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

Qi, Yonghe; Gao, Zhenchao; Xu, Guangwei; Peng, Bo; Liu, Chenxuan; Yan, Huan; Yao, Qiyan; Sun, Guoliang; Yang, Liu; Tang, Dingbin; Song, Zilin; Wang, He; Sun, Yinyan; Guo, Ju‐Tao; Li, Wenhui

doi:10.1371/journal.ppat.1005893

Cited by 166 publications

(188 citation statements)

References 65 publications

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“…At least some of the RC-DNA to cccDNA conversion steps could be performed by P protein, foremostly filling-in the gap in plus-strand DNA. However, confirming earlier studies in animal models [50,51,52,53], inhibition of HBV P protein’s DNA polymerase activity did not block cccDNA formation in HepaRG cells [54], HepG2-NTCP [46] cells or stem cell derived hepatocytes [55]. …”

Section: From P Protein-linked Rc-dna To Cccdna In Multiple Steps—supporting

confidence: 85%

A Role for the Host DNA Damage Response in Hepatitis B Virus cccDNA Formation—and Beyond?

Schreiner

Nassal

2017

Viruses

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Chronic hepatitis B virus (HBV) infection puts more than 250 million people at a greatly increased risk to develop end-stage liver disease. Like all hepadnaviruses, HBV replicates via protein-primed reverse transcription of a pregenomic (pg) RNA, yielding an unusually structured, viral polymerase-linked relaxed-circular (RC) DNA as genome in infectious particles. Upon infection, RC-DNA is converted into nuclear covalently closed circular (ccc) DNA. Associating with cellular proteins into an episomal minichromosome, cccDNA acts as template for new viral RNAs, ensuring formation of progeny virions. Hence, cccDNA represents the viral persistence reservoir that is not directly targeted by current anti-HBV therapeutics. Eliminating cccDNA will thus be at the heart of a cure for chronic hepatitis B. The low production of HBV cccDNA in most experimental models and the associated problems in reliable cccDNA quantitation have long hampered a deeper understanding of cccDNA molecular biology. Recent advancements including cccDNA-dependent cell culture systems have begun to identify select host DNA repair enzymes that HBV usurps for RC-DNA to cccDNA conversion. While this list is bound to grow, it may represent just one facet of a broader interaction with the cellular DNA damage response (DDR), a network of pathways that sense and repair aberrant DNA structures and in the process profoundly affect the cell cycle, up to inducing cell death if repair fails. Given the divergent interactions between other viruses and the DDR it will be intriguing to see how HBV copes with this multipronged host system.

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Section: From P Protein-linked Rc-dna To Cccdna In Multiple Steps—supporting

confidence: 85%

A Role for the Host DNA Damage Response in Hepatitis B Virus cccDNA Formation—and Beyond?

Schreiner

Nassal

2017

Viruses

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“…The latter was shown to involve Ku80, a protein of the host's DNA repair pathway (31). In addition, polymerase was found to be crucial for cccDNA formation (35), and the observation that a host cell polymerase is involved in the conversion from rcDNA to cccDNA is in line with our results that NA treatment did not inhibit cccDNA formation.…”

Section: Fig 4 Legend (Continued)supporting

confidence: 84%

Capsid Assembly Modulators Have a Dual Mechanism of Action in Primary Human Hepatocytes Infected with Hepatitis B Virus

Berke

Dehertogh

Vergauwen

et al. 2017

Antimicrob Agents Chemother

122

129

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Hepatitis B virus (HBV) capsid assembly is a critical step in the propagation of the virus and is mediated by the core protein. Due to its multiple functions in the viral life cycle, core became an attractive target for new antiviral therapies. Capsid assembly modulators (CAMs) accelerate the kinetics of capsid assembly and prevent encapsidation of the polymerase-pregenomic RNA (Pol-pgRNA) complex, thereby blocking viral replication. CAM JNJ-632 is a novel and potent inhibitor of HBV replication in vitro across genotypes A to D. It induces the formation of morphologically intact viral capsids, as demonstrated by size exclusion chromatography and electron microscopy studies. Antiviral profiling in primary human hepatocytes revealed that CAMs prevented formation of covalently closed circular DNA in a dosedependent fashion when the compound was added together with the viral inoculum, whereas nucleos(t)ide analogues (NAs) did not. This protective effect translated into a dose-dependent reduction of intracellular HBV RNA levels as well as reduced HBe/cAg and HBsAg levels in the cell culture supernatant. The same observation was made with another CAM (BAY41-4109), suggesting that mechanistic rather than compound-specific effects play a role. Our data show that CAMs have a dual mechanism of action, inhibiting early and late steps of the viral life cycle. These effects clearly differentiate CAMs from NAs and may translate into higher functional cure rates in a clinical setting when given alone or in combination with the current standard of care.

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“…Unlike DNA polymerase inhibitors, treatment of capsid assembly modulators may suppress the selection of drug resistance. Due to the lack of cell culture systems supporting efficient multiple rounds of HBV infection and spread (22,69), this unique therapeutic feature of capsid assembly modulators can be examined only in future clinical trials.…”

Section: Discussionmentioning

confidence: 99%

“…Rather, it is structurally metastable, functionally participates in the process of reverse transcriptional replication of viral DNA, and plays critical roles in the regulation of virion assembly and egress (15,16) as well as rcDNA nuclear import, nucleocapsid uncoating, and cccDNA formation via de novo infection and intracellular amplification pathways (17)(18)(19). It has also been speculated that HBV core proteins may associate with the cccDNA minichromosome, in an as-yet-undefined structural nature, to modulate its structure and transcription activity (20)(21)(22). Intriguingly, the core protein had also been shown to recruit cytokine-induced DNA cytosine deaminase APOBEC3A or APOBEC3B to cccDNA minichromosome, which results in cytosine deamination and decay of cccDNA (23).…”

mentioning

confidence: 99%

Discovery and Mechanistic Study of Benzamide Derivatives That Modulate Hepatitis B Virus Capsid Assembly

Zhao

Zhang

et al. 2017

J Virol

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Chronic hepatitis B virus (HBV) infection is a global public health problem. Although the currently approved medications can reliably reduce the viral load and prevent the progression of liver diseases, they fail to cure the viral infection. In an effort toward discovery of novel antiviral agents against HBV, a group of benzamide (BA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA were discovered. The initial lead optimization efforts identified two BA derivatives with improved antiviral activity for further mechanistic studies. Interestingly, similar to our previously reported sulfamoylbenzamides (SBAs), the BAs promote the formation of empty capsids through specific interaction with HBV core protein but not other viral and host cellular components. Genetic evidence suggested that both SBAs and BAs inhibited HBV nucleocapsid assembly by binding to the heteroaryldihydropyrimidine (HAP) pocket between core protein dimer-dimer interfaces. However, unlike SBAs, BA compounds uniquely induced the formation of empty capsids that migrated more slowly in native agarose gel electrophoresis from A36V mutant than from the wild-type core protein.Moreover, we showed that the assembly of chimeric capsids from wild-type and drug-resistant core proteins was susceptible to multiple capsid assembly modulators. Hence, HBV core protein is a dominant antiviral target that may suppress the selection of drug-resistant viruses during core protein-targeting antiviral therapy. Our studies thus indicate that BAs are a chemically and mechanistically unique type of HBV capsid assembly modulators and warranted for further development as antiviral agents against HBV.IMPORTANCE HBV core protein plays essential roles in many steps of the viral replication cycle. In addition to packaging viral pregenomic RNA (pgRNA) and DNA polymerase complex into nucleocapsids for reverse transcriptional DNA replication to take place, the core protein dimers, existing in several different quaternary structures in infected hepatocytes, participate in and regulate HBV virion assembly, capsid uncoating, and covalently closed circular DNA (cccDNA) formation. It is anticipated that small molecular core protein assembly modulators may disrupt one or multiple steps of HBV replication, depending on their interaction with the distinct quaternary structures of core protein. The discovery of novel core protein-targeting antivirals, such as benzamide derivatives reported here, and investigation of their antiviral mechanism may lead to the identification of antiviral therapeutics for the cure of chronic hepatitis B.KEYWORDS antiviral agents, capsid assembly, hepatitis B virus

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

Cited by 166 publications

References 65 publications

A Role for the Host DNA Damage Response in Hepatitis B Virus cccDNA Formation—and Beyond?

A Role for the Host DNA Damage Response in Hepatitis B Virus cccDNA Formation—and Beyond?

Capsid Assembly Modulators Have a Dual Mechanism of Action in Primary Human Hepatocytes Infected with Hepatitis B Virus

Discovery and Mechanistic Study of Benzamide Derivatives That Modulate Hepatitis B Virus Capsid Assembly

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