cccDNA Amplification Research Articles

Hepatitis B Virus Capsid: The Core in Productive Entry and Covalently Closed Circular DNA Formation.

Hepatitis B virus (HBV) relies on the core protein (HBc) to establish productive infection, as defined by the formation of the covalently closed circularized DNA (cccDNA), as well as to carry out almost every step of the lifecycle following cccDNA formation. Multiple copies of HBc form an icosahedral capsid shell that encapsidates the viral pregenomic RNA (pgRNA) and facilitates the reverse transcription of pgRNA to a relaxed circular DNA (rcDNA) within the capsid. During infection, the complete HBV virion, which contains an outer envelope layer in addition to the internal nucleocapsid containing rcDNA, enters human hepatocytes via endocytosis and traffics through the endosomal compartments and the cytosol to deliver its rcDNA to the nucleus to produce cccDNA. In addition, progeny rcDNA, newly formed in cytoplasmic nucleocapsids, is also delivered to the nucleus in the same cell to form more cccDNA in a process called intracellular cccDNA amplification or recycling. Here, we focus on recent evidence demonstrating differential effects of HBc in affecting cccDNA formation during de novo infection vs. recycling, obtained using HBc mutations and small molecule inhibitors. These results implicate a critical role of HBc in determining HBV trafficking during infection, as well as in nucleocapsid disassembly (uncoating) to release rcDNA, events essential for cccDNA formation. HBc likely functions in these processes via interactions with host factors, which contributes critically to HBV host tropism. A better understanding of the roles of HBc in HBV entry, cccDNA formation, and host species tropism should accelerate ongoing efforts to target HBc and cccDNA for the development of an HBV cure and facilitate the establishment of convenient animal models for both basic research and drug development.

Heat Shock Protein Family A Member 1 Promotes Intracellular Amplification of Hepatitis B Virus Covalently Closed Circular DNA.

Hepatitis B virus (HBV) contains a partially double-stranded relaxed circular DNA (rcDNA) genome that is converted into a covalently closed circular DNA (cccDNA) in the nucleus of the infected hepatocyte by cellular DNA repair machinery. cccDNA associates with nucleosomes to form a minichromosome that transcribes RNA to support the expression of viral proteins and reverse transcriptional replication of viral DNA. In addition to the de novo synthesis from incoming virion rcDNA, cccDNA can also be synthesized from rcDNA in the progeny nucleocapsids within the cytoplasm of infected hepatocytes via the intracellular amplification pathway. In our efforts to identify cellular DNA repair proteins required for cccDNA synthesis using a chemogenetic screen, we found that B02, a small-molecule inhibitor of DNA homologous recombination repair protein RAD51, significantly enhanced the synthesis of cccDNA via the intracellular amplification pathway in human hepatoma cells. Ironically, neither small interfering RNA (siRNA) knockdown of RAD51 expression nor treatment with another structurally distinct RAD51 inhibitor or activator altered cccDNA amplification. Instead, it was found that B02 treatment significantly elevated the levels of multiple heat shock protein mRNA, and siRNA knockdown of HSPA1 expression or treatment with HSPA1 inhibitors significantly attenuated B02 enhancement of cccDNA amplification. Moreover, B02-enhanced cccDNA amplification was efficiently inhibited by compounds that selectively inhibit DNA polymerase α or topoisomerase II, the enzymes required for cccDNA intracellular amplification. Our results thus indicate that B02 treatment induces a heat shock protein-mediated cellular response that positively regulates the conversion of rcDNA into cccDNA via the authentic intracellular amplification pathway. IMPORTANCE Elimination or functional inactivation of cccDNA minichromosomes in HBV-infected hepatocytes is essential for the cure of chronic hepatitis B virus (HBV) infection. However, lack of knowledge of the molecular mechanisms of cccDNA metabolism and regulation hampers the development of antiviral drugs to achieve this therapeutic goal. Our findings reported here imply that enhanced cccDNA amplification may occur under selected pathobiological conditions, such as cellular stress, to subvert the dilution or elimination of cccDNA and maintain the persistence of HBV infection. Therapeutic inhibition of HSPA1-enhanced cccDNA amplification under these pathobiological conditions should facilitate the elimination of cccDNA and cure of chronic hepatitis B.

Constrained evolution of overlapping genes in viral host adaptation: Acquisition of glycosylation motifs in hepadnaviral precore/core genes.

Hepadnaviruses use extensively overlapping genes to expand their coding capacity, especially the precore/core genes encode the precore and core proteins with mostly identical sequences but distinct functions. The precore protein of the woodchuck hepatitis virus (WHV) is N-glycosylated, in contrast to the precore of the human hepatitis B virus (HBV) that lacks N-glycosylation. To explore the roles of the N-linked glycosylation sites in precore and core functions, we substituted T77 and T92 in the WHV precore/core N-glycosylation motifs (75NIT77 and 90NDT92) with the corresponding HBV residues (E77 and N92) to eliminate the sequons. Conversely, these N-glycosylation sequons were introduced into the HBV precore/core gene by E77T and N92T substitutions. We found that N-glycosylation increased the levels of secreted precore gene products from both HBV and WHV. However, the HBV core (HBc) protein carrying the E77T substitution was defective in supporting virion secretion, and during infection, the HBc E77T and N92T substitutions impaired the formation of the covalently closed circular DNA (cccDNA), the critical viral DNA molecule responsible for establishing and maintaining infection. In cross-species complementation assays, both HBc and WHV core (WHc) proteins supported all steps of intracellular replication of the heterologous virus while WHc, with or without the N-glycosylation sequons, failed to interact with HBV envelope proteins for virion secretion. Interestingly, WHc supported more efficiently intracellular cccDNA amplification than HBc in the context of either HBV or WHV. These findings reveal novel determinants of precore secretion and core functions and illustrate strong constraints during viral host adaptation resulting from their compact genome and extensive use of overlapping genes.

Regulatory function of interferon-inducible 44-like for hepatitis B virus covalently closed circular DNA in primary human hepatocytes.

Curing hepatitis B virus (HBV) infection requires elimination of covalently closed circular DNA (cccDNA). Interferon (IFN)-γ has noncytolytic antiviral potential; however, elimination of cccDNA could not be achieved. To enhance the regulatory effect, we comprehensively analyzed the host factors associated with cccDNA amplification and IFN-γ and IFN-α effects using an in vitro HBV infection system showing various transcription levels. Primary human hepatocytes were infected with HBV using genomic plasmids carrying the basic core promoter mutation A1762T/G1764A and/or the precore mutation G1896A and treated with IFN-γ and IFN-α. Comprehensive and functional studies involving microarray and small interfering RNA analysis revealed the host factors related to cccDNA regulation. The HBV infection system reproduced the HBV life cycle and showed various propagation levels. Microarray analysis revealed 53 genes correlated with the cccDNA levels. Of the 53 genes, expression of IFN-induced protein 44-like (IFI44L) was significantly upregulated by IFN-γ and IFN-α. The anti-HBV effect of IFI44L is exerted regardless of IFN-γ or IFN-α by inhibiting the activation of nuclear factor-κB and signal transducer and activator of transcription 1 pathways. Using the in vitro HBV infection system, an IFN-inducible molecule, IFI44L, associated with cccDNA amplification, was identified. These results suggest an innovative molecular strategy for the regulation of HBV cccDNA by controlling a novel host factor, IFI44L.

Hepatitis B virus nucleocapsid uncoating: biological consequences and regulation by cellular nucleases

ABSTRACT Upon infection of hepatocyte, Hepatitis B virus (HBV) genomic DNA in nucleocapsid is transported into the nucleus and converted into a covalently closed circular (ccc) DNA to serve as the template for transcription of viral RNAs. Viral DNA in the cytoplasmic progeny nucleocapsid is another resource to fuel cccDNA amplification. Apparently, nucleocapsid disassembly, or viral genomic DNA uncoating, is an essential step for cccDNA synthesis from both de novo infection and intracellular amplification pathways, and has a potential to activate DNA sensors and induce an innate immune response in infected hepatocytes. However, where and how the nucleocapsid disassembly occurs is not well understood. The work reported herein showed that the enhanced disassembly of progeny mature nucleocapsids in the cytoplasm supported cccDNA intracellular amplification, but failed to activate the cGAS-STING-mediated innate immune response in hepatocytes. Interestingly, while expression of a cytoplasmic exonuclease TREX1 in human hepatoma cells supporting HBV replication significantly reduced the amounts of cccDNA as well as its precursor, deproteinized relaxed circular (rc) DNA, expression of TREX1 in sodium taurocholate cotransporting polypeptide-expressing human hepatoma cells did not inhibit cccDNA synthesis from de novo HBV infection. The results from this cytoplasmic nuclease protection assay imply that the disassembly of progeny mature nucleocapsids and removal of viral DNA polymerase covalently linked to the 5′ end of minus strand of rcDNA take place in the cytoplasm. On the contrary, the disassembly of virion-derived nucleocapsids during de novo infection may occur at a different subcellular compartment and possibly via distinct mechanisms.

Discovery and structure activity relationship of glyoxamide derivatives as anti-hepatitis B virus agents

Chronic hepatitis B viral infection is a significant health problem world-wide, and currently available antiviral agents suppress HBV infections, but rarely cure this disease. It is presumed that antiviral agents that target the viral nuclear reservoir of transcriptionally active cccDNA may eliminate HBV infection. Through a series of chemical optimization, we identified a new series of glyoxamide derivatives affecting HBV nucleocapsid formation and cccDNA maintenance at low nanomolar levels. Among all the compounds synthesized, GLP-26 displays a major effect on HBV DNA, HBeAg secretion and cccDNA amplification. In addition, GLP-26 shows a promising pre-clinical profile and long-term effect on viral loads in a humanized mouse model.

DNA Polymerase alpha is essential for intracellular amplification of hepatitis B virus covalently closed circular DNA.

Persistent hepatitis B virus (HBV) infection relies on the establishment and maintenance of covalently closed circular (ccc) DNA, a 3.2 kb episome that serves as a viral transcription template, in the nucleus of an infected hepatocyte. Although evidence suggests that cccDNA is the repair product of nucleocapsid associated relaxed circular (rc) DNA, the cellular DNA polymerases involving in repairing the discontinuity in both strands of rcDNA as well as the underlying mechanism remain to be fully understood. Taking a chemical genetics approach, we found that DNA polymerase alpha (Pol α) is essential for cccDNA intracellular amplification, a genome recycling pathway that maintains a stable cccDNA pool in infected hepatocytes. Specifically, inhibition of Pol α by small molecule inhibitors aphidicolin or CD437 as well as silencing of Pol α expression by siRNA led to suppression of cccDNA amplification in human hepatoma cells. CRISPR-Cas9 knock-in of a CD437-resistant mutation into Pol α genes completely abolished the effect of CD437 on cccDNA formation, indicating that CD437 directly targets Pol α to disrupt cccDNA biosynthesis. Mechanistically, Pol α is recruited to HBV rcDNA and required for the generation of minus strand covalently closed circular rcDNA, suggesting that Pol α is involved in the repair of the minus strand DNA nick in cccDNA synthesis. Our study thus reveals that the distinct host DNA polymerases are hijacked by HBV to support the biosynthesis of cccDNA from intracellular amplification pathway compared to that from de novo viral infection, which requires Pol κ and Pol λ.

Duck Hepatitis B Virus cccDNA Amplification Efficiency in Natural Infection Is Regulated by Virus Secretion Efficiency.

Previous mutation based studies showed that ablating synthesis of viral envelope proteins led to elevated hepadnaviral covalently closed circular DNA (cccDNA) amplification, but it remains unknown how cccDNA amplification is regulated in natural hepadnaviral infection because of a lack of research system. In this study we report a simple procedure to prepare two identical duck hepatitis B virus inocula, but they possess 10-100-fold difference in cccDNA amplification in infected cell culture. We demonstrate that the infected cells with higher cccDNA amplification significantly reduce the virus secretion efficiency that results in higher accumulation of relaxed circular DNA (rcDNA) and DHBsAg in the cells. The infected cells with lower cccDNA amplification significantly increase the virus secretion efficiency that leads to lower intracellular rcDNA and DHBsAg accumulation. In contrast with the findings generated in the mutation based experimental system, the regulation of cccDNA amplification in natural hepadnaviral infection bypasses direct regulation of the cellular envelope proteins concentration, instead it modulates virus secretion efficiency that ultimately impacts the intracellular rcDNA concentration, an important factor determining the destination of the synthesized rcDNA in infected cells.

Towards the elimination and eradication of hepatitis B

Despite the introduction of vaccination, chronic hepatitis B remains a major cause of liver-related morbidity and mortality including cirrhosis, decompensated cirrhosis and hepatocellular carcinoma. Maintenance antiviral therapy is required for most people, as low rates of cure occur. The stated aim of therapy presently is HBV DNA suppression; effective suppression of viral replication is associated with significant reductions in morbidity from end-stage liver failure and to an extent, hepatocellular carcinoma. Unfortunately, major barriers to cure, such as a reservoir of episomal covalently closed circular DNA (cccDNA) (the HBV minichromosome), and a dysfunctional immune response, pose challenges. These barriers will need to be overcome to ensure higher rates of cure than can be achieved presently.Quantitative and diagnostic testing for HBV DNA is not generally available, hampering effective monitoring and treatment in low-income countries. The majority of patients in resource-constrained countries are not identified before the onset of cirrhosis. Without coordinated action, and transfer of new diagnostic technologies and treatments to low-income countries, recent therapeutic advances will have little effect on the global burden of disease.A shift to curative treatment for the majority would be a major advance in the elimination of hepatitis B. New and improved molecular therapeutics and immunological strategies for the treatment of chronic hepatitis are emerging, however. A number of promising lines of development are in progress. A curative regimen may require a combination of viral suppression via nucleoside analogue therapy to prevent cccDNA amplification and viral propagation, safe selective cccDNA inhibitors to deplete, silence or degrade cccDNA, agents to block the entry of HBV into the hepatocyte plus compounds to prevent capsid assembly and cccDNA interactions. Targeted immune activation could restore the exhausted immune cell repertoire.

Inhibition of Hepatitis B Virus cccDNA by siRNA in Transgenic Mice

The elimination of viral covalently closed circular DNA (cccDNA) from the nucleus of infected hepatocytes is an obstacle to achieving sustained viral clearance during antiviral therapy of chronic hepatitis B virus (HBV) infection. The aim of our study was to determine whether treatment with siRNA is able to suppress viral cccDNA amplification using a HBV-transgenic mice model. The experimental results revealed that siRNAs can serve as efficient alternative anti-HBV agents, because they showed better inhibitory effect on viral replication and antigen expression in transgenic mice. More importantly, the siRNA markedly inhibited HBV cccDNA amplification.

Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

Persistence of hepatitis B virus (HBV) infection requires covalently closed circular (ccc)DNA formation and amplification, which can occur via intracellular recycling of the viral polymerase-linked relaxed circular (rc) DNA genomes present in virions. Here we reveal a fundamental difference between HBV and the related duck hepatitis B virus (DHBV) in the recycling mechanism. Direct comparison of HBV and DHBV cccDNA amplification in cross-species transfection experiments showed that, in the same human cell background, DHBV but not HBV rcDNA converts efficiently into cccDNA. By characterizing the distinct forms of HBV and DHBV rcDNA accumulating in the cells we find that nuclear import, complete versus partial release from the capsid and complete versus partial removal of the covalently bound polymerase contribute to limiting HBV cccDNA formation; particularly, we identify genome region-selectively opened nuclear capsids as a putative novel HBV uncoating intermediate. However, the presence in the nucleus of around 40% of completely uncoated rcDNA that lacks most if not all of the covalently bound protein strongly suggests a major block further downstream that operates in the HBV but not DHBV recycling pathway. In summary, our results uncover an unexpected contribution of the virus to cccDNA formation that might help to better understand the persistence of HBV infection. Moreover, efficient DHBV cccDNA formation in human hepatoma cells should greatly facilitate experimental identification, and possibly inhibition, of the human cell factors involved in the process.

Combination of small interfering RNAs mediates greater suppression on hepatitis B virus cccDNA in HepG2.2.15 cells

To observe the inhibition of hepatitis B virus (HBV) replication and expression in HepG2.2.15 cells by combination of small interfering RNAs (siRNAs). Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells. At 48 h, 72 h and 96 h after transfection, culture media were collected and cells were harvested for HBV replication assay. HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay (ELISA). Intracellular viral DNA and covalently closed circular DNA (cccDNA) were quantified by real-time polymerase chain reaction (PCR). HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR (RT-PCR). siRNAs showed marked anti-HBV effects. siRNAs could specifically inhibit the expression of HBsAg and the replication of HBV DNA in a dose-dependent manner. Furthermore, combination of siRNAs, compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication. More importantly, combination of siRNAs significantly suppressed HBV cccDNA amplification. Combination of siRNAs mediates a stronger inhibition on viral replication and antigen expression in HepG2.2.15 cells, especially on cccDNA amplification.

Recombinant baculovirus expressing anti‐HBsAg shRNA inhibits HBV cccDNA amplification in HepG2 cells

A recombinant baculovirus (bac) expressing U6 promoter-encoded anti-HBV surface antigen (sAg) short hairpin RNAs (U6S) was generated. HepG2 cells were transduced with U6S 24hrs prior to bac-mediated HBV infection and the effect of U6S on HBV replicative intermediates (RI), extracellular DNA (EC), and covalently closed circular DNA (cccDNA) was analyzed. U6S significantly inhibited HBV RI, EC, and cccDNA levels through day 6 post-HBV infection compared to control treated cells. The effect of U6S on a persistent HBV infection was analyzed using a novel HBV infected HepG2 cell subculture system. HBV infected HepG2 cells subcultured 1:4 10 days post bac-mediated HBV infection, a time in which HBV transcription is primarily driven off cccDNA, continue to produce constant levels of HBV RI, EC, and cccDNA through 20 days post-subculture while retaining the ability to be reproducibly transduced at 100% efficiency by recombinant bac. HBV RI and EC were significantly inhibited through day 8 post-U6S treatment in HBV infected HepG2 cells transduced with U6S 24 hrs post-subculture; however, HBV cccDNA levels were unaffected compared to control treated cells. Bac expressed shRNA U6S is capable of reducing HBV RI, EC and cccDNA levels when administered prior to HBV infection; however, during a persistent infection, U6S can reduce only HBV RI and EC and not cccDNA levels. Research supported by RO1-CA 023931 to H.C.I. from N.I.H.

Intrahepatic Hepatitis B Virus Covalently Closed Circular DNA Can Be a Predictor of Sustained Response to Therapy

This study aimed to determine whether intrahepatic hepatitis B virus (HBV) covalently closed circular (ccc) DNA and total HBV DNA levels at the end of therapy would predict sustained response to therapy. Hepatitis B e antigen (HBeAg)-positive chronic hepatitis B patients receiving either lamivudine monotherapy or combination of peginterferon and lamivudine had liver biopsy at the end of 1 year therapy and were followed for 52 more weeks after cessation of therapy. Serum HBV DNA, intrahepatic HBV ccc DNA, and total HBV DNA levels were determined. Forty-seven patients, including 34 males and 13 females, were studied. Twenty-seven patients received combination therapy, and 20 patients received lamivudine monotherapy. Twenty-nine patients had end-of-treatment virologic response, and 15 patients had sustained response 52 weeks after therapy. At the end of treatment, log serum HBV DNA levels correlated well with log intrahepatic HBV cccDNA and log intrahepatic total HBV DNA levels. Log intrahepatic cccDNA and log intrahepatic total DNA levels were significantly lower among patients with sustained virologic response. The adjusted odds ratio for log cccDNA was 5.3 (95% CI: 1.5-18.2, P = .009) and, for log intrahepatic HBV DNA, was 4.4 (95% CI: 1.3-14.7, P = .015) to predict sustained virologic response. Using log cccDNA at -0.80 copies/genome equivalent as cutoff, the sensitivity, specificity, and positive and negative predictive values and accuracy of predicting sustained virologic response were 73%, 78%, 56%, 86%, and 77% respectively. Intrahepatic HBV cccDNA and intrahepatic total HBV DNA levels at the end of therapy are superior to serum HBV DNA as surrogates of sustained virologic response.

Inhibitory effect of adefovir on viral DNA synthesis and covalently closed circular DNA formation in duck hepatitis B virus-infected hepatocytes in vivo and in vitro.

The elimination of viral covalently closed circular DNA (CCC DNA) from the nucleus of infected hepatocytes is an obstacle to achieving sustained viral clearance during antiviral therapy of chronic hepatitis B virus (HBV) infection. The aim of our study was to determine whether treatment with adefovir, a new acyclic nucleoside phosphonate, the prodrug of which, adefovir dipivoxil, is in clinical evaluation, is able to suppress viral CCC DNA both in vitro and in vivo using the duck HBV (DHBV) model. First, the effect of adefovir on viral CCC DNA synthesis was examined with primary cultures of DHBV-infected fetal hepatocytes. Adefovir was administered for six consecutive days starting one day before or four days after DHBV inoculation. Dose-dependent inhibition of both virion release in culture supernatants and synthesis of intracellular viral DNA was observed. Although CCC DNA amplification was inhibited by adefovir, CCC DNA was not eliminated by antiviral treatment and the de novo formation of CCC DNA was not prevented by pretreatment of the cells. Next, preventive treatment of experimentally infected ducklings with lamivudine or adefovir revealed that both efficiently suppressed viremia and intrahepatic DNA. However, persistence of viral DNA even when detectable only by PCR was associated with a recurrence of viral replication following drug withdrawal. Taken together, our results demonstrate that adefovir is a potent inhibitor of DHBV replication that inhibits CCC DNA amplification but does not effectively prevent the formation of CCC DNA from incoming viral genomes.

N-Butyrate, a cell cycle blocker, inhibits early amplification of duck hepatitis B virus covalently closed circular DNA after in vitro infection of duck hepatocytes.

During chronic hepadnavirus infection, virus persistence depends on the regulation of the pool of covalently closed circular DNA (cccDNA), which is the template for transcription of viral RNA species. The development of in vitro infection of duck hepatocyte primary cultures by duck hepatitis B virus (DHBV) provides a unique opportunity to study the regulation of cccDNA synthesis. After DHBV in vitro infection, cccDNA is detected 1 day later and is amplified to a high copy number after 1 week in culture. We studied whether this amplification occurs during cell cycle progression of duckling hepatocytes. By using [3H]thymidine incorporation, we found that hepatocytes obtained from 3-week-old ducklings spontaneously entered the S phase of the cell cycle when cultured in serum-free medium without added growth factors. Bromodeoxyuridine labeling confirmed that cellular DNA synthesis took place in more than 50% of parenchymal cells. Cytofluorometry analysis revealed the presence of asynchronous populations and polyploidization processes. The addition of a cell cycle blocker, n-butyrate, completely inhibited [3H]thymidine incorporation and blocked duckling hepatocytes in the G1 phase of the cell cycle. Simultaneously, butyrate inhibited cccDNA amplification and allowed the establishment of DHBV infection, as demonstrated by the detection of a basal level of cccDNA in treated hepatocytes. Both effects were reversible since active cell DNA synthesis was restored and cccDNA accumulated after drug withdrawal.

Morphogenetic and regulatory effects of mutations in the envelope proteins of an avian hepadnavirus

The envelope gene of the avian hepadnavirus, duck hepatitis B virus, was mutated in order to dissect the functions of the two major envelope proteins pre-S/S and S. Both envelope proteins were found to be required for virus particle assembly and secretion. The placement of stop codons after each of the first three AUG codons in the pre-S region allowed efficient translational initiation at downstream AUG codons to produce novel N-terminally truncated pre-S/S proteins. These proteins could substitute for pre-S/S protein in the production of enveloped virus production, but not in the production infectious virus. A mutant defective in myristylation of the pre-S/S protein produced reduced amounts of enveloped virus, and this virus was not infectious. Mutants defective in the pre-S/S protein accumulated high levels of covalently closed circular viral DNA (cccDNA) compared with the wild type or with a mutant defective in only the S protein. Hyperamplification of cccDNA resulted in high levels of viral RNA, consistent with the proposed role of cccDNA as the transcriptional template. Myristylation of the pre-S/S protein was not required for control of cccDNA amplification, and mutants that produced N-terminally truncated pre-S/S proteins displayed higher levels of cccDNA. We concluded that the pre-S/S protein, but not the S protein, is required for control of cccDNA amplification and persistent infection.