNA as the substrates (Fig. 1). Although DP-rcDNA had been
demonstrated to be the direct precursor of cccDNA synthesis in hepatocytes (1), accumulating
evidence suggests that the DP-rcDNA represents a structurally heterogeneous population of
protein-free DNA species containing not only the authentic cccDNA precursor(s) and intermediates,
but also mis-repaired species that are functionally dead end products for cccDNA synthesis (2). In
addition to increasing the efficiency of cccDNA synthesis, utilization of the RrcDNA with defined
terminal structures as the substrates can also facilitate the biochemical analysis of DNA end
processing and fill-in reactions during cccDNA synthesis.

Taking advantages of the relative simplicity or less redundancy of yeast DNA repair pathways
and robust biochemical/genetic systems, Wei and Ploss first demonstrated that incubation of RrcDNA
with either cytoplasmic or nuclear yeast extracts led to efficient cccDNA formation. Using genetic
inactivation and antibody depletion assays, five of the eight yeast proteins involving in DNA lagging
strand synthesis were identified as essential for cccDNA formation in the yeast cell extracts. The
involvement of proliferating cell nuclear antigen (PCNA) in cccDNA formation was further
confirmed in HepG2-NTCP cell extracts and the requirement of Pol δ in cccDNA synthesis was
further validated in HBV-infected HepG2-NTCP cells by aphidicolin treatment. Intriguingly,
reconstitution of the in vitro cccDNA synthesis reaction with the five purified human proteins,
including PCNA, replication factor C (RFC) complex, Pol δ, FEN-1 and LIG1, is sufficient to convert
the recombinant rcDNA to cccDNA. Therefore, this study has identified the minimal set of host
cellular proteins required for cccDNA synthesis. In fact, among the five core components, FEN-1,
LIG 1 and Pol δ have been shown to involve in cccDNA synthesis in hepatoma cells in previous
cell-based studies (3-5). However, the involvement of PCNA and RFC, which usually act as scaffold
proteins in recruiting essential proteins in DNA replication, DNA repair and chromatin remodeling, in
cccDNA synthesis in hepatocytes remains to be determined. Interestingly, the finding that both
cytoplasmic and nuclear extracts can efficiently catalyze cccDNA formation implies that rcDNA to
cccDNA conversion occurs in the nucleus of infected hepatocyte probably because rcDNA is shielded
from cytoplasmic factors by the capsids.

The break and gap in the two strands of rcDNA are structurally different (Fig. 1). While the
break in the minus strand of rcDNA has a short flap sequence with viral DNA polymerase attached at
its 5’ terminus, the gap in the plus-strand of rcDNA varies in length and has a short capped RNA
(primer) linked to its 5’ terminus. It is thus speculated that the two strands of rcDNA require different
end processing and may be repaired by distinct DNA repair pathways (proteins). In support of this
hypothesis, a rcDNA species with covalently closed minus-strand DNA, i.e., cc(-)rcDNA, had been
identified in HBV infected cells (2). We demonstrated recently that Pol α and DNA topoisomerase I
involve in the repair of minus strand DNA, whereas topoisomerase II most likely involves in the
repair of plus strand DNA during cccDNA synthesis (5, 6). It will be interesting to see whether the
cc(-)rcDNA can be detected in the in vitro biochemical system. It is also interesting to find out how
FEN1 processes the ends of both strands of RrcDNA.

It is also important to point out that several cellular proteins reported to be required for cccDNA
synthesis in hepatocytes were not identified as essential components of the in vitro cccDNA synthesis
(Fig. 1). For instance, it had been demonstrated in cell-based studies that DNA polymerases are
distinctly required for the cccDNA synthesis in de novo HBV infection and intracellular amplification
pathways. While Pol κ or λ is required for de novo cccDNA synthesis, Pol α, δ or ɛ is required for
intracellular cccDNA amplification (5). However, only Pol δ is required for in vitro cccDNA
synthesis. In addition, it is rather interesting that the supercoiled cccDNA is formed in vitro without
DNA topoisomerases. The requirement of topoisomerases in cccDNA formation in hepatocytes
indicates the cccDNA synthesis involves several topological changes of its precursor and/or
intermediates. Moreover, the efficient conversion of RrcDNA to cccDNA in the biochemical system
reconstituted with the five purified core components of lagging strand DNA synthesis indicates that
histones are not required in cccDNA synthesis in vitro. However, the studies by others and us suggest
that upon reaching the nucleus, HBV rcDNA or retroviral episomal cDNA quickly associate with
nucleosomes to form minichromosomes and thereby the repair reaction for cccDNA formation may
not take place on naked rcDNA, but rcDNA minichromosomes (5).

Apparently, cccDNA synthesis in the nucleus of hepatocyte is much more complicated than the
in vitro repair reaction of RrcDNA. Because the recruitment of DNA repair enzymes is usually
determined by the lesion recognition DNA binding scaffold proteins, it is possible that binding of
PCNA and/or RFC to the RrcDNA dominates the recruitment of DNA repair components required for
the in vitro cccDNA synthesis, which may or may not recapitulate the rcDNA repair process in
hepatocytes. PCNA usually serves as an interacting platform for specialized polymerases, whose
ubiquitination play a critical role in the selection and switch of DNA polymerases (7). In addition,
FEN-1 is able to bind to PCNA by a PCNA-interacting protein (PIP) motif and could also be activated
by PCNA. Therefore, it is important to validate whether PCNA and RFC involve in cccDNA
synthesis in hepatocytes.

Taken together, while the in vitro cccDNA formation system provides an excellent platform for
biochemical analyses of cccDNA biosynthesis, further cross-examinational study with the
comprehensive cell-based HBV infection systems and well-defined biochemical assays should resolve
the discrepancy described above and define the role and mechanism of cellular DNA repair proteins
and pathways in cccDNA biosynthesis.