Regulation Of Hepatitis B Virus Replication Research Articles

Perilipin2 inhibits the replication of hepatitis B virus deoxyribonucleic acid by regulating autophagy under high-fat conditions.

Chronic hepatitis B virus (HBV) infection is often associated with increased lipid deposition in hepatocytes. However, when combined with non-alcoholic fatty liver disease or hyperlipidemia, it tends to have a lower HBV deoxyribonucleic acid (DNA) load. The relationship between lipid metabolism and HBV DNA replication and its underlying mechanisms are not well understood. To investigate the relationship between lipid metabolism and HBV DNA replication and its underlying mechanisms. 1603 HBsAg-seropositive patients were included in the study. We first explored the relationship between patients' lipid levels, hepatic steatosis, and HBV DNA load. Also, we constructed an HBV infection combined with a hepatic steatosis cell model in vitro by fatty acid stimulation of HepG2.2.15 cells to validate the effect of lipid metabolism on HBV DNA replication in vitro. By knocking down and overexpressing Plin2, we observed whether Plin2 regulates autophagy and HBV replication. By inhibiting both Plin2 and cellular autophagy under high lipid stimulation, we examined whether the Plin2-autophagy pathway regulates HBV replication. The results revealed that serum triglyceride levels, high-density lipoprotein levels, and hepatic steatosis ratio were significantly lower in the HBV-DNA high load group. Logistic regression analysis indicated that hepatic steatosis and serum triglyceride levels were negatively correlated with HBV-DNA load. Stratified analysis by HBeAg showed significant negative correlations between HBV-DNA load and hepatic steatosis ratio in both HBeAg-positive and HBeAg-negative groups. An in vitro cell model was developed by stimulating HepG2.2.15 cells with palmitic acid and oleic acid to study the relationship between HBV-DNA load and lipid metabolism. The results of the in vitro experiments suggested that fatty acid treatment increased lipid droplet deposition and decreased the expression of cell supernatant HBsAg, HBeAg, and HBV DNA load. Western blot and polymerase chain reaction analysis showed that fatty acid stimulation significantly induced Plin2 protein expression and inhibited the expression of hepatocyte autophagy proteins. Inhibition of Plin2 protein expression under fatty acid stimulation reversed the reduction in HBsAg and HBeAg expression and HBV DNA load induced by fatty acid stimulation and the inhibition of cellular autophagy. Knocking down Plin2 and blocking autophagy with 3-methyladenine (3-MA) inhibited HBV DNA replication. In conclusion, lipid metabolism is a significant factor affecting HBV load in patients with HBV infection. The in vitro experiments established that fatty acid stimulation inhibits HBV replication via the Plin2-autophagy pathway.

NSUN2-mediated m5C modification of HBV RNA positively regulates HBV replication.

Chronic hepatitis B virus (HBV) infection is a major cause of liver cirrhosis and liver cancer, despite strong prevention and treatment efforts. The study of the epigenetic modification of HBV has become a research hotspot, including the N6-methyladenosine (m6A) modification of HBV RNA, which plays complex roles in the HBV life cycle. In addition to m6A modification, 5-methylcytosine (m5C) is another major modification of eukaryotic mRNA. In this study, we explored the roles of m5C methyltransferase and demethyltransferase in the HBV life cycle. The results showed that m5C methyltransferase NSUN2 deficiency could negatively regulate the expression of HBV while m5C demethyltransferase TET2 deficiency positively regulates the expression of HBV. Subsequently, we combined both in vitro bisulfite sequencing and high-throughput bisulfite sequencing methods to determine the distribution and stoichiometry of m5C modification in HBV RNA. Two sites: C2017 and C131 with the highest-ranking methylation rates were identified, and mutations at these two sites could lead to the decreased expression and replication of HBV, while the mutation of the "fake" m5C site had no effect. Mechanistically, NSUN2-mediated m5C modification promotes the stability of HBV RNA. In addition, compared with wild-type HepG2-NTCP cells and primary human hepatocytes, the replication level of HBV after NSUN2 knockdown decreased, and the ability of the mutant virus to infect and replicate in wild-type HepG2-NTCP cells and PHHs was substantially impaired. Similar results were found in the experiments using C57BL/6JGpt-Nsun2+/- mice. Interestingly, we also found that HBV expression and core protein promoted the endogenous expression of NSUN2, which implied a positive feedback loop. In summary, our study provides an accurate and high-resolution m5C profile of HBV RNA and reveals that NSUN2-mediated m5C modification of HBV RNA positively regulates HBV replication by maintaining RNA stability.

SPOP inhibits HBV transcription and replication by ubiquitination and degradation of HNF1α.

Hepatitis B virus (HBV) infection remains a significant public health burden worldwide. The persistence of covalently closed circular DNA (cccDNA) within the nucleus of infected hepatocytes is responsible for the failure of antiviral treatments. The ubiquitin proteasome system (UPS) has emerged as a promising antiviral target, as it can regulate HBV replication by promoting critical protein degradation in steps of viral life cycle. Speckle-type POZ protein (SPOP) is a critical adaptor for Cul3-RBX1 E3 ubiquitin ligase complex, but the effect of SPOP on HBV replication is less known. Here, we identified SPOP as a novel host antiviral factor against HBV infection. SPOP overexpression significantly inhibited the transcriptional activity of HBV cccDNA without affecting cccDNA level in HBV-infected HepG2-NTCP and primary human hepatocyte cells. Mechanism studies showed that SPOP interacted with hepatocyte nuclear factor 1α (HNF1α), and induced HNF1α degradation through host UPS pathway. Moreover, the antiviral role of SPOP was also confirmed in vivo. Together, our findings reveal that SPOP is a novel host factor which inhibits HBV transcription and replication by ubiquitination and degradation of HNF1α, providing a potential therapeutic strategy for the treatment of HBV infection.

Baicalin inhibits the replication of the hepatitis B virus by targeting TRIM25.

Baicalin, which is a key bioactive constituent obtained from Scutellaria baicalensis, has been utilized in traditional Chinese medicine for many centuries. Although it has been reported that Baicalin (BA) can inhibit the replication of the Hepatitis B virus (HBV), the exact mechanism behind this process remains unclear. Interferon-stimulated genes (ISGs) are crucial in the process of antiviral defense. We aim to investigate whether BA can regulate the expression of ISGs, and thereby potentially modulate the replication of HBV. The study involved the use of CRISPR/Cas9 technology to perform knockout experiments on TRIM25 and IFIT3 genes. The expression of these genes was confirmed through techniques such as immunoblotting or Q-PCR. The levels of HBsAg and HBeAg were measured using ELISA, and the expression of interferon-stimulated genes was detected using a luciferase assay. It is interesting to note that several ISGs belonging to the TRIM family, including TRIM5, TRIM25, and TRIM14, were induced after BA treatment. On the other hand, members of the IFIT family were reduced by BA stimulation. Additionally, BA-mediated HBV inhibition was found to be significantly restored in HepG2 cells where TRIM25 was knocked out. Additional research into the mechanism of action of BA found that prolonged treatment with BA activated the JAK/STAT signaling pathway while simultaneously inhibiting the NF-kB pathway. The findings of our study indicate that TRIM25 has a significant impact on the regulation of HBV replication following BA treatment, providing additional insight into the mechanisms by which BA exerts its antiviral effects.

LncRNA NKILA inhibits HBV replication by repressing NF-κB signalling activation

Hepatitis B virus (HBV) infection results in liver cirrhosis and hepatocellular carcinoma (HCC). HBx/nuclear factor (NF)-κB pathway plays a role in HBV replication. However, whether NF-κB-interacting long noncoding RNA (NKILA), a suppressor of NF-κB activation, regulates HBV replication remains largely unknown. In this study, gain-and-loss experiments showed that NKILA inhibited HBV replication by inhibiting NF-κB activity. In turn, HBV infection down-regulated NKILA expression. In addition, expression levels of NKILA were lower in the peripheral blood-derived monocytes (PBMCs) of HBV-positive patients than in healthy individuals, which were correlated with HBV viral loads. And a negative correlation between NKILA expression level and HBV viral loads was observed in blood serum from HBV-positive patients. Lower levels of endogenous NKILA were also observed in HepG2 cells expressing a 1.3-fold HBV genome, HBV-infected HepG2-NTCP cells, stable HBV-producing HepG2.2.15 and HepAD38 ​cells, compared to those HBV-negative cells. Furthermore, HBx was required for NKILA-mediated inhibition on HBV replication. NKILA decreased HBx-induced NF-κB activation by interrupting the interaction between HBx and p65, whereas NKILA mutants lack of essential domains for NF-ĸB inhibition, lost the ability to inhibit HBV replication. Together, our data demonstrate that NKILA may serve as a suppressor of HBV replication via NF-ĸB signalling.

RNA binding protein TIAR modulates HBV replication by tipping the balance of pgRNA translation

The pregenomic RNA (pgRNA) of hepatitis B virus (HBV) serves not only as a bicistronic message RNA to translate core protein (Cp) and DNA polymerase (Pol), but also as the template for reverse transcriptional replication of viral DNA upon packaging into nucleocapsid. Although it is well known that pgRNA translates much more Cp than Pol, the molecular mechanism underlying the regulation of Cp and Pol translation efficiency from pgRNA remains elusive. In this study, we systematically profiled HBV nucleocapsid- and pgRNA-associated cellular proteins by proteomic analysis and identified TIA-1-related protein (TIAR) as a novel cellular protein that binds pgRNA and promotes HBV DNA replication. Interestingly, loss- and gain-of-function genetic analyses showed that manipulation of TIAR expression did not alter the levels of HBV transcripts nor the secretion of HBsAg and HBeAg in human hepatoma cells supporting HBV replication. However, Ribo-seq and PRM-based mass spectrometry analyses demonstrated that TIAR increased the translation of Pol but decreased the translation of Cp from pgRNA. RNA immunoprecipitation (RIP) and pulldown assays further revealed that TIAR directly binds pgRNA at the 5’ stem-loop (ε). Moreover, HBV replication or Cp expression induced the increased expression and redistribution of TIAR from the nucleus to the cytoplasm of hepatocytes. Our results thus imply that TIAR is a novel cellular factor that regulates HBV replication by binding to the 5’ ε structure of pgRNA to tip the balance of Cp and Pol translation. Through induction of TIAR translocation from the nucleus to the cytoplasm, Cp indirectly regulates the Pol translation and balances Cp and Pol expression levels in infected hepatocytes to ensure efficient viral replication.

Hydrogen Peroxide Inhibits Hepatitis B Virus Replication by Downregulating HBx Levels via Siah-1-Mediated Proteasomal Degradation in Human Hepatoma Cells.

The hepatitis B virus (HBV) is constantly exposed to significant oxidative stress characterized by elevated levels of reactive oxygen species (ROS), such as H2O2, during infection in hepatocytes of patients. In this study, we demonstrated that H2O2 inhibits HBV replication in a p53-dependent fashion in human hepatoma cell lines expressing sodium taurocholate cotransporting polypeptide. Interestingly, H2O2 failed to inhibit the replication of an HBV X protein (HBx)-null HBV mutant, but this defect was successfully complemented by ectopic expression of HBx. Additionally, H2O2 upregulated p53 levels, leading to increased expression of seven in absentia homolog 1 (Siah-1) levels. Siah-1, an E3 ligase, induced the ubiquitination-dependent proteasomal degradation of HBx. The inhibitory effect of H2O2 was nearly abolished not only by treatment with a representative antioxidant, N-acetyl-L-cysteine but also by knockdown of either p53 or Siah-1 using specific short hairpin RNA, confirming the role of p53 and Siah-1 in the inhibition of HBV replication by H2O2. The present study provides insights into the mechanism that regulates HBV replication under conditions of oxidative stress in patients.

Inhibition of HBV replication by EVA1A via enhancing cellular degradation of HBV components and its potential therapeutic application

Hepatitis B virus (HBV) DNA is much higher during HBeAg-positive chronic HBV infection (EP-CBI) than during HBeAg-negative chronic HBV infection (EN-CBI), although the necroinflammation in liver is minimal and the adaptive immune response is similar in both phases. We previously reported that mRNA levels of EVA1A were higher in EN-CBI patients. In this study, we aimed to investigate whether EVA1A inhibits HBV gene expression and examine the underlying mechanisms. The available cell models for HBV replication and model HBV mice were used to investigate how EVA1A regulates HBV replication and the antiviral activity based on gene therapy. The signaling pathway was determined through RNA sequencing analysis. The results demonstrated that EVA1A can inhibit HBV gene expression in vitro and in vivo. In particular, EVA1A overexpression resulted in accelerated HBV RNA degradation and activation of the PI3K-Akt-mTOR pathway, two processes that directly and indirectly inhibiting HBV gene expression. EVA1A is a promising candidate for treating chronic hepatitis B (CHB). In conclusion, EVA1A is a new host restriction factor that regulates the HBV life cycle via a nonimmune process.

Long non-coding RNA WAC antisense RNA 1 mediates hepatitis B virus replication in vitro by reinforcing miR-192-5p/ATG7-induced autophagy.

Long non-coding RNA WAC antisense RNA 1 (lncRNA WAC-AS1) is involved in the replication of the hepatitis B virus (HBV). The purpose of this study was to determine its functions and specific mechanism. The levels of lncRNA WAC-AS1, RNA (miR)-192-5p and were examined in serum of HBV-infected patients and in HepG2.2.15 cells using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blotting. Using the database starBase, the target binding sites of lncRNA WAC-AS1 and miR-192-5p were predicted and confirmed by dual-luciferase reporter assay and RNA pull-down assay. The expression of pgRNA and HBV DNA was determined by qRT-PCR, while the levels of HBeAg and HBsAg were measured by enzymelinked immunosorbent assay (ELISA). Using laser scanning confocal microscopy, the light chain 3 (LC3) expression was analyzed. qRT-PCR and Western blotting were used to assess the expression of beclin-1, p62, and LC3I/II. Overexpression of lncRNA WAC-AS1, upregulation of ATG7. and downregulation of miR-192-5p were observed in the serum of HBV-infected patients and the in vitro model. miR-192-5p directly targets lncRNA WAC-AS1. LncRNA WAC-AS1 was downregulated in lncRNA WAC-AS1-shRNA‒transfected cells. miR-192-5p was upregulated in lncRNA WAC-AS1-shRNA-transfected cells and downregulated in cells transfected with a miR-192-5p inhibitor. In HepG2 2.15 cells, the downregulation of lncRNA WAC-AS1 inhibited HBV replication and autophagy. In contrast, the miR-192-5p inhibitor-transfected group exhibited the opposite results, and ATG7 overexpression reversed the effects of miR-192-5p mimic or lncRNA WAC-AS1-shRNA on HBV replication and cell autophagy. Our findings indicate that lncRNA WAC-AS1 regulates HBV replication by reinforcing the autophagy induced by miR-192-5p/ATG7. Consequently, lncRNA WAC-AS1 may serve as a therapeutically- promising target in HBV patients.

Sac1 phosphatidylinositol 4-phosphate phosphatase is a novel host cell factor regulating hepatitis B virus particles assembly and release.

The life-cycle of the Hepatitis B Virus (HBV), an enveloped DNA virus affecting the lives of more than 296 million chronicallyinfected people, is tightly dependent on the lipid metabolism of the host cell. Fatty acids and cholesterol are among the lipid factors with documented roles in regulating HBV replication and infection, respectively, but little is known about the phosphoinositide metabolism in these processes. In this study, we investigated the role of Sac1, a highly conserved phosphatidylinositol-4-phosphate (PI4P) phosphatase, with essential functions in phospholipid metabolism, in HBV assembly, and release. PI4P is one of the most abundant cellular phosphoinositide with complex functions at the level of the secretory pathway. Owing to the highly specific phosphatase activity toward PI4P, Sac1 controls the levels and restricts the localization of this lipid particularly at the trans-Golgi network, where it regulates sphingolipid synthesis, proteins sorting, and vesicles budding, by recruiting specific adaptor proteins. As a complete loss of Sac1 function compromises cell viability, in this work, we first developed and characterized several HBV replication-permissive cellular models with a moderate, transient, or stable downregulation of Sac1 expression. Our results show that Sac1 depletion in hepatic cells results in increased levels and redistribution of intracellular PI4P pools and impaired trafficking of the HBV envelope proteins to the endosomal vesicular network. Importantly, virus envelopment and release from these cells are significantly inhibited, revealing novel roles for Sac1, as a key host cell factor regulating morphogenesis of a DNA virus.

Nicotinamide N-Methyltransferase inhibits HBV replication by suppressing NR5A1 expression invitro

Chronic hepatitis B virus (HBV) infection can lead to fibrosis, liver cirrhosis, and primary hepatocellular carcinoma. Investigating host factors that regulate HBV replication helps to identify antiviral targets. In the current study, we identified Nicotinamide N-Methyltransferase gene (NNMT) as a novel factor that regulates HBV transcription. NNMT is up-regulated at both the mRNA and protein levels in HepG2.2.15 cells compared to HepG2 cells. Overexpression of NNMT reduces HBV replication in several cell models, while knockdown of NNMT enhances HBV DNA levels. Mechanistically, NNMT suppresses HBV DNA replication by inhibiting HBV RNA transcription. The region required for the inhibitory effect of NNMT was narrowed to nt 1672–1708 in enhancer II by luciferase assays. On the other hand, ChIP assays and EMSA results showed that NNMT does not bind to this region substantially, either directly or indirectly. Next, a collection of hepatic nuclear receptor transcription factors was screened to determine whether they were affected by NNMT overexpression. NR5A1, a positive regulator of HBV replication, decreased significantly after NNMT overexpression. Collectively, the findings of this study shed light on the regulation of HBV transcription.

Interaction between the Hepatitis B Virus and Cellular FLIP Variants in Viral Replication and the Innate Immune System.

During viral evolution and adaptation, many viruses have utilized host cellular factors and machinery as their partners. HBx, as a multifunctional viral protein encoded by the hepatitis B virus (HBV), promotes HBV replication and greatly contributes to the development of HBV-associated hepatocellular carcinoma (HCC). HBx interacts with several host factors in order to regulate HBV replication and evolve carcinogenesis. The cellular FADD-like IL-1β-converting enzyme (FLICE)-like inhibitory protein (c-FLIP) is a major factor that functions in a variety of cellular pathways and specifically in apoptosis. It has been shown that the interaction between HBx and c-FLIP determines HBV fate. In this review, we provide a comprehensive and detailed overview of the interplay between c-FLIP and HBV in various environmental circumstances. We describe strategies adapted by HBV to establish its chronic infection. We also summarize the conventional roles of c-FLIP and highlight the functional outcome of the interaction between c-FLIP and HBV or other viruses in viral replication and the innate immune system.

Sirtuins as Potential Therapeutic Targets for Hepatitis B Virus Infection.

Sirtuins (SIRTs) are well-known histone deacetylases that are capable of modulating various cellular processes in numerous diseases, including the infection of hepatitis B virus (HBV), which is one of the primary pathogenic drivers of liver cirrhosis and hepatocellular carcinoma. Mounting evidence reveals that HBV can alter the expression levels of all SIRT proteins. In turn, all SIRTs regulate HBV replication via a cascade of molecular mechanisms. Furthermore, several studies suggest that targeting SIRTs using suitable drugs is a potential treatment strategy for HBV infection. Here, we discuss the molecular mechanisms associated with SIRT-mediated upregulation of viral propagation and the recent advances in SIRT-targeted therapy as potential therapeutic modalities against HBV infection.

Hypoxia inducible factors regulate hepatitis B virus replication by activating the basal core promoter

Background & AimsHypoxia inducible factors (HIFs) are a hallmark of inflammation and are key regulators of hepatic immunity and metabolism, yet their role in HBV replication is poorly defined. HBV replicates in hepatocytes within the liver, a naturally hypoxic organ, however most studies of viral replication are performed under conditions of atmospheric oxygen, where HIFs are inactive. We therefore investigated the role of HIFs in regulating HBV replication.MethodsUsing cell culture, animal models, human tissue and pharmacological agents inhibiting the HIF-prolyl hydroxylases, we investigated the impact of hypoxia on the HBV life cycle.ResultsCulturing liver cell-based model systems under low oxygen uncovered a new role for HIFs in binding HBV DNA and activating the basal core promoter, leading to increased pre-genomic RNA and de novo HBV particle secretion. The presence of hypoxia responsive elements among all primate members of the hepadnaviridae highlights an evolutionary conserved role for HIFs in regulating this virus family.ConclusionsIdentifying a role for this conserved oxygen sensor in regulating HBV transcription suggests that this virus has evolved to exploit the HIF signaling pathway to persist in the low oxygen environment of the liver. Our studies show the importance of considering oxygen availability when studying HBV-host interactions and provide innovative routes to better understand and target chronic HBV infection.Lay summaryViral replication in host cells is defined by the cellular microenvironment and one key factor is local oxygen tension. Hepatitis B virus (HBV) replicates in the liver, a naturally hypoxic organ. Hypoxia inducible factors (HIFs) are the major sensors of low oxygen; herein, we identify a new role for these factors in regulating HBV replication, revealing new therapeutic targets.

Research advances in hepatitis B virus and ubiquitination of related proteins

Protein ubiquitination is widely observed in cells and is a modification after protein translation. Hepatitis B virus (HBV) and ubiquitination of related proteins have attracted more and more attention. This article reviews HBV and the ubiquitination of related proteins, so as to provide a reference for further research on the regulation of HBV replication and the ubiquitination of related proteins, as well as new ideas and methods for curing chronic HBV infection.

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.

AMPK and Akt/mTOR signalling pathways participate in glucose-mediated regulation of hepatitis B virus replication and cellular autophagy.

A growing consensus indicates that host metabolism plays a vital role in viral infections. Hepatitis B virus (HBV) infection occurs in hepatocytes with active glucose metabolism and may be regulated by cellular metabolism. We addressed the question whether and how glucose regulates HBV replication in hepatocytes. The low glucose concentration at 5 mM significantly promoted HBV replication via enhanced transcription and autophagy when compared with higher glucose concentrations (10 and 25 mM). At low glucose concentration, AMPK activity was increased and led to ULK1 phosphorylation at Ser 555 and LC3-II accumulation. By contrast, the mTOR pathway was activated by high glucose concentrations, resulting in reduced HBV replication. mTOR inhibition by rapamycin reversed negative effects of high glucose concentrations on HBV replication, suggesting that low glucose concentration promotes HBV replication by stimulating the AMPK/mTOR-ULK1-autophagy axis. Consistently, we found that glucose transporters inhibition using phloretin also enhanced HBV replication via increased AMPK/mTOR-ULK1-induced autophagy. Surprisingly, the glucose analogue 2-deoxy-D-glucose reduced HBV replication through activating the Akt/mTOR signalling pathway also at the low glucose concentrations. Our study reveals that glucose is an important factor for the HBV life cycle by regulating HBV transcription and posttranscriptional steps of HBV replication via cellular autophagy.

Long noncoding RNA GAS5 does not regulate HBV replication.

Hepatitis B virus (HBV) infection remains a severe health burden worldwide. Emerging long noncoding RNAs (lncRNAs) are hijacked to enhance virus replication or employed by the host to stimulate immune responses to clear the virus. LncRNA growth arrest-specific transcript 5 (GAS5) can regulate RNA virus by suppressing the replication of both hepatitis C virus and human immunodeficiency virus. In this study, we explored the changes of HBV replication by overexpressing or knocking down GAS5 in HepAD38 cell and HepG2 cell transfected with pHBV1.2. We found HBV can induce the expression of GAS5. However, GAS5 had no effect on extracellular HBsAg and HBeAg, nor intracellular HBV RNA and HBV DNA. In addition, GAS5 possessed similar expression levels between stable HBV-producing cell lines and hepatoma cell lines. Furthermore, GAS5 showed no difference between healthy subjects and patients with chronic HBV in multiple GEO microarray data sets by GEO2R analysis. Taken together these results, GAS5 does not modulate the replication of HBV but it inhibits cell proliferation in HepAD38. This provides insights into the possible roles of GAS5 in HBV infection.

MicroRNA‑20 induces methylation of hepatitis B virus covalently closed circular DNA in human hepatoma cells.

Methylation was suggested to suppress the transcriptional activity of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in hepatocytes. This may be associated with its low replicative activity during the inactive stage of chronic HBV infection; however, the exact mechanisms of methylation in HBV infection remain unknown. We have previously shown that short hairpin RNAs induced the methylation of the HBV genome in hepatoma cell lines. We also reported that the microRNA (miR) 17–92 cluster negatively regulates HBV replication in human hepatoma cells. In addition, miR-20a, a member of the miR 17–92 cluster, has sequence homology with the short hairpin RNA that induces HBV methylation. In the present study, we investigated whether miR-20a can function as an endogenous effector of HBV DNA methylation. The results indicated that overexpression of miR-20a could suppress the replicative activity of HBV and increased the degree of methylation of HBV cccDNA in the HepAD38 hepatoma cell line. Argonaute (AGO)1 and AGO2, effectors of the RNA-induced silencing complex, were detected in the nucleus of HepAD38 cells; however, only AGO2 was bound to HBV cccDNA. In addition, intranuclear AGO2 was determined to be bound with miR-20a. In conclusion, miR-20a may be loaded onto AGO2, prior to its translocation into the nucleus, inducing the methylation of HBV DNA in human hepatoma cells, leading to the suppression of HBV replication.

RNA-Binding Motif Protein 24 (RBM24) Is Involved in Pregenomic RNA Packaging by Mediating Interaction between Hepatitis B Virus Polymerase and the Epsilon Element.

Encapsidation of pregenomic RNA (pgRNA) is a crucial step in hepatitis B virus (HBV) replication. Binding by viral polymerase (Pol) to the epsilon stem-loop (ε) on the 5'-terminal region (TR) of pgRNA is required for pgRNA packaging. However, the detailed mechanism is not well understood. RNA-binding motif protein 24 (RBM24) inhibits core translation by binding to the 5'-TR of pgRNA. Here, we demonstrate that RBM24 is also involved in pgRNA packaging. RBM24 directly binds to the lower bulge of ε via RNA recognition submotifs (RNPs). RBM24 also interacts with Pol in an RNA-independent manner. The alanine-rich domain (ARD) of RBM24 and the reverse transcriptase (RT) domain of Pol are essential for binding between RBM24 and Pol. In addition, overexpression of RBM24 increases Pol-ε interaction, whereas RBM24 knockdown decreases the interaction. RBM24 was able to rescue binding between ε and mutant Pol lacking ε-binding activity, further showing that RBM24 mediates the interaction between Pol and ε by forming a Pol-RBM24-ε complex. Finally, RBM24 significantly promotes the packaging efficiency of pgRNA. In conclusion, RBM24 mediates Pol-ε interaction and formation of a Pol-RBM24-ε complex, which inhibits translation of pgRNA and results in pgRNA packing into capsids/virions for reverse transcription and DNA synthesis.IMPORTANCE Hepatitis B virus (HBV) is a ubiquitous human pathogen, and HBV infection is a major global health burden. Chronic HBV infection is associated with the development of liver diseases, including fulminant hepatitis, hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. A currently approved vaccine can prevent HBV infection, and medications are able to reduce viral loads and prevent liver disease progression. However, current treatments rarely achieve a cure for chronic infection. Thus, it is important to gain insight into the mechanisms of HBV replication. In this study, we found that the host factor RBM24 is involved in pregenomic RNA (pgRNA) packaging and regulates HBV replication. These findings highlight a potential target for antiviral therapeutics of HBV infection.