Study on the role of histone epigenetic modification in replication of hepatitis B virus.

Hepatitis B virus (HBV) infection is a global health problem and lacks effective therapies in clinic. This study attempted to investigate the role of histone deacetylase 3 (HDAC3) in HBV replication. Cells were treated with 1.3 folds of HBV genome. The expression patterns of HDAC3, miR-29a-3p, and nuclear factor of activated T-cells 5 (NFAT5) in cells were determined by real-time quantitative polymerase chain reaction and Western blot analysis. HBV replication was assessed by measurements of HBV DNA, HBV RNA, hepatitis B surface antigen, and hepatitis B E antigen. After chromatin immunoprecipitation and RNA pull-down assays to testify gene interactions, rescue experiments and animal experiments were performed to assess the role of miR-29a-3p/NFAT5 in HBV replication and the role of HDAC3 in vivo. HDAC3 level was decreased by pHBV1.3 plasmid in a concentration-dependent manner. HDAC3 overexpression can inhibit HBV replication, which was neutralized by miR-29a-3p overexpression or NFAT5 downregulation. Mechanically, HDAC3 overexpression reduced the enrichment of histone 3 lysine 9 acetylation on the miR-29a-3p promoter to inhibit miR-29a-3p expression and then promote NFAT5 transcription. In vivo, HDAC3 restrained HBV replication through the miR-29a-3p/NFAT5 axis. Overall, HDAC3 downregulation was associated with HBV replication and HDAC3 overexpression inhibited HBV replication through H3K9ac/miR-29a-3p/NFAT5.

Hepatocystin/80K-H inhibits replication of hepatitis B virus through interaction with HBx protein in hepatoma cell.

Hepatitis B virus (HBV) X protein (HBx) is a key player in HBV replication as well as HBV-induced hepatocellular carcinoma (HCC). However, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. In this study, a combination of affinity purification and mass spectrometry was applied to identify the host factors interacting with HBx in hepatoma cells. Thirteen proteins were identified as HBx binding partners. Among them, we first focused on determining the functional significance of the interaction between HBx and hepatocystin. A physical interaction between HBx and hepatocystin was confirmed by co-immunoprecipitation and Western blotting. Immunocytochemistry demonstrated that HBx and hepatocystin colocalized in the hepatoma cells. Domain mapping of both proteins revealed that the HBx C-terminus (amino acids 110-154) was responsible for binding to the mannose 6-phosphate receptor homology domain (amino acids, 419-525) of hepatocystin. Using translation and proteasome inhibitors, we found that hepatocystin overexpression accelerated HBx degradation via a ubiquitin-independent proteasome pathway. We demonstrated that this effect was mediated by an interaction between both proteins using a HBx deletion mutant. Hepatocystin overexpression significantly inhibited HBV DNA replication and expression of HBs antigen concomitant with HBx degradation. Using the hepatocystin mutant constructs that bind HBx, we also confirmed that hepatocystin inhibited HBx-dependent HBV replication. In conclusion, we demonstrated for the first time that hepatocystin functions as a chaperon-like molecule by accelerating HBx degradation, and thereby inhibits HBV replication. Our results suggest that inducing hepatocystin may provide a novel therapeutic approach to control HBV infection.

The novel HBx mutation F30V correlates with hepatocellular carcinoma in vivo, reduces hepatitis B virus replicative efficiency and enhances anti-apoptotic activity of HBx N terminus in vitro.

OBJECTIVE: We aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and their impact on (a) HBV replicative efficiency, (b) HBx binding to circular covalently closed DNA (cccDNA), (c) apoptosis and cell-cycle progression, and (d) HBx structural stability. METHODS: This study included 123 individuals chronically infected with HBV: 27 with HCC (77.9% (21/27) genotype D; 22.1% (6/27) genotype A) and 96 without HCC (75% (72/96) genotype D; 25.0% (24/96) genotype A). HepG2 cells were transfected by wild-type or mutated linear HBV genome to assess pre-genomic RNA (pgRNA) and core-associated HBV-DNA levels, HBx-binding onto cccDNA by chromatin immunoprecipitation-based quantitative assay, and rate of apoptosis and cell-cycle progression by cytofluorimetry. RESULTS: F30V was the only HBx mutation correlated with HCC (18.5% (5/27) in HCC patients versus 1.0% (1/96) in non-HCC patients, p 0.002); a result confirmed by multivariate analysis. In vitro, F30V determined a 40% and 60% reduction in pgRNA and core-associated HBV-DNA compared with wild-type (p <0.05), in parallel with a significant decrease of HBx binding to cccDNA and decreased HBx stability. F30V also decreased the percentage of apoptotic cells compared with wild-type (14.8 ± 6.8% versus 19.1 ± 10.1%, p <0.01, without affecting cell-cycle progression) and increased the probability of HBx-Ser-31 being phosphorylated by PI3K-Akt kinase (known to promote anti-apoptotic activity). CONCLUSIONS: F30V was closely correlated with HBV-induced HCC in vivo, reduced HBV replicative efficiency by affecting HBx-binding to cccDNA and increased anti-apoptotic HBx activity in vitro. This suggests that F30V (although hampering HBV's replicative capacity) may promote hepatocyte survival, so potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Investigation of viral genetic markers associated with HCC is crucial to identify those patients at higher risk of HCC, who hence deserve intensive liver monitoring and/or early anti-HBV therapy.

Cisplatin induces autophagy to enhance hepatitis B virus replication via activation of ROS/JNK and inhibition of the Akt/mTOR pathway.

Chronic hepatitis B virus (HBV) infection remains a serious global health concern. Cisplatin is a chemotherapeutic agent commonly used to treat various cancers. However, HBV-infected patients receiving chemotherapy are at risk of HBV reactivation via unknown mechanisms, which we aimed to elucidate in this study. We found that autophagy plays a central role in cisplatin-induced HBV replication. Cisplatin treatment induced autophagy in both HBV-replicating cells and an HBV-transgenic mouse model as evident from marked upregulation of microtubule-associated protein 1 light chain 3 (LC3)-II and the accumulation of red fluorescent protein (RFP)-LC3 puncta. Cisplatin induced complete autophagic flux, which was detected via monitoring of p62 degradation and RFP-GFP-LC3 expression. Inhibition of autophagy by chloroquine, 3-methyladenine, or Atg5 knockdown significantly attenuated cisplatin-induced HBV replication. Additionally, cisplatin-induced autophagy could be significantly attenuated by using the ROS scavenger N-acetyl-l-cysteine. Mechanically, cisplatin promoted HBV replication and autophagy through ROS/JNK and AKT/mTOR signaling. Inhibition of JNK or activation of Akt/mTOR signaling reversed cisplatin-mediated autophagy and HBV replication promotion. In contrast, suppression of Akt/mTOR signaling further promoted cisplatin-induced HBV replication. Finally, pharmacotherapeutic inhibition of autophagy or ROS production impaired HBV production induced by cisplatin in vivo. Together, our results indicate that ROS/JNK and mTOR/AKT-mediated autophagy plays an important role in cisplatin-induced HBV reactivation.

Identification of TRIM14 as a Type I IFN-Stimulated Gene Controlling Hepatitis B Virus Replication by Targeting HBx.

Hepatitis B virus (HBV) remains a major cause of hepatic disease that threatens human health worldwide. Type I IFN (IFN-I) therapy is an important therapeutic option for HBV patients. The antiviral effect of IFN is mainly mediated via upregulation of the expressions of the downstream IFN-stimulated genes. However, the mechanisms by which IFN induces ISG production and inhibits HBV replication are yet to be clarified. TRIM14 was recently reported as a key molecule in the IFN-signaling pathway that regulates IFN production in response to viral infection. In this study, we sought to understand the mechanisms by which IFN restricts HBV replication. We confirmed that TRIM14 is an ISG in the hepatic cells, and that the pattern-recognition receptor ligands polyI:C and polydAdT induce TRIM14 dependent on IFN-I production. In addition, IFN-I-activated STAT1 (but not STAT3) directly bound to the TRIM14 promoter and mediated the induction of TRIM14. Interestingly, TRIM14 played an important role in IFN-I-mediated inhibition of HBV, and the TRIM14 SPRY domain interacted with the C-terminal of HBx, which might block the role of HBx in facilitating HBV replication by inhibiting the formation of the Smc-HBx-DDB1 complex. Thus, our study clearly demonstrates that TRIM14 is a STAT1-dependent ISG, and that the IFN-I-TRIM14-HBx axis shows an alternative way to understand the mechanism by which IFN-I inhibits virus replication.

Expression of microRNA let-7a positively correlates with hepatitis B virus replication in hepatocellular carcinoma tissues.

Let-7a miRNA is downregulated in various cancers. However, in hepatocellular carcinoma (HCC) patients infected with hepatitis B virus (HBV), the relationship between let-7a and HBV replication has not been fully elucidated. Liver specimens were collected from 23 HCC patients with chronically active HBV. The serum levels of the HBV antigens hepatitis B surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg), and the HBV antibodies, anti-HBs, anti-HBe and anti-hepatitis B core antigen (anti-HBc) were measured using the microparticle enzyme immunoassay. Let-7a levels and HBV DNA copy numbers were measured by quantitative real-time PCR (qRT-PCR) and analyzed statistically. A let-7a specific antisense oligonucleotide was introduced to the HBV-producing cell line HepG2.2.15 and a change in HBV DNA copy numbers was assessed by qRT-PCR. HCC patients with highly active HBV replication (>106 DNA copies/mL) showed higher levels of serum HBsAg and anti-HBc than patients with less active HBV replication (<103 DNA copies/mL). The level of let-7a was lower in malignant tissues than in adjacent normal tissues. However, patients with highly active HBV replication demonstrated a significantly higher level of let-7a in hepatocarcinoma tissue than patients with less active HBV replication ( P < 0.05). A higher level of let-7a was observed in the HBV-producing cell line HepG2.2.15 than in HepG2 cells ( P < 0.05), and let-7a down-regulation by antisense oligonucleotides led to a reduction in HBV DNA copy numbers ( P < 0.05), indicating a correlation between the let-7a level and HBV replication. Down-regulation of let-7a reduces HBV replication and could prevent the development of HCC, suggesting it could be an effective therapeutic treatment for HBV infection. Impact statement Although interferon and nucleic acid analogues effectively suppress HBV replication in HBV patients, there is no treatment which eradicates the virus. Moreover, the therapeutic effect can be reduced by virus mutations or drug resistance. Let-7a is a miRNA initially found in the nematode as a master regulator of developmental processes, but also exists in humans. It has been reported that the transcription of let-7a was much lower in HCC than in normal liver tissues and specific miRNA could directly promote virus replication. Therefore we hypothesized that transcription of let-7a promotes HBV replication, which might compromise the therapeutic effects of antivirus treatments. In our present study, we demonstrated a correlation between let-7a transcription and HBV replication in surgical specimens obtained from patients with HCC, as well as in HCC cell lines. Our finding might be the base for a new approach to improve HBV infection treatments in the future.

miR-29a promotes hepatitis B virus replication and expression by targeting SMARCE1 in hepatoma carcinoma.

AIM: To investigate the functional role and underlying molecular mechanism of miR-29a in hepatitis B virus (HBV) expression and replication. METHODS: The levels of miR-29a and SMARCE1 in HBV-infected HepG2.2.15 cells were measured by quantitative real-time PCR and western blot analysis. HBV DNA replication was measured by quantitative PCR and Southern blot analysis. The relative levels of hepatitis B surface antigen and hepatitis B e antigen were detected by enzyme-linked immunosorbent assay. The Cell Counting Kit-8 (CCK-8) was used to detect the viability of HepG2.2.15 cells. The relationship between miR-29a and SMARCE1 were identified by target prediction and luciferase reporter analysis. RESULTS: miR-29a promoted HBV replication and expression, while SMARCE1 repressed HBV replication and expression. Cell viability detection indicated that miR-29a transfection had no adverse effect on the host cells. Moreover, SMARCE1 was identified and validated to be a functional target of miR-29a. Furthermore, restored expression of SMARCE1 could relieve the increased HBV replication and expression caused by miR-29a overexpression. CONCLUSION: miR-29a promotes HBV replication and expression through regulating SMARCE1. As a potential regulator of HBV replication and expression, miR-29a could be a promising therapeutic target for patients with HBV infection.

Intrahepatic hepatitis B virus replication and liver histology in subjects with occult hepatitis B infection.

We studied the intrahepatic hepatitis B virus (HBV) replicative status in 40 people with occult hepatitis B infection (OBI) and 40 patients with chronic hepatitis B (CHB). Intrahepatic HBV DNA, covalently closed circular DNA (cccDNA), and pre-genomic RNA (pgRNA) were quantified. Patients with OBI had median necroinflammation and fibrosis scores of 1 and 0, respectively. Intrahepatic total HBV DNA, cccDNA and pgRNA were detectable in 30 (77%), one (3%) and five (13%) of the participants with OBI, respectively. People with OBI had lower median intrahepatic total HBV DNA than the patients with CHB (p < 0.0001). They had nearly normal liver histology and low intrahepatic HBV replication.

Hepatitis B virus and microRNAs: Complex interactions affecting hepatitis B virus replication and hepatitis B virus-associated diseases.

Chronic infection with the hepatitis B virus (HBV) is the leading risk factor for the development of hepatocellular carcinoma (HCC). With nearly 750000 deaths yearly, hepatocellular carcinoma is the second highest cause of cancer-related death in the world. Unfortunately, the molecular mechanisms that contribute to the development of HBV-associated HCC remain incompletely understood. Recently, microRNAs (miRNAs), a family of small non-coding RNAs that play a role primarily in post-transcriptional gene regulation, have been recognized as important regulators of cellular homeostasis, and altered regulation of miRNA expression has been suggested to play a significant role in virus-associated diseases and the development of many cancers. With this in mind, many groups have begun to investigate the relationship between miRNAs and HBV replication and HBV-associated disease. Multiple findings suggest that some miRNAs, such as miR-122, and miR-125 and miR-199 family members, are playing a role in HBV replication and HBV-associated disease, including the development of HBV-associated HCC. In this review, we discuss the current state of our understanding of the relationship between HBV and miRNAs, including how HBV affects cellular miRNAs, how these miRNAs impact HBV replication, and the relationship between HBV-mediated miRNA regulation and HCC development. We also address the impact of challenges in studying HBV, such as the lack of an effective model system for infectivity and a reliance on transformed cell lines, on our understanding of the relationship between HBV and miRNAs, and propose potential applications of miRNA-related techniques that could enhance our understanding of the role miRNAs play in HBV replication and HBV-associated disease, ultimately leading to new therapeutic options and improved patient outcomes.

Negative regulation of hepatitis B virus replication by forkhead box protein A in human hepatoma cells.

Hepatitis B virus (HBV) replication is controlled by liver-enriched transcriptional factors, including forkhead box protein A (FOXA) members. Here, we found that FOXA members are directly and indirectly involved in HBV replication in human hepatic cells. HBV replication was elevated in HuH-7 treated with individual FOXA members-specific siRNA. Reciprocally, the downregulation of HBV replication was observed in FOXA-induced HuH-7. However, the mechanism of downregulation is different among FOXA members at the level of HBV RNA transcription, such as precore/pg RNA and 2.1 kb RNA. In addition, FOXA1 and FOXA2 suppressed nuclear hormone receptors, such as HNF4α, that are related to HBV replication.

Serum microRNA-30c levels are correlated with disease progression in Xinjiang Uygur patients with chronic hepatitis B

We aimed to investigate the potential role and mechanism of microRNA-30c (miR-30c) in the pathological development of chronic hepatitis B (CHB). The serum levels of miR-30c in hepatitis B virus (HBV) carrier Xinjiang Uygur patients with inactive, low-replicative, high-replicative and HBe antigen-positive CHB were investigated. HepG2 cells were co-transfected with pHBV1.3 and miR-30c mimic or inhibitor or scramble RNA. The effects of miR-30c dysregulation on HBV replication and gene expression, cell proliferation and cell cycle were then investigated. miR-30c was down-regulated in Xinjiang Uygur patients with CHB compared to healthy controls and its expression level discriminated HBV carrier patients with inactive, low-replicative, high-replicative and HBe antigen-positive risk for disease progression. Overexpression of miR-30c significantly inhibited HBV replication and the expressions of HBV pgRNA, capsid-associated virus DNA and Hbx in hepatoma cells. Moreover, overexpression of miR-30c significantly inhibited cell proliferation and delayed G1/S phase transition in hepatoma cells. Opposite effects were obtained after suppression of miR-30c. Our results indicate that miR-30c was down-regulated in Xinjiang Uygur patients with CHB, and miR-30c levels could serve as a marker for risk stratification of HBV infection. Down-regulation of miR-30c may result in the progression of CHB via promoting HBV replication and cell proliferation.