Inhibit Hepatitis B Virus Replication Research Articles

Advances in Targeting the Innate and Adaptive Immune Systems to Cure Chronic Hepatitis B Virus Infection.

“Functional cure” is being pursued as the ultimate endpoint of antiviral treatment in chronic hepatitis B (CHB), which is characterized by loss of HBsAg whether or not anti-HBs antibodies are present. “Functional cure” can be achieved in <10% of CHB patients with currently available therapeutic agents. The dysfunction of specific immune responses to hepatitis B virus (HBV) is considered the major cause of persistent HBV infection. Thus, modulating the host immune system to strengthen specific cellular immune reactions might help eliminate HBV. Strategies are needed to restore/enhance innate immunity and induce HBV-specific adaptive immune responses in a coordinated way. Immune and resident cells express pattern recognition receptors like TLRs and RIG I/MDA5, which play important roles in the induction of innate immunity through sensing of pathogen-associated molecular patterns (PAMPs) and bridging to adaptive immunity for pathogen-specific immune control. TLR/RIG I agonists activate innate immune responses and suppress HBV replication in vitro and in vivo, and are being investigated in clinical trials. On the other hand, HBV-specific immune responses could be induced by therapeutic vaccines, including protein (HBsAg/preS and HBcAg), DNA, and viral vector-based vaccines. More than 50 clinical trials have been performed to assess therapeutic vaccines in CHB treatment, some of which display potential effects. Most recently, using genetic editing technology to generate CAR-T or TCR-T, HBV-specific T cells have been produced to efficiently clear HBV. This review summarizes the progress in basic and clinical research investigating immunomodulatory strategies for curing chronic HBV infection, and critically discusses the rather disappointing results of current clinical trials and future strategies.

Hepatitis C virus infection suppresses hepatitis B virus replication via the RIG-I-like helicase pathway

Mechanisms of hepatitis B virus (HBV) reactivation after hepatitis C virus (HCV) elimination by direct-acting antiviral (DAA) treatment in HBV/HCV-co-infected patients remain unclear. We examined RIG-I-like helicase (RLH) pathway activation by HBV mono-infection, HCV mono-infection or HBV/HCV co-infection and interference between HBV and HCV in primary human hepatocytes. Interference between HBV and HCV and HBV reactivation after DAA treatment in humanized-liver mice were assessed. HCV infection activated RLH pathway, as evidenced by RIG-I, ISG15 and ISG56 expression induction; HBV caused only RIG-I induction in vitro. RLH activation was also found in HBV/HCV-co-infected cells, and HBV replication were suppressed in HBV/HCV-co-infected than in HBV-mono-infected cells. siRNA-mediated double knockdown of ISG15 and ISG56 increased HBV replication in HBV/HCV-co-infected cells. HCV infection activated RLH pathway and suppressed HBV replication in humanized-liver mice. Subsequent elimination of HCV by DAA administration downregulated RLH pathway and upregulated HBV replication in mice. RLH pathway was activated in livers of chronic hepatitis C patients compared to those of chronic hepatitis B or non-B, non-C patients. The RLH pathway activation was downregulated by HCV elimination. In conclusion, HCV infection activated RLH pathway and suppressed HBV replication in human hepatocytes. HCV elimination upregulated HBV replication, probably through RLH pathway downregulation.

MCPIP1 inhibits Hepatitis B virus replication by destabilizing viral RNA and negatively regulates the virus-induced innate inflammatory responses

Monocyte chemotactic protein-induced protein 1 (MCPIP1) is an inflammatory regulator in immune response. Recently, MCPIP1 has also been identified as a host antiviral factor against certain virus infection including human immunodeficiency virus, dengue virus and hepatitis C virus. However, whether MCPIP1 could restrict the replication of hepatitis B virus (HBV), a DNA pararetrovirus belonging to Hepadnaviridae family, has not been investigated. In this study, we found that MCPIP1 expression was up-regulated in mouse livers upon acute HBV replication and in HBV-replicated hepatoma cells or HBV-stimulated macrophages. Enforced MCPIP1 expression by hydrodynamic DNA injection in vivo significantly inhibited HBV replication in the mouse livers. Then in vitro studies by overexpression or knockdown assays in cell-lines identified the direct antiviral effect of MCPIP1 on HBV replication. RNA immunoprecipitation and decay assay further suggested that MCPIP1 potently restricted HBV replication through directly binding viral RNA and degrading RNA via its RNase activity, but not deubiquitinase activity. Moreover, we further verified that MCPIP1 negatively regulated HBV-induced proinflammatory cytokines, such as IL-1β, TNF-α and IL-6 in macrophages. Taken together, our data expand MCPIP1's range of viral targets to DNA virus and also demonstrate the negative regulatory role of MCPIP1 in suppressing virus-induced inflammatory response, suggesting MCPIP1 as a potential therapeutic target for treating HBV-related diseases via inducing a host defense against HBV and reducing inflammatory injury meanwhile.

MicroRNA-185 reduces the expression of hepatitis B virus surface antigen by targeting PRKCH in HepG2 2.2.15 cells

MicroRNAs (miRNAs) are single-stranded noncoding RNAs with 18 to 25 nucleotides and play critical roles in a wide spectrum of biological processes. We repored that miR-185 inhibited hepatitis B surface antigen (HBsAg) expression and hepatitis B virus (HBV) replication without affecting the proliferation of HepG2 2.2.15 cells, compared with the controls. We identified that protein kinase C eta (PRKCH) is a direct target gene of miR-185 that affects HBV replication and protein expression and that the miR-185 may suppress HBV replication. Our results provide more information for gene therapy in HBV infection. Keywords: miR-185; HBV; HBV surface antigen; viral replication; PRKCH.

DHX9 interacts with APOBEC3B and attenuates the anti-HBV effect of APOBEC3B

ABSTRACT Hepatitis B virus (HBV) is a partially double-stranded DNA virus that replicates by reverse transcription. We previously demonstrated that the host restriction factor-APOBEC3B (A3B) inhibited HBV replication which was dependent on its deaminase activity during reverse transcription. However, the host factors involved in the process of regulating the anti-HBV function of A3B are less known. In this research, to obtain a comprehensive understanding of the interaction networks of A3B, we conducted coimmunoprecipitation and mass spectrometry to identify A3B-interacting proteins in the presence of HBV. By this approach, we determined that DExD/H-box helicase 9 (DHX9) suppressed the anti-HBV effect of A3B, and this suppression was dependent on their interaction. Although DHX9 did not affect the deamination activity of A3B in vitro assay or the viral DNA editing of A3B in HepG2-NTCP cells that support HBV infection, it inhibited the binding of A3B with pgRNA. These data suggest that DHX9 can interact with A3B and attenuate the anti-HBV efficacy of A3B. Abbreviations: 3D-PCR: differential DNA denaturation PCR; APOBEC3: apolipoprotein B mRNA-editing catalytic polypeptide 3; cccDNA: covalently closed circular DNA; co-IP: coimmunoprecipitation; DDX: DExD-box RNA helicases; HBc: HBV core protein; HBV: hepatitis B virus; HepAD38: HepG2 cell line stably transfected with HBV DNA; HepG2-NTCP: HepG2 cell line stably transfected with Na+/taurocholate cotransporter polypeptide; Huh7: human hepatoma cell line; pgRNA: pregenomic RNA; PPI: protein–protein interactions; RC DNA: relaxed circular DNA.

Coevolution analysis of amino-acids reveals diversified drug-resistance solutions in viral sequences: a case study of hepatitis B virus.

The study of mutational landscapes of viral proteins is fundamental for the understanding of the mechanisms of cross-resistance to drugs and the design of effective therapeutic strategies based on several drugs. Antiviral therapy with nucleos(t)ide analogues targeting the hepatitis B virus (HBV) polymerase protein (Pol) can inhibit disease progression by suppression of HBV replication and makes it an important case study. In HBV, treatment may fail due to the emergence of drug-resistant mutants. Primary and compensatory mutations have been associated with lamivudine resistance, whereas more complex mutational patterns are responsible for resistance to other HBV antiviral drugs. So far, all known drug-resistance mutations are located in one of the four Pol domains, called reverse transcriptase. We demonstrate that sequence covariation identifies drug-resistance mutations in viral sequences. A new algorithmic strategy, BIS2TreeAnalyzer, is designed to apply the coevolution analysis method BIS2, successfully used in the past on small sets of conserved sequences, to large sets of evolutionary related sequences. When applied to HBV, BIS2TreeAnalyzer highlights diversified viral solutions by discovering thirty-seven positions coevolving with residues known to be associated with drug resistance and located on the four Pol domains. These results suggest a sequential mechanism of emergence for some mutational patterns. They reveal complex combinations of positions involved in HBV drug resistance and contribute with new information to the landscape of HBV evolutionary solutions. The computational approach is general and can be applied to other viral sequences when compensatory mutations are presumed.

CON: All Patients With Immune-Tolerated Hepatitis B Virus Do Not Need to Be Treated.

http://aasldpubs.onlinelibrary.wiley.com/hub/journal/10.1002/(ISSN)2046-2484/video/15-1-reading-attar a video presentation of this article.

Host Gene SEL1L Involved in Endoplasmic Reticulum-Associated Degradation Pathway Could Inhibit Hepatitis B Virus at RNA, DNA, and Protein Levels.

Hepatitis B virus (HBV) belongs to the Hepadnaviridae family of enveloped DNA viruses. Recent studies have found that host factors can suppress HBV replication. HBV envelope proteins are reported to be degraded by the endoplasmic reticulum-associated degradation (ERAD) pathway. As a component of the ERAD pathway, suppressor of lin-12-like 1 (SEL1L) was earlier found to be upregulated in the inactive carrier phase of chronic HBV infection relative to that in the immune tolerant phase. However, the role of SEL1L in regulating HBV replication remains largely unknown. In this study, we found the levels of HBV RNA, DNA, and core and envelope proteins to be significantly downregulated by SEL1L overexpression and upregulated by SEL1L silencing in Huh7 cells transiently transfected with an overlength HBV genome. Similar upregulation was observed in HepG2.2.15 cells as well. SEL1L co-localized with HBV surface antigen (HBsAg), which changed its staining pattern. Treatment with an inhibitor of ERAD pathway remarkably increased intracellular S protein. Surprisingly, silencing SEL1L to block the ERAD pathway activated an alternative ER quality control (ERQC)-autophagy pathway, which might account for the increased HBV RNAs and core protein. Together, our results demonstrate that SEL1L is a host restriction factor that exerts anti-HBV effect through ERAD and alternative ERQC-autophagy pathway.

Non-nucleoside hepatitis B virus polymerase inhibitors identified by an in vitro polymerase elongation assay.

Hepatitis B virus (HBV) polymerase is the only virus-encoded enzyme essential for producing the HBV genome and is regarded as an attractive drug target. However, the difficulty of synthesizing and purifying recombinant HBV polymerase protein has hampered the development of new drugs targeting this enzyme, especially compounds unrelated to the nucleoside structure. We recently have developed a technique for the synthesis and purification of recombinant HBV polymerase containing the reverse transcriptase (RT) domain that carried DNA elongation activity in vitro. We used the overproduced protein to establish an in vitro high-throughput screening system to identify compounds that inhibit the elongation activity of HBV polymerase. We screened 1120 compounds and identified a stilbene derivative, piceatannol, as a potential anti-HBV agent. Derivative analysis identified another stilbene derivative, PDM2, that was able to inhibit HBV replication with an IC50 of 14.4 ± 7.7μM. An infection experiment suggested that the compounds inhibit the replication of HBV rather than the entry process, as expected. Surface plasmon resonance analysis demonstrated a specific interaction between PDM2 and the RT domain. Importantly, PDM2 showed similar inhibitory activity against the replication of both wild-type HBV and a lamivudine/entecavir-resistant HBV variant. Furthermore, PDM2 showed an additive effect in combination with clinically used nucleos(t)ide analogs. We report the development of a screening system that is useful for identifying non-nucleos(t)ide RT inhibitors.

HBV Genome and Life Cycle.

Chronic hepatitis B virus (HBV) infection remains to be a serious threat to public health and is associated with many liver diseases including chronic hepatitis B (CHB), liver cirrhosis, and hepatocellular carcinoma. Although nucleos(t)ide analogues (NA) and pegylated interferon-α (Peg-IFNα) have been confirmed to be efficient in inhibiting HBV replication, it is difficult to eradicate HBV and achieve the clinical cure of CHB. Therefore, long-term therapy has been recommended to CHB treatment under the current antiviral therapy. In this context, the new antiviral therapy targeting one or multiple critical steps of viral life cycle may be an alternative approach in future. In the last decade, the functional receptor [sodium-taurocholate cotransporting polypeptide (NTCP)] of HBV entry into hepatocytes has been discovered, and the immature nucleocapsids containing the non- or partially reverse-transcribed pregenomic RNA, the nucleocapsids containing double-strand linear DNA (dslDNA), and the empty particles devoid of any HBV nucleic acid have been found to be released into circulation, which have supplemented the life cycle of HBV. The understanding of HBV life cycle may offer a new instruction for searching the potential antiviral targets, and the new viral markers used to monitor the efficacy of antiviral therapy for CHB patients in the future.

Replenishment of Hepatitis B Virus cccDNA Pool Is Restricted by Baseline Expression of Host Restriction Factors In Vitro.

Background: Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major cause of viral persistence in patients with chronic HBV infection. Understanding the mechanisms underlying stability and persistence of HBV cccDNA in hepatocytes is critical for developing novel therapeutics and managing chronic hepatitis B. In this study, we observed an unexpected increase in HBV cccDNA levels upon suppression of transcription by de novo DNA methyltransferase DNMT3A and uncovered additional mechanisms potentially involved in HBV cccDNA maintenance. Methods: HBV-expressing cell lines were transfected with a DNMT3A-expressing plasmid. Real-time PCR and HBsAg assays were used to assess the HBV replication rate. Cell cycling was analyzed by fluorescent cell sorting. CRISPR/Cas9 was utilized to abrogate expression of APOBEC3A and APOBEC3B. Alterations in the expression of target genes were measured by real-time PCR. Results: Similar to previous studies, HBV replication induced DNMT3A expression, which in turn, led to reduced HBV transcription but elevated HBV cccDNA levels (4- to 6-fold increase). Increased levels of HBV cccDNA were not related to cell cycling, as DNMT3A accelerated proliferation of infected cells and could not contribute to HBV cccDNA expansion by arresting cells in a quiescent state. At the same time, DNMT3A suppressed transcription of innate immunity factors including cytidine deaminases APOBEC3A and APOBEC3B. CRISPR/Cas9-mediated silencing of APOBEC3A and APOBEC3B transcription had minor effects on HBV transcription, but significantly increased HBV cccDNA levels, similar to DNMT3A. In an attempt to further analyze the detrimental effects of HBV and DNMT3A on infected cells, we visualized γ-H2AX foci and demonstrated that HBV inflicts and DNMT3A aggravates DNA damage, possibly by downregulating DNA damage response factors. Additionally, suppression of HBV replication by DNMT3A may be related to reduced ATM/ATR expression. Conclusion: Formation and maintenance of HBV cccDNA pools may be partially suppressed by the baseline expression of host inhibitory factors including APOBEC3A and APOBEC3B. HBV inflicts DNA damage both directly and by inducing DNMT3A expression.

2210 Resolution of Hepatocellular Carcinoma in a Patient With Chronic Hepatitis B Treated With Entecavir Therapy

INTRODUCTION: Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC) globally. Prolonged HBV DNA suppression with nucleos(t)ide analogues is known to regress hepatic fibrosis and reduce HCC risk. This is an unusual presentation of a patient with hepatitis B-associated HCC whose liver lesion resolved on antiviral therapy alone. CASE DESCRIPTION/METHODS: Patient is a 64-year-old Chinese male with HBeAg negative chronic hepatitis B with persistent normal serum aminotransferases and was antiviral treatment naïve. He had a positive family history of HCC and was first noted to have asymptomatic elevation of alpha-fetoprotein (AFP) to 648.9 ng/ml 6 months prior to his referral to Hepatology. During his initial Liver Center visit, the AFP increased to 1,900 ng/ml with elevated AFP-L3 at 18.4%. His hepatitis B remained inactive with normal ALT and minimal detectable HBV DNA at 1.6 log IU/ml (Table 1). He was started on entecavir 0.5 mg daily in view of his age and suspicious HCC complication. Initial MRI of the abdomen revealed lesions in segment 4 (2 cm) and segment 7 (2.1 cm) of the liver with arterial enhancement, venous washout and pseudocapsule. Targeted liver biopsy of segment 7 lesion and adjacent liver was performed within 2 weeks on entecavir therapy. The targeted liver specimen revealed a necrotic focus with loss of reticulin with strong immunoreactivity for cytokeratin Hep Par1 and Glypican-3; the findings were highly suspicious for HCC. The adjacent liver had only mild inflammation (grade 0-1) and minimal fibrosis (stage 0-1). Six weeks later, AFP decreased to 19 ng/dl but AFP-L3 remained elevated at 17%. On repeat CT scan, the segment 7 lesion decreased to 1.2 cm and no longer met OPTN HCC criteria. Patient remained clinically stable with optimal HBV DNA suppression. By 7-month follow-up, both AFP and AFP-L3 normalized and the liver lesions remained stable in size (Table 1). He continued to have regular monitoring and the segment 7 liver lesion was no longer visualized on abdominal MRI after 2 years. DISCUSSION: Nucleos(t)ide analogue therapy is known to suppress HBV replication but has not been reported to have direct effect on HCC. This HCC case is unique as the liver lesion regressed and resolved on entecavir therapy alone. While the molecular mechanism leading to the HCC resolution is currently unknown, this case highlights the beneficial role of prolonged HBV DNA suppression with antiviral therapy.

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice.

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of liver cancer. This is a major concern, especially in developing countries. Failure of the immune system to mount an effective response against HBV leads to chronic infection. Although HBV vaccine is present and novel antiviral medicines are being created, eradication of virus-reservoir cells remains a major health topic. Described here is a method for the generation of viral antigen (Ag) -specific CD8+ cytotoxic T lymphocytes (CTLs) derived from induced pluripotent stem cells (iPSCs) (i.e., iPSC-CTLs), which have the ability to suppress HBV replication. HBV replication is efficiently induced in mice through hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver. Then, HBV surface Ag-specific mouse iPSC-CTLs are adoptively transferred, which greatly suppresses HBV replication in the liver and blood as well as prevents HBV surface Ag expression in hepatocytes. This method demonstrates HBV replication in mice after hydrodynamic injection and that stem cell-derived viral Ag-specific CTLs can suppress HBV replication. This protocol provides a useful method for HBV immunotherapy.

Human apolipoprotein E promotes hepatitis B virus infection and production.

Hepatitis B virus (HBV) is a common cause of liver diseases, including chronic hepatitis, steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). HBV chronically infects about 240 million people worldwide, posing a major global health problem. The current standard antiviral therapy effectively inhibits HBV replication but does not eliminate the virus unlike direct-acting antivirals (DAA) for curing hepatitis C. Our previous studies have demonstrated that human apolipoprotein E (apoE) plays important roles in hepatitis C virus infection and morphogenesis. In the present study, we have found that apoE is also associated with HBV and is required for efficient HBV infection. An apoE-specific monoclonal antibody was able to capture HBV similar to anti-HBs. More importantly, apoE monoclonal antibody could effectively block HBV infection, resulting in a greater than 90% reduction of HBV infectivity. Likewise, silencing of apoE expression or knockout of apoE gene by CRISPR/Cas9 resulted in a greater than 90% reduction of HBV infection and more than 80% decrease of HBV production, which could be fully restored by ectopic apoE expression. However, apoE silencing or knockout did not significantly affect HBV DNA replication or the production of nonenveloped (naked) nucleocapsids. These findings demonstrate that human apoE promotes HBV infection and production. We speculate that apoE may also play a role in persistent HBV infection by evading host immune response similar to its role in the HCV life cycle and pathogenesis. Inhibitors interfering with apoE biogenesis, secretion, and/or binding to receptors may serve as antivirals for elimination of chronic HBV infection.

Pyrimidotriazine derivatives as selective inhibitors of HBV capsid assembly

Chronic hepatitis B virus (HBV) infection is currently treated with nucleoside/nucleotides analogs. They are potent inhibitors of HBV DNA polymerase, which also functions as reverse transcriptase. Although nucleoside/nucleotide analogs efficiently suppress HBV replication in liver cells, they cannot eradicate HBV DNA from liver cells and cure the disease. Therefore, it is still mandatory to identify and develop effective inhibitors that target a step other than reverse transcription in the viral replication cycle. HBV capsid assembly is a critical step for viral replication and an attractive target for inhibition of HBV replication. We conducted in silico screening of compounds expected to bind to the HBV capsid dimer-dimer interaction site. The selected compounds were further examined for their anti-HBV activity in vitro. Among the test compounds, novel pyrimidotriazine derivatives were found to be selective inhibitors of HBV replication in HepG2.2.15.7 cells. Among the compounds, 2-[(2,3-dichlorophenyl)amino]-4-(4-tert-butylphenyl)-8-methyl-4H,9H-pyrimido[1,2-a][1,3,5]triazin-6-one was the most active against HBV replication. Studies on its mechanism of action revealed that the compound interfered with HBV capsid assembly determined by a cell-free capsid assembly system. Thus, the pyrimidotriazine derivatives are considered to be potential leads for novel HBV capsid assembly inhibitors.

Structural and functional analyses of hepatitis B virus X protein BH3-like domain and Bcl-xL interaction

Hepatitis B virus (HBV) X protein, HBx, interacts with anti-apoptotic Bcl-2 and Bcl-xL proteins through its BH3-like motif to promote HBV replication and cytotoxicity. Here we report the crystal structure of HBx BH3-like motif in complex with Bcl-xL where the BH3-like motif adopts a short α-helix to snuggle into a hydrophobic pocket in Bcl-xL via its noncanonical Trp120 residue and conserved Leu123 residue. This binding pocket is ~2 Å away from the canonical BH3-only binding pocket in structures of Bcl-xL with proapoptotic BH3-only proteins. Mutations altering Trp120 and Leu123 in HBx impair its binding to Bcl-xL in vitro and HBV replication in vivo, confirming the importance of this motif to HBV. A HBx BH3-like peptide, HBx-aa113-135, restores HBV replication from a HBx-null HBV replicon, while a shorter peptide, HBx-aa118-127, inhibits HBV replication. These results provide crucial structural and functional insights into drug designs for inhibiting HBV replication and treating HBV patients.

&amp;lt;italic&amp;gt;In vitro&amp;lt;/italic&amp;gt; experimental models for the study of hepatitis B virus cccDNA

Chronic infection of hepatitis B virus (HBV) remains a global public health problem and affects at least 240 million people worldwide. HBV is a small enveloped DNA virus belonging to the Hepadnaviridae family. The virus has a narrow host range, limited to humans and chimpanzees. In the nucleus of infected hepatocytes, the partially double-stranded DNA (rcDNA) HBV genome is converted to covalent closed circular DNA (cccDNA), which serves as the sole template for HBV transcription. HBV cccDNA exists as an episomal minichromosome in the nucleus, with a turnover rate that correlates with the mitosis or death of virus-infected hepatocytes. It is thus regarded as a primary molecular mechanism for viral persistence. In the past decade, significant progress has been made in HBV research and the related antiviral development. Sodium taurocholate co-transporting polypeptide (NTCP) has been identified as a receptor for HBV entry into hepatocytes. It was recently shown that HBV x (HBx), an enigmatic viral protein, achieves its regulatory function by hijacking the cellular DDB1-containing E3 ubiquitin ligase to target the Smc5/6 complex for degradation. Although still controversial, type I interferon has been implicated to either affect the epigenetic control of cccDNA minichromosome, or induce cytidine deaminases-mediated specific degradation of the episomal cccDNA. Of particular note, core protein allosteric modulator (CpAM) has been most widely investigated. These studies represent a new frontier in the development of antivirals against HBV infection. Despite continuing progress in HBV research, a complete cure of HBV infection is still not achievable. Current antiviral therapies with nucleos(t)ide analogs (NUC) and pegylated IFN-α can efficiently inhibit HBV replication, but the treatments fail to eliminate the preexisting cccDNA pool that is responsible for a viral rebound after therapy cessation. Therefore, it is widely held that cccDNA elimination is a prerequisite for complete cure of chronic HBV infection. To this end, it is essential to understand the mechanisms involved in cccDNA metabolism and transcriptional regulation. Studies of HBV cccDNA have often been hampered by the low copy number of cccDNA per cell and the lack of convenient techniques with high sensitivity and specificity for cccDNA detection. Development of appropriate experimental models is thus fundamental for HBV cccDNA studies. Herein, we reviewed recent advances in cell culture models for the study of HBV cccDNA, including models of HBV de novo infection and cccDNA biogenesis, cccDNA formation (rcDNA repair-related molecular events), HBV replicon-based transfection models to study established cccDNA, and the surrogate model of cccDNA produced from site-specific DNA recombination. Animal models to study HBV cccDNA will be further discussed by other authors in this issue. We hope this review may help clinicians and researchers who are interested in the study of cccDNA and antiviral development against HBV.

An interferon-like small chemical compound CDM-3008 suppresses hepatitis B virus through induction of interferon-stimulated genes.

Oral administration of nucleotide analogues and injection of interferon-α (IFNα) are used to achieve immediate suppression in replication of hepatitis B virus (HBV). Nucleotide analogs and IFNα inhibit viral polymerase activity and cause long-term eradication of the virus at least in part through removing covalently closed circular DNA (cccDNA) via induction of the APOBEC3 deaminases family of molecules, respectively. This study aimed to explore whether the orally administrable low molecular weight agent CDM-3008 (RO8191), which mimics IFNα through the binding to IFNα/β receptor 2 (IFNAR2) and the activation of the JAK/STAT pathway, can suppress HBV replication and reduce cccDNA levels. In primary cultured human hepatocytes, HBV DNA levels were decreased after CDM-3008-treatment in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) value of 0.1 μM, and this was accompanied by significant reductions in cellular cccDNA levels, both HBeAg and HBsAg levels in the cell culture medium. Using a microarray we comprehensively analyzed and compared changes in gene (mRNA) expression in CDM-3008- and IFNα-treated primary cultured human hepatocytes. As reported previously, CDM-3008 mimicked the induction of genes that participate in the interferon signaling pathway. OAS1 and ISG20 mRNA expression was similarly enhanced by both CDM-3008 and IFNα. Thus, CDM-3008 could suppress pgRNA expression to show anti-HBV activity. APOBEC3F and 3G mRNA expression was also induced by CDM-3008 and IFNα treatments, suggesting that cccDNA could be degraded through induced APOBEC3 family proteins. We identified the genes whose expression was specifically enhanced in CDM-3008-treated cells compared to IFNα-treated cells. The expression of SOCS1, SOCS2, SOCS3, and CISH, which inhibit STAT activation, was enhanced in CDM-3008-treated cells suggesting that a feedback inhibition of the JAK/STAT pathway was enhanced in CDM-3008-treated cells compared to IFNα-treated cells. In addition, CDM-3008 showed an additive effect with a clinically-used nucleoside entecavir on inhibition of HBV replication. In summary, CDM-3008 showed anti-HBV activity through activation of the JAK/STAT pathway, inducing the expression of interferon-stimulated genes (ISGs), with greater feedback inhibition than IFNα.

The cyclophilin inhibitor CRV431 inhibits liver HBV DNA and HBsAg in transgenic mice.

Hepatitis B virus (HBV) infection is a major health burden worldwide with 240 million chronically infected individuals. Nucleos(t)ide analogs and interferons are the current standards of care due to their suppression of HBV replication, but the treatments rarely eradicate HBV from individuals. Similar to current treatments for human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV) patients, improved HBV therapies will require the combination of multiple drugs which target distinct steps of the HBV life cycle. In this study, we tested the potential of a cyclophilin inhibitor, CRV431, to affect HBV replication in transgenic mice. We found that oral treatment with CRV431 (50 mg/kg/day) for a period of 16 days significantly reduced liver HBV DNA levels and moderately decreased serum HBsAg levels. We observed an additive inhibitory effect on liver HBV DNA levels in mice treated with a combination of low doses of CRV431 (10 mg/kg/day) and the nucleotide prodrug, tenofovir exalidex (TXL), (5 mg/kg/day). No toxicity was observed in CRV431-treated mice. Although it is well known that CRV431 neutralizes the peptidyl-prolyl isomerase activity of cyclophilins, its anti-HBV mechanism(s) of action remains unknown. Nevertheless, this study provides the first demonstration of a beneficial effect of a cyclophilin inhibitor in vivo in an HBV transgenic mouse model. Altogether our data reveal the potential of CRV431 to be part of improved new therapies for HBV patients.

The Stem Cell-Derived Viral Antigen-Specific T Cells Suppress HBV Replication Through Production of IFN-γ and TNF-⍺

The viral antigen (Ag)-specific CD8+ cytotoxic T lymphocytes (CTLs) derived from pluripotent stem cells (PSCs), i.e., PSC-CTLs, have the ability to suppress the hepatitis B virus (HBV) infection. After adoptive transfer, PSC-CTLs can be programmed to be HBV-specific and to infiltrate into the liver to suppress HBV replication. Nevertheless, the mechanisms by which the viral Ag-specific PSC-CTLs provoke the antiviral response remain to be fully elucidated. In this study, we generated the functional HBV Ag-specific CTLs from the induced PSC (iPSCs), i.e., iPSC-CTLs, and investigated the underlying mechanisms of the CTL-mediated antiviral replication in a murine model of HBV infection. A hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver induced HBV replication efficiently in mice. We show that adoptive transfer of HBV Ag-specific iPSC-CTLs greatly suppressed the HBV replication in the liver and blood in the animal model and prevented the HBV surface Ag expression on hepatocytes. We further demonstrate that the adoptive transfer significantly increased the migration of CD8+ T cells and the production of the antiviral cytokines (IFN-γ, TNF-α) in the liver. Moreover, using neutralizing antibody to IFN-r or/and TNF-a dramatically dismissed the suppression of the HBV replication mediated by the HBV Ag-specific iPSC-CTLs. These results indicate that the stem cell-derived tissue-associated CTLs can robustly accumulate in the liver and suppress the HBV replication through producing the antiviral cytokines IFN-r and TNF-a.