HBV Surface Antigen Expression Research Articles

The N-linked glycosylation modifications in the hepatitis B surface protein impact cellular autophagy, HBV replication, and HBV secretion.

N-linked glycosylation is a pivotal post-translational modification that significantly influences various aspects of protein biology. Autophagy, a critical cellular process, is instrumental in cell survival and maintenance. The hepatitis B virus (HBV) has evolved mechanisms to manipulate this process to ensure its survival within host cells. Significantly, post-translational N-linked glycosylation in the large surface protein of HBV (LHBs) influences virion assembly, infectivity, and immune evasion. This study investigated the role of N-linked glycosylation of LHBs in autophagy, and its subsequent effects on HBV replication and secretion. LHBs plasmids were constructed by incorporating single-, double-, and triple-mutated N-linked glycosylation sites through amino acid substitutions at N4, N112, and N309. In comparison to the wild-type LHBs, N-glycan mutants, including N309Q, N4-309Q, N112-309Q, and N4-112-309Q, induced autophagy gene expression and led to autophagosome accumulation in hepatoma cells. Acridine orange staining of cells expressing LHBs mutations revealed impaired lysosomal acidification, suggesting potential blockage of autophagic flux at later stages. Furthermore, N-glycan mutants increased the mRNA expression of HBV surface antigen (HBsAg). Notably, N309Q significantly elevated HBx oncogene level. The LHBs mutants, particularly N309Q and N112-309Q, significantly enhanced HBV replication, whereas N309Q, N4-309Q, and N4-112-309Q markedly increased HBV progeny secretion. Remarkably, our findings demonstrated that autophagy is indispensable for the impact of N-linked glycosylation mutations in LHBs on HBV secretion, as evidenced by experiments with a 3-methyladenine (3-MA) inhibitor. Our study provides pioneering insights into the interplay between N-linked glycosylation mutations in LHBs, host autophagy, and the HBV life cycle. Additionally, we offer a new clue for further investigation into carcinogenesis of hepatocellular carcinoma (HCC). These findings underscore the potential of targeting either N-linked glycosylation modifications or the autophagic pathway for the development of innovative therapies against HBV and/or HCC.

Hepatitis B Virus Envelope Antigen and Hepatitis B Virus Surface Antigen Both Contribute to the Innate Immune Response During Persistent Hepatitis B Virus Infection.

This study aimed to investigate the changes of toll-like receptor 4 (TLR4), proinflammatory cytokine expression, hepatitis B virus surface antigen (HBsAg), and hepatitis B virus envelope antigen (HBeAg) expression as well as innate immune cell percentages in a mouse model of persistent hepatitis B virus (HBV) infection to better understand the innate immune response. Mouse models of persistent HBV infection, HBsAg expression, and HBeAg expression were developed using high-pressure tail-vein injection of recombinant adeno-associated viruses. Enzyme-linked immunosorbent assays (ELISAs) were used to determine the serum proinflammatory cytokine levels. Immunohistochemistry and western blot assays were used to detect TLR4 expression. Flow cytometric analysis was used to assess the percentage of innate immune cells in the whole blood. Persistent HBV infection, HBsAg expression, and HBeAg expression each significantly decreased the expression of TLR4. Persistent HBV infection significantly increased the percentages of T cells and monocytes, whereas it decreased the percentage of natural killer (NK) cells. Persistent HBeAg expression also decreased the percentage of NK cells, whereas persistent HBsAg expression increased the percentage of NK cells. Both persistent HBsAg and HBeAg expression increased the percentage of monocytes. However, both persistent HBsAg and HBeAg expression decreased the percentage of T cells. HBV as well as HBsAg and HBeAg showed similar effects on the expression of TLR4 and proinflammatory cytokines as well as the percentage of monocytes. Persistent HBV infection increased the percentage of T cells and decreased the percentage of NK cells, whereas only persistent HBeAg expression contributed to a decreased percentage of NK cells.

Decreased expression of HBV surface antigen (HBsAg) with sK122R and sV96A co-mutation is associated with an ineffective antibody response in a chronic hepatitis B patient.

Emergence and predominance of hepatitis B virus (HBV) variants carrying S gene mutations frequently occur in HBV-infected individuals. Here, coexistent serum anti-HBsAg antibody (HBsAb) and HBV surface antigen (HBsAg) were detected in a chronic HBV patient. The patient's HBsAg proteins possessed amino acid substitutions sK122R and sV96A. We reported this case and conducted relevant studies to investigate differences in expression levels and antibody neutralization of HBsAg proteins bearing sK122R and sV96A amino acid substitutions to explore causes of antigen-antibody coexistence in a chronic hepatitis B patient. We first sequenced the S gene from HBV present within the patient's serum. Based on the S gene sequence, we cloned wild-type and mutated S gene sequences via site-directed mutagenesis to construct expression plasmids pJW4303-WT (wild-type), pJW4303-sV96A, pJW4303-sK122R, and pJW4303-sV96A-sK122R. Plasmids were transfected into HEK 293T cells then culture supernatants and cells were collected. Collected cells and supernatants were next subjected to a series of quantitative and functional tests to assess expression and neutralization characteristics of wild-type and mutant HBsAg proteins. Based on quantification of HBsAg expression in cells transfected with the four plasmids, HBsAg-sK122R-sV96A was more intracellularly retained and less secreted than HBsAg-sV96A single-mutant protein and WT. Neutralization ability of serum from chronic HBV patient against culture supernatants containing recombinant HBsAg proteins were ranked from highest to lowest as HBsAg-sV96A, HBsAg-sV96A-sK122R, and HBsAg-sK122R. However, no significant differences of neutralization efficiency by high-potency antibodies from HBV-vaccinees against these three mutant proteins were observed. The levels of HBsAg proteins with amino acid substitutions sV96A-sK122R were greatly reduced in culture supernatants but were apparently increased in the intracellular fraction. This may account for the higher levels of HBV replication in patients. HBsAg neutralization by HBsAb in this patient may have been compromised by the HBsAg sK122R amino acid substitution, suggesting that antibodies produced by the patient had lost their HBV-neutralizing effect.

Novel anti-hepatitis B virus-active catechin and epicatechin from Rhus tripartita.

Bioactive natural or phytoproducts have emerged as a potential source of antiviral agents. Of the Rhus spp., R. coriaria and R. succedanea have been reported for their antiviral activities against hepatitis B virus (HBV), while the anti-HBV efficacy of R. tripartita has remained elusive. In the present study, the anti-HBV activities of R. tripartita-derived novel catechin [3,5,13,14-flavantetrol-catechin or rhuspartin (RPT)] and epicatechin-3-O-rhamnoside (ECR), were assessed using the HBV-reporter cell line HepG2.2.15. RPT and ECR proved to efficiently inhibit HBV surface antigen (HBsAg) synthesis by 68.8 and 71.3%, respectively, and HBV pre-core antigen (HBeAg) production by 62.3 and 71.2%, respectively, after 5 days of treatment. Of note, RPT had a lower anti-HBV activity than ECR. In comparison, the reference drug lamivudine (LAM) inhibited HBsAg and HBeAg expression by 83.6 and 85.4%, respectively. Further molecular docking analysis revealed formations of strong complexes of RPT, ECR and LAM with HBV polymerase through interactions with binding pocket residues. Taken together, the present results demonstrated promising therapeutic potential of the novel R. tripartita-derived catechin and epicatechin for HBV, warranting their further molecular and pharmacological evaluation.

Molecular and Serological Characterization of Hepatitis B Virus (HBV)-Positive Samples with Very Low or Undetectable Levels of HBV Surface Antigen.

Background: Gaps remain in the detection of nucleic acid test (NAT) yield and occult hepatitis B virus (HBV) infection (OBI) by current HBV surface antigen (HBsAg) assays. The lack of detection may be due to HBsAg levels below current assay detection limits, mutations affecting HBsAg assays or HBsAg levels, or the masking of HBsAg by antibody to HBsAg (anti-HBs). In this study, we evaluate the incremental detection of NAT yield and OBI from five diverse geographic areas by an improved sensitivity HBsAg assay and characterize the samples relative to the viral load, anti-HBs status, and PreS1–S2–S mutations. Included is a comparison population with HBV DNA levels comparable to OBI, but with readily detectable HBsAg (High Surface–Low DNA, HSLD). Methods: A total of 347 samples collected from the USA, South Africa, Spain, Cameroon, Vietnam, and Cote D’Ivoire representing NAT yield (HBsAg(−), antibody to HBV core antigen (anti-HBc)(−), HBV DNA(+), N = 131), OBI (HBsAg(−), anti-HBc(+), HBV DNA(+), N = 188), and HSLD (HBsAg(+), anti-HBc(+), HBV DNA(+), N = 28) were tested with ARCHITECT HBsAg NEXT (HBsAgNx) (sensitivity 0.005 IU/mL). The sequencing of the PreS1–S2–S genes from a subset of 177 samples was performed to determine the genotype and assess amino acid variability, particularly in anti-HBs(+) samples. Results: HBsAgNx detected 44/131 (33.6%) NAT yield and 42/188 (22.3%) OBI samples. Mean HBV DNA levels for NAT yield and OBI samples were lower in HBsAgNx(−) (50.3 and 25.9 IU/mL) than in HBsAgNx(+) samples (384.1 and 139.5 IU/mL). Anti-HBs ≥ 10 mIU/mL was present in 28.6% HBsAgNx(+) and 45.2% HBsAgNx(−) OBI, and in 3.6% HSLD samples. The genotypes were A1, A2, B, C, D, E, F, and H. There was no significant difference between HBsAgNx(−) and HBsAgNx(+) in the proportion of samples harboring substitutions or in the mean number of substitutions per sample in PreS1, PreS2, or S for the NAT yield or OBI (p range: 0.1231 to >0.9999). A total of 21/27 (77.8%) of HBsAgNx(+) OBI carried S escape mutations, insertions, or stop codons. HSLD had more PreS1 and fewer S substitutions compared to both HBsAgNx(−) and HBsAgNx(+) OBI. Mutations/deletions associated with impaired HBsAg secretion were observed in the OBI group. Conclusions: HBsAgNx provides the improved detection of NAT yield and OBI samples. Samples that remain undetected by HBsAgNx have exceptionally low HBsAg levels below the assay detection limit, likely due to low viremia or the suppression of HBsAg expression by host and viral factors.

Havachoobe (Onosma dichroanthum Boiss) Root Extract Decreases the Hepatitis B Virus Surface Antigen Secretion in the PLC/PRF/5 Cell Line

Background: Many efforts are currently focused on functional treatment of the hepatitis B virus (HBV). This can be done by suppressing the secretion of HBV surface antigen (HBsAg). Scientific communities are very interested in natural products in that respect. Objective: Use of root extract of Havachoobe (Onosma dichroanthum BoissI), a Northern Iranian native medical herb, for assessment of its anti-HBsAg secretion activity. Methods: Havachoobe had been bought at a nearby apothecary store. Plant root extract was obtained using a hydroalcoholic process. Cytotoxic activity of the extract was examined on PLC/PRF/5 cells using MTT assay. ELISA has been used to measure HBsAg in the treated cell line supernatants. In addition, real-time PCR analysis was performed to evaluate the expression of HBsAg before and after treatment of Onosma in vitro. Results: The results showed very low root extract cytotoxicity at concentrations under 8 μg/mL. Tissue culture infectious dose 50 was obtained at 63.78 μg/mL. In a dose-dependent and time-dependent manner, a significantly reduced HBsAg secretion was observed at a concentration of 8 ppm at 12 h post-treatment. The real-time PCR result showed relative decreased HBsAg expression at all doses at 12 h post-treatment time. Discussion: In this study, we first reported anti-HBsAg activity on an Iranian herbal medicine. Havachoobe root extract was shown to be able to inhibit HBsAg in a dose-dependent and time-dependent manner. We find the extract exerts its inhibitory effect of HBsAg by targeting transcription of HBsAg.

Cre/LoxP-HBV plasmids generating recombinant covalently closed circular DNA genome upon transfection

New therapies against hepatitis B virus (HBV) require the elimination of covalently closed circular DNA (cccDNA), the episomal HBV genome. HBV plasmids containing an overlength 1.3-mer genome and bacterial backbone (pHBV1.3) are used in many different models, but do not replicate the unique features of cccDNA. Since the stable cccDNA pool is a barrier to HBV eradication in patients, we developed a recombinant circular HBV genome (rcccDNA) to mimic the cccDNA using Cre/LoxP technology. We validated four LoxP insertion sites into the HBV genome using hydrodynamic tail vein injection into murine liver, demonstrating high levels of HBV surface antigen (HBsAg) and HBV DNA expression with rcccDNA formation. HBsAg expression from rcccDNA was >30,000 ng/mL over 78 days, while HBsAg-expression from pHBV1.3 plasmid DNA declined from 2753 ng/mL to 131 ng/mL over that time in immunodeficient mice (P < 0.001), reflective of plasmid DNA silencing. We then cloned Cre-recombinase in cis on the LoxP-HBV plasmids, achieving plasmid stability in bacteria with intron insertion into Cre and demonstrating rcccDNA formation after transfection in vitro and in vivo. These cis-Cre/LoxP-HBV plasmids were then used to create HBx-mutant and GFP reporter plasmids to further probe cccDNA biology and antiviral strategies against cccDNA. Overall, we believe these auto-generating rcccDNA plasmids will be of great value to model cccDNA for testing new therapies against HBV infection.

Analysis of clinical characteristics and S gene sequences in chronic asymptomatic HBV carriers with low-level HBsAg

During the natural hepatitis B virus (HBV) infection process, some infected subjects are characterized by a sustained low serum HBV surface antigen (HBsAg) expression level. Most members in this population are chronic asymptomatic HBV carriers (ASCs). To elucidate the mechanism underlying low-level HBsAg expression in ASCs, we sequenced the HBV S gene in these patients to reveal specific sequence characteristics. Overall, 1308 cases of chronic ASCs were grouped according to their HBsAg serum expression levels (10 IU/mL). The clinical characteristics of the population were analysed in detail. The HBV S gene was sequenced from 276 ASC cases with low-level HBsAg expression. Additionally, 100 of 1032 ASC cases with high-level HBsAg expression were randomly selected for HBV S gene sequencing based on age matching according to the low-level HBsAg group. A comparative analysis was conducted with the HBV S gene sequences from ASCs with low HBsAg expression and the HBV reference S gene sequences from ASCs with high HBsAg expression. The population with low-level HBsAg expression displayed the following primary clinical characteristics: mostly chronic asymptomatic HBV carriers, older age (mean age 55.09 years), HBsAg/anti-HBe/anti-HBc (core) positivity as the main serological pattern (97.1%), low HBV DNA replication (1.32 ± 1.60 log10 IU/mL), a low HBV-DNA positive rate (45.65%) and primarily genotype B (82.54%) and serotype adw (84.13%). The comparative analysis of the HBV S gene sequences from ASCs with low-level HBsAg showed significant mutations (including co-mutations) on both sides of the main hydrophilic region (MHR). Significant mutations in multiple regions and at multiple sites (including co-mutations) on both sides of the MHR may be one cause of the low HBsAg expression level in this population.

Curcumin inhibits hepatitis B virus infection by down-regulating cccDNA-bound histone acetylation.

AIMTo investigate the potential effect of curcumin on hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and the underlying mechanism.METHODSA HepG2.2.15 cell line stably transfected with HBV was treated with curcumin, and HBV surface antigen (HBsAg) and e antigen (HBeAg) expression levels were assessed by ELISA. Intracellular HBV DNA replication intermediates and cccDNA were detected by Southern blot and real-time PCR, respectively. The acetylation levels of histones H3 and H4 were measured by Western blot. H3/H4-bound cccDNA was detected by chromatin immunoprecipitation (ChIP) assays. The deacetylase inhibitors trichostatin A and sodium butyrate were used to study the mechanism of action for curcumin. Additionally, short interfering RNAs (siRNAs) targeting HBV were tested along with curcumin.RESULTSCurcumin treatment led to time- and dose-dependent reductions in HBsAg and HBeAg expression and significant reductions in intracellular HBV DNA replication intermediates and HBV cccDNA. After treatment with 20 μmol/L curcumin for 2 d, HBsAg and cccDNA levels in HepG2.2.15 cells were reduced by up to 57.7% (P < 0.01) and 75.5% (P < 0.01), respectively, compared with levels in non-treated cells. Meanwhile, time- and dose-dependent reductions in the histone H3 acetylation levels were also detected upon treatment with curcumin, accompanied by reductions in H3- and H4-bound cccDNA. Furthermore, the deacetylase inhibitors trichostatin A and sodium butyrate could block the effects of curcumin. Additionally, transfection of siRNAs targeting HBV enhanced the inhibitory effects of curcumin.CONCLUSIONCurcumin inhibits HBV gene replication via down-regulation of cccDNA-bound histone acetylation and has the potential to be developed as a cccDNA-targeting antiviral agent for hepatitis B.

Dual oncogenic and tumor suppressor roles of the promyelocytic leukemia gene in hepatocarcinogenesis associated with hepatitis B virus surface antigen.

Proteasome-mediated degradation of promyelocytic leukemia tumor suppressor (PML) is upregulated in many viral infections and cancers. We previously showed that PML knockdown promotes early-onset hepatocellular carcinoma (HCC) in hepatitis B virus surface antigen (HBsAg)-transgenic mice. Here we report the effects of PML restoration on late-onset HBsAg-induced HCC. We compared protein expression patterns, genetic mutations and the effects of pharmacologically targeting PML in wild-type, PML−/−, PML+/+HBsAgtg/o and PML−/−HBsAgtg/o mice. PML−/− mice exhibited somatic mutations in DNA repair genes and developed severe steatosis and proliferative disorders, but not HCC. PML−/−HBsAgtg/o mice exhibited early mutations in cancer driver genes and developed hyperplasia, fatty livers and indolent adipose-like HCC. In PML+/+HBsAg-transgenic mice, HBsAg expression declined over time, and HBsAg-associated PML suppression was concomitantly relieved. Nevertheless, these mice accumulated mutations in genes contributing to oxidative stress pathways and developed aggressive late-onset angiogenic trabecular HCC. PML inhibition using non-toxic doses of arsenic trioxide selectively killed long-term HBsAg-affected liver cells in PML+/+HBsAgtg/o mice with falling HBsAg and rising PML levels, but not normal liver cells or early-onset HCC cells in PML−/−HBsAgtg/0 mice. These findings suggest dual roles for PML as a tumor-suppressor lost in early-onset HBsAg-induced hepatocarcinogenesis and as an oncogenic promoter in late-onset HBsAg-related HCC progression.

Effect of the cytoplasmic DNA sensor DAI on replication of hepatitis B virus

To explore the effect of the cytoplasmic DNA sensor DAI on replication of hepatitis B virus (HBV) and its possible mechanism. The hepatocyte-derived cell line HepG2 was co-transfected with DAI siRNA and the HBV1.3 replicative plasmid PHY106, and the cells were divided into two experimental groups. Six hours later, total RNA was extracted from the first group of cells and expression of IFIT1 and IL-6 were detected by real-time RT-PCR. The second group of cells was incubated for 4 days, after which the cell supernatant was collected and the HBV surface antigen (HBsAg) and envelope antigen (HBeAg) were detected by ELISA. In addition, HBV core particles were extracted and applied to southern blot assay to detect the intracellular HBV replication intermediates (rcDNA, dlDNA and ssDNA). Next, the HepG2 cells were triple transfected with siRNA targeting the type I interferon pathway molecule TBK1 and DAI simultaneously and HBV1.3, after which HBV viral proteins were detected. Two-group comparisons were made using the independent sample t-test, and more-than-2-group comparisons were made using ANOVA. DAI gene expression was down-regulated in response to DAI siRNA transfection. Cells with down-regulated DAI showed inhibited HBV replication (in a dose-dependent manner), accompanied by reduced levels of HBsAg (0.0195+/-0.0050 vs. 0.3150+/-0.0200, P less than 0.05, t = 14.77) and HBeAg (0.0140+/-0.0040 vs. 0.01235+/-0.0135, P less than 0.05, t = 7.777). No effect of down-regulated DAI was observed for the expression of IFIT1 of IL-6. siRNA-mediated down-regulation of TBK1 and DAI simultaneously led to reduced expression of HBsAg and HBeAg. Down-regulation of DAI gene expression inhibited HBV replication and HBV protein expression, but the underlying mechanism was not related to the type I interferon or NF-kB signaling pathway.

HBV-DNA levels predict overall mortality in HIV/HBV coinfected individuals.

The coinfection of Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) has been associated with increased death rates. However, the relevant research has mostly relied on serologic HBV testing [HBV surface antigen (HBsAg)]. The aim of this work was to explore the relationship of HBV viraemia with overall mortality among HIV/HBV coinfected individuals. The analysis included 1,609 HIV seropositives of a previously described cohort (1984-2003) with limited exposure to tenofovir (12%) and a median follow-up of approximately 5 years. Those with persistent expression of HBsAg were further tested for HBV-DNA. The data were analyzed using Poisson regression models. Totally, 101 participants were chronic carriers of HBsAg (6.28%). Of these, 81 were tested for HBV-DNA. The median HBV-DNA levels were 3.81 log (base-10) International Units (IU)/ml. A third (31%) of those tested for HBV-DNA had received tenofovir. Before developing acquired immune deficiency syndrome (AIDS), the adjusted incidence rate ratio (IRR) for all-cause mortality of coinfected patients with HBV viraemia above the median value versus the HIV monoinfected group was 3.44 [95% confidence interval (CI): 1.05-11.27]. Multivariable regressions in the coinfected group only (n = 81) showed that one log-10 increase in HBV-DNA levels was associated with an elevated risk for death (IRR: 1.24, 95%CI: 1.03-1.49). HBV-DNA levels predict overall mortality in the setting of HIV/HBV coinfection, especially during the period before developing AIDS, and could thus help prioritize needs and determine the frequency of medical monitoring.

Gluconeogenesis, lipogenesis, and HBV replication are commonly regulated by PGC-1α-dependent pathway

PGC-1α, a major metabolic regulator of gluconeogenesis and lipogenesis, is strongly induced to coactivate Hepatitis B virus (HBV) gene expression in the liver of fasting mice. We found that 8-Br-cAMP and glucocorticoids synergistically induce PGC-1α and its downstream targets, including PEPCK and G6Pase. Also, HBV core promoter activity was synergistically enhanced by 8-Br-cAMP and glucocorticoids. Graptopetalum paraguayense (GP), a herbal medicine, is commonly used in Taiwan to treat liver disorders. Partially purified fraction of GP (named HH-F3) suppressed 8-Br-cAMP/glucocorticoid-induced G6Pase, PEPCK and PGC-1α expression and suppressed HBV core promoter activity. HH-F3 blocked HBV core promoter activity via inhibition of PGC-1α expression. Ectopically expressed PGC-1α rescued HH-F3-inhibited HBV surface antigen expression, HBV mRNA production, core protein levels, and HBV replication. HH-F3 also inhibited fatty acid synthase (FASN) expression and decreased lipid accumulation by down-regulating PGC-1α. Thus, HH-F3 can inhibit HBV replication, gluconeogenesis and lipogenesis by down-regulating PGC-1α. Our study indicates that targeting PGC-1α may be a therapeutic strategy for treatment of HBV infections. HH-F3 may have potential use for the treatment of chronic hepatitis B patients with associated metabolic syndrome.

Clonal expansion of hepatocytes with a selective advantage occurs during all stages of chronic hepatitis B virus infection.

Hepatocyte clone size was measured in liver samples of 21 patients in various stages of chronic hepatitis B virus (HBV) infection and from 21 to 76 years of age. Hepatocyte clones containing unique virus-cell DNA junctions formed by the integration of HBV DNA were detected using inverse nested PCR. The maximum hepatocyte clone size tended to increase with age, although there was considerable patient-to-patient variation in each age group. There was an upward trend in maximum clone size with increasing fibrosis, inflammatory activity and with seroconversion from HBV e-antigen (HBeAg)-positive to HBeAg-negative, but these differences did not reach statistical significance. Maximum hepatocyte clone size did not differ between patients with and without a coexisting hepatocellular carcinoma. Thus, large hepatocyte clones containing integrated HBV DNA were detected during all stages of chronic HBV infection. Using laser microdissection, no significant difference in clone size was observed between foci of HBV surface antigen (HBsAg)-positive and HBsAg-negative hepatocytes, suggesting that expression of HBsAg is not a significant factor in clonal expansion. Laser microdissection also revealed that hepatocytes with normal-appearing histology make up a major fraction of the cells undergoing clonal expansion. Thus, preneoplasia does not appear to be a factor in the clonal expansion detected in our assays. Computer simulations suggest that the large hepatocyte clones are not produced by random hepatocyte turnover but have an as-yet-unknown selective advantage that drives increased clonal expansion in the HBV-infected liver.

MicroRNA-581 promotes hepatitis B virus surface antigen expression by targeting Dicer and EDEM1.

Hepatitis B virus surface antigen (HBsAg) is an important risk factor for hepatocellular carcinoma (HCC) and is downregulated during hepatocarcinogenesis. MicroRNAs (miRNAs) are frequently deregulated in HCC tissues. However, whether the deregulation of certain miRNAs in HCC has an impact on HBsAg expression remains unclear. We found here that microRNA-581 (miR-581), which is deregulated during hepatocarcinogenesis, promoted HBsAg expression. Additionally, miR-581 targeted Dicer and endoplasmic reticulum degradation-enhancing alpha-mannosidase-like protein 1 (EDEM1) and repressed their expression. Although Dicer cannot process HBV transcripts, Dicer knockdown led to increased HBsAg secretion, most likely due to a reduction in the levels of Dicer-processed 7SL RNA fragments. Moreover, Dicer-processed 7SL RNA fragments partially inhibited the ability of miR-581 to stimulate HBsAg expression. Furthermore, we found that forced EDEM1 expression inhibited miR-581-mediated induction of HBsAg. Finally, transfection of miR-581 into HepG2.2.15 cells promoted cell proliferation and led to upregulation of genes involved in development, cell proliferation and protein secretion. Altogether, we conclude that miR-581 promotes HBsAg expression by targeting Dicer and EDEM1. Our findings suggest that downregulation of miR-581 during hepatocarcinogenesis may lead to a reduction in HBsAg expression and impede HCC development.

Functional interplay between hepatitis B virus X protein and human miR-125a in HBV infection

The hepatitis B virus (HBV) is a widespread human pathogen and chronic HBV infection is a major risk factor for hepatocellular carcinoma (HCC). Some cellular microRNAs are emerging as important regulators of virus–host interaction, indirectly or directly modulating HBV replication and pathogenesis. miR-125a binds the viral transcript encoding the surface antigen and interferes with its expression, thus inhibiting viral replication. Intriguingly, liver miR-125a expression has been found increased in patients with high levels of hepatic HBV-DNA. The present study investigates the mechanism by which liver exposure to HBV induces the expression of miR-125a. The analyses were first performed on liver biopsies from HBV patients, showing that the expression of the viral transactivator X protein (HBx) paralleled the increase of miR-125a expression. Then, transfection of HCC cell lines with an HBx-expressing vector showed a substantial increase of miR-125a expression. Overall, the available data depict a self-inhibitory feedback loop in which HBV, through HBx, increases the expression of miR-125a, that in turn interferes with expression of HBV surface antigen, thus repressing viral replication.

Effects of a clinic-initiated behavioral nutrition intervention emphasizing the dash diet on blood pressure control in adolescents with elevated blood pressure

The hepatitis B virus (HBV) is a widespread human pathogen and chronic HBV infection is a major risk factor for hepatocellular carcinoma (HCC). Some cellular microRNAs are emerging as important regulators of virus–host interaction, indirectly or directly modulating HBV replication and pathogenesis. miR-125a binds the viral transcript encoding the surface antigen and interferes with its expression, thus inhibiting viral replication. Intriguingly, liver miR-125a expression has been found increased in patients with high levels of hepatic HBV-DNA. The present study investigates the mechanism by which liver exposure to HBV induces the expression of miR-125a. The analyses were first performed on liver biopsies from HBV patients, showing that the expression of the viral transactivator X protein (HBx) paralleled the increase of miR-125a expression. Then, transfection of HCC cell lines with an HBx-expressing vector showed a substantial increase of miR-125a expression. Overall, the available data depict a self-inhibitory feedback loop in which HBV, through HBx, increases the expression of miR-125a, that in turn interferes with expression of HBV surface antigen, thus repressing viral replication.

Metformin inhibits hepatitis B virus protein production and replication in human hepatoma cells

Hepatitis B virus surface antigen (HBsAg) plays an important role in maintaining the tolerance and may interfere with host innate and adaptive immune responses; therefore, novel therapeutic strategies to reduce HBsAg loads in patients infected with hepatitis B virus (HBV) are emerging as an attractive but challenging issue. Metformin could regulate hepatic metabolism while the latter interacts with HBV infection. We hypothesized that metformin could affect HBsAg expression and HBV replication and may work synergistically when combined with current antivirals. In our study, a notably inhibitory effect on HBsAg production, as well as a moderate inhibition in HBV replication and HBeAg expression was observed following metformin treatment. The 50% effective concentration (EC50) for extracellular HBsAg and intracellular HBsAg in HBV-producing HepG2.2.15 cells was 2.85 mm and 2.75 mm, respectively, with a similarly selective index of about 18. When administered in combination, metformin enhanced the inhibitory effects of interferon-α2b on HBsAg expression and HBV replication and provided a complimentary role in HBsAg expression for lamivudine (LMV). This novel action of metformin derives partially from its inhibition on multiple HBV cis-acting elements. By the virtues of preferably hepatocyte distribution and safety profile, collectively, our results suggest that metformin would be potentially clinically helpful as an HBsAg production inhibitor.

Synthesis and biological evaluation of nucleoside analogues than contain silatrane on the basis of the structure of acyclovir (ACV) as novel inhibitors of hepatitis B virus (HBV)

Hepatitis B virus (HBV) infection causes major public health problems worldwide. Acyclovir (ACV) is mainly used to inhibit herpes simplex virus (HSV) rather than HBV. In this study, we used the combination principle to design and synthesize nucleoside analogues that contain silatrane on the basis of the structure of ACV. We found that the compounds were effective inhibitors of HBV, both in vitro and in vivo. All of the compounds showed suppressive activity on the expression of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in the HepG2.2.15 cell line with low cytotoxicity. One of compounds was studied in HBV transgenic mice model, and the test results showed its ability to reduce the levels of HBsAg, HBeAg and HBV DNA by ELASE and qPCR. Furthermore, significant improvement of T lymphocyte was observed after treatment, as evaluated by flow cytometry (FCM).

Heat shock protein 90-mediated inhibition of hepatitis B virus replication in hepatic cells

To evaluate the effect of heat shock protein 90 (HSP90) on hepatitis B virus (HBV) replication in hepatocytes and to investigate the related molecular mechanism. A eukaryotic plasmid expressing human HSP90 was constructed (designated as HA-HSP90). HepG2 cells were co-transfected with HA-HSP90 and the HBV replicative plasmid HBV1.3. Expression of the exogenous HSP90 was assessed by Western blotting. Expression of the HBV surface antigen (HBsAg) was determined by enzyme-linked immunosorbent assay, and HBV replicative intermediates were detected by Southern blotting. Small interfering (si)RNAs were designed against HSP90 and TBK1 and transfected into the HepG2 cells to further assess the effects of HSP90 and its underlying mechanism. HSP90-mediated effects on the expression of interleukins IL-1b and IL-6 and the interferon response gene IFIT1 were assessed by quantitating mRNA levels with real time RT-PCR. The HA-HSP90 plasmid successfully expressed exogenous HSP90 protein in HepG2 cells. The exogenous HSP90 was able to inhibit HBV replication and HBsAg expression. IFIT1 expression was up-regulated after HA-HSP90 transfection, but neither IL-1b nor IL-6 were affected. The siRNA-mediated TBK1 down-regulation had no effect on the HSP90-inhibited HBV replication. HSP90 can inhibit HBV replication and TBK1 is not involved in this process.