HBeAg In HepG2 Research Articles

Hepatitis B doubly spliced protein (HBDSP) promotes hepatocellular carcinoma cell apoptosis via ETS1/GATA2/YY1-mediated p53 transcription.

Hepatitis B virus (HBV) spliced variants are associated with viral persistence or pathogenicity. Hepatitis B doubly spliced protein (HBDSP), which has been previously reported as a pleiotropic transactivator protein, can potentially serve as an HBV virulence factor. However, the underlying mechanisms of HBDSP in HBV-associated liver diseases remain to be elucidated. In this study, we revealed that HBDSP promotes cellular apoptosis and induces wt-p53-dependent apoptotic signaling pathway in wt-p53 hepatocellular cells by transactivating p53 transcription, and increases the release of HBV progeny. Therefore, HBDSP may promote the HBV particles release through wt-p53-dependent hepatocellular apoptosis. Our findings suggest that blocking HBDSP-induced wt-p53-dependent apoptosis might have therapeutic values for chronic hepatitis B.

Transcriptomics based identification of S100A3 as the key anti-hepatitis B virus factor of 16F16

More than 250 million people worldwide have chronic hepatitis B virus (HBV) infections, resulting in over 1 million annual fatalities because HBV cannot be adequately treated with current antivirals. Hepatocellular carcinoma (HCC) risk is elevated in the presence of the HBV. Novel and powerful medications that specifically target the persistent viral components are needed to remove infection. This study aimed to use HepG2.2.15 cells and the rAAV-HBV1.3 C57BL/6 mouse model established in our laboratory to examine the effects of 16F16 on HBV. The transcriptome analysis of the samples was performed to examine the impact of 16F16 therapy on host factors. We found that the HBsAg and HBeAg levels significantly decreased in a dose-dependent manner following the 16F16 treatment. 16F16 also showed significant anti-hepatitis B effects in vivo. The transcriptome analysis showed that 16F16 regulated the expression of several proteins in HBV-producing HepG2.2.15 cells. As one of the differentially expressed genes, the role of S100A3 in the anti-hepatitis B process of 16F16 was further investigated. The expression of the S100A3 protein significantly decreased following the 16F16 therapy. And upregulation of S100A3 caused an upregulation of HBV DNA, HBsAg, and HBeAg in HepG2.2.15 cells. Similarly, knockdown of S100A3 significantly reduced the levels of HBsAg, HBeAg, and HBV DNA. Our findings proved that S100A3 might be a new target for combating HBV pathogenesis. 16F16 can target several proteins involved in HBV pathogenesis, and may be a promising drug precursor molecule for the treatment of HBV.

Effect of lncRNA pvt1 on HBV replication in HepG2.2.15 cells, and its possible mechanism of action

Purpose: To investigate the influence of lncRNA pvt1 on hepatitis B virus replication in HepG2.2.15 cells and the underlying mechanism of action. 
 Methods: Concentrations of lncRNA pvt1 in HBV-positive liver cancer cell lines (HepAD38+, HepG2.2.15+, HepG2+, and HepaRG+), and 3 HBV-negative liver cancer cell lines were determined and compared. Differences in HBV DNA content, levels of HBsAg and HBeAg, and STAT3 axis signal pathway among groups were evaluated. 
 Results: LncRNA pvt1 levels in HBV-positive hepatoma cells were significantly higher than the corresponding expression levels in HBV-negative hepatoma cells. after si-pvt1 treatment in HepG2.2.15 cells, the 3.2K HBV DNA content increased significantly (p < 0.05), and the expression levels of HBsAg and HBeAg in HepG2.2.15 cells also significantly increased. In all groups, STAT3 protein level was comparable, but p-STAT3 protein expression in si-pvt1 group was significantly reduced, relative to those in Si NC and control groups (p < 0.05). 
 Conclusion: Levels of lncRNA pvt1 expression in HBV-positive and HBV-negative hepatoma cells differ significantly. Treatment with si-pvt1 decrease HBV DNA content in HepG2.2.15 cells through a mechanism that relates to the STAT3 axis signal pathway. This finding may be beneficial for follow-up treatment of HBV.

Effects of lamivudine on cell proliferation of liver cancer and expressions of HBsAg, HBeAg, and MMP-9 in patients.

To explore the effects of lamivudine on cell proliferation of liver cancer and expressions of HBsAg, HBeAg, and MMP-9 in hepatoma cells. In the intervention group, HepG2.2.15 cells were cultured with lamivudine at 100, 200, and 300 μmol/L for 24 hours, 48 hours, and 72 hours. In the control group, HepG2.2.15 cells were cultured without lamivudine. MTT assay was used to assess the proliferative activity of cells after the intervention by lamivudine for 24 hours, 48 hours, and 72 hours. ELISA was used to measure the expression levels of HBsAg, HBeAg, and MMP-9 after the intervention by lamivudine for 48 hours and 72 hours. There was no significant difference between the intervention group and the control group in the proliferation activity of cells (p>0.05). After 48 hours and 72 hours of intervention by lamivudine, the expressions of MMP-9, HBsAg, and HBeAg in the control group were statistically lower than those in the intervention groups with lamivudine at 100 μmol/L, 200 μmol/L, and 300 μmol/L (p<0.05). The expressions of MMP-9, HBsAg, and HBeAg in HepG2.2.15 gradually decreased with the increase of intervention concentration and intervention time of lamivudine (p<0.05). Lamivudine cannot directly inhibit the proliferation of liver cancer cells, but it can reduce the expressions of MMP-9, HBsAg, and HBeAg in hepatoma cells, inhibit the replication of HBV disease in hepatoma cells, and suppress tumor growth.

Structural Elucidation of a Novel Pectin-Polysaccharide from the Petal of Saussurea laniceps and the Mechanism of its Anti-HBV Activity

A novel polysaccharide designated SLP-4 with the Mw of 19681 Da was purified from the petal of Saussurea laniceps. Monosaccharide composition analysis indicated that SLP-4 was composed of mannose, rhamnose, galacturonic acid, glucose, galactose, xylose and arabinose in a molar ratio of 0.825:2.030:14.998:0.841:8.260:4.039:6.009. Structural features indicated that SLP-4 was a typical pectin polysaccharide with a backbone containing →3,6)-Galp-(1→, →4)-GalpA-(1→, →6)-Galp-(1→, →4, 6)-Galp-(1→ and →2, 4)-Rhap-(1→ with the branches of →4)-Galp-(1→, T-Galp-(1→, →3)-Galp-(1→, T-Rhap-(1→, T-Araf-(1→, →5)-Araf-(1→, T-Glcp-(1→, →4)-Xylp-(1→ and →4)-Manp-(1→. Additionally, SLP-4 could effectively inhibit the secretion of HBsAg and HBeAg in HepG2.2.15 cells, but had little effect on the replication of HBV DNA. This inhibition didn’t involve cellular pathways, and was due to the interaction between SLP-4 and HBsAg or HBeAg, which may block the ELISA detection of HBsAg and HBeAg. The present study may provide useful information for further study of SLP-4 and understanding of anti-HBV activity of polysaccharides.

Phenolic Constituents with Antioxidant and Antiviral Activities from Phyllanthus urinaria Linnea

Phyllanthus urinaria Linnea is commonly used in folk medicine with considerable therapeutic utility. Sixteen phenols of Phyllanthus urinaria were isolated from ethanol extract and their structures were identified by spectroscopic data. Oxygen radical absorbance capacity, radical scavenging assay and HBsAg and HBeAg ELISA kits were used to evaluate antioxidant and antiviral activities. All the isolates showed significant antioxidant activity. Especially, isolates 4 and 5 had the oxygen radical absorbance capacity of 5.12 and 8.13 U/mol and strong radical scavenging activity with IC50 values of 5.50 and 5.55 μM, respectively. Additionally, these studies also demonstrated that the compounds 1, 2, 11, 12, and 16 inhibited secretion of HBsAg and HBeAg in HepG2.2.15 cells. This study suggested that the phenolic constituents of Phyllanthus urinaria possessed antioxidant activities and these might play an important role in antihepatitic effects.

Identification of anti-HBV activities in Paeonia suffruticosa Andr. using GRP78 as a drug target on Herbochip\xae

BackgroundHerbochip® technology is a high throughput drug screening platform in a reverse screening manner, in which potential chemical leads in herbal extracts are immobilized and drug target proteins can be used as probes for screening process [BMC Complementary and Alternative Medicine (2015) 15:146]. While herbal medicines represent an ideal reservoir for drug screenings, here a molecular chaperone GRP78 is demonstrated to serve as a potential target for antiviral drug discovery.MethodsWe cloned and expressed a truncated but fully functional form of human GRP78 (hGRP781-508) and used it as a probe for anti-HBV drug screening on herbochips. In vitro cytotoxicity and in vitro anti-HBV activity of the herbal extracts were evaluated by MTT and ELISA assays, respectively. Finally, anti-HBV activity was confirmed by in vivo assay using DHBV DNA levels in DHBV-infected ducklings as a model.ResultsPrimary screenings using GRP78 on 40 herbochips revealed 11 positives. Four of the positives, namely Dioscorea bulbifera, Lasiosphaera fenzlii, Paeonia suffruticosa and Polygonum cuspidatum were subjected to subsequent assays. None of the above extracts was cytotoxic to AML12 cells, but P. cuspidatum extract (PCE) was found to be cytotoxic to HepG2 2.2.15 cells. Both PCE and P. suffruticosa extract (PSE) suppressed secretion of HBsAg and HBeAg in HepG2 2.2.15 cells. The anti-HBV activity of PSE was further confirmed in vivo.ConclusionWe have demonstrated that GRP78 is a valid probe for anti-HBV drug screening on herbochips. We have also shown that PSE, while being non-cytotoxic, possesses in vitro and in vivo anti-HBV activities. Taken together, our data suggest that PSE may be a potential anti-HBV agent for therapeutic use.

Baicalin benefits the anti-HBV therapy via inhibiting HBV viral RNAs

BackgroundAlthough current antiviral treatments (nucleoside analogs, NAs) for chronic hepatitis B virus (HBV) infection are effective in suppressing HBV-DNA replication, their clinical outcomes can be compromised by the increasing drug resistance and the inefficiency in promoting HBsAg/HBeAg seroconversion. ObjectivesIn this study, we will explore possible effects and mechanism of a natural product baicalin (BA) with the anti-HBV efficacy of entecavir (ETV), a first-line anti-HBV drug, in HBV-DNA, HBsAg/HBeAg seroconversion and drug-resistance. MethodsThe co-effects of BA and ETV were conducted in wild-type/NA-resistance mutant HBV cell lines and DHBV-infected duckling models. HBV-DNA/RNAs, HBsAg/HBeAg, host factors (hepatocyte nuclear factors) were explored for possible anti-HBV mechanism. Results and discussionBA could significantly enhance and reduced HBsAg and HBeAg in hepG2.2.15, a wild-type HBV cell line. Co-treatment of BA and ETV had a more dramatic effect in NA-resistant HBVrtM204V/rtLl80M transfected hepG2 cells. Our study further revealed that BA mainly inhibited the production of HBV RNAs (3.5, 2.4, 2.1kb), the templates for viral proteins and HBV-DNA synthesis. BA blocked HBV RNAs transcription possibly by down-regulating transcription and expression of HBV replication dependent hepatocyte nuclear factors (HNF1α and HNF4α). Thus, BA may benefit the anti-HBV therapy via inhibiting HBV viral RNAs.

Anti-HBV activity and mechanism of marine-derived polyguluronate sulfate (PGS) in vitro

Polyguluronate sulfate (PGS) is a low molecular-weight sulfated derivative, which has a structure of 2,3-O-disulfated-1,4-poly-l-guluronic acid (PG) with about 1.5 sulfate per sugar residue. Herein, our results showed that PGS effectively inhibited the expression and secretion of HBsAg and HBeAg in HepG2.2.15 cells. PGS could bind and enter into HepG2.2.15 cells to interfere with HBV transcription rather than blocking HBV DNA replication. Moreover, PGS also enhanced the production and secretion of interferon beta (IFN-β) in HepG2.2.15 cells. Cellular NF-κB and Raf/MEK/ERK signaling pathways were also involved in the anti-HBV actions of PGS. Thus, PGS may inhibit HBV replication through upregulating the NF-κB and Raf/MEK/ERK pathways to enhance the interferon system. In summary, PGS merits further investigation as a novel anti-HBV agent aimed at modulating the host innate immune system in the future.

Study on the Chemical Constituents and anti-HBV Activity ofHypericum japonica

The most common cause of chronic viral hepatitis worldwide is from the Hepatitis B virus (HBV). Screening the safe and effective anti-HBV compounds in traditional Chinese medicine provides a large opportunity for the development of anti-HBV drugs. We found the ethanol extract of H. japonica had the besr inhibitory effects on HBeAg secretion from 20 species of medicinal plants In this study, silica gelSephadex LH-20. and a thin layer of chromatography were used to screen and isolate active anti-hepatitis B virus compounds from Hypericum japonica that was extracted with 60% ethanol. Four compounds were separated from the ethyl acetate extract of H•japonica and their structures were identified by spectral chemical and structural analyses. In addition, using HepG2 2.2.15 cell line with the cytotoxicity and inhibitory effecting on the secretion of HBeAg and HBsAg of the 4 isolated compounds were examined by MTT and enzyme-linked immunosorbent assays. The four isolated compounds were determined and 1,3,6,7-tetrahydroxy-9H-xanthen-9-one (Compound 4) exhibited the highest activity in inhibiting secretion of HBeAg in HepG2 2.2.15cells. The results indicated a 67.66% of inhibition rate, 40.25 μg /mL of IC50 value and TI＞2. We conducted a qualitatively evaluated research using duck hepatitis B virus model in vivo, the results indicated that ethanol extracts of H. japonica had anti-HBV effects and almost no sign of disease rebound after drug withdrawing.But the compound 4 has no direct inhibitory effect on HBV-DNA in duck experiment.

Effect of Radix Trichosanthis and Trichosanthin on Hepatitis B Virus in HepG2.2.15 Cells.

Radix Trichosanthis is a Chinese herbal medicine that has great medical value and pharmacological actions. There is already a long history of using the plant Radix Trichosanthis as treatment for hepatitis B virus in China. This research mainly focused on investigating the therapeutic effect of different extracts from Radix Trichosanthis on hepatitis B virus, on a cellular level (ex vivo). Cell survival rate of HepG2.2.15 cells was detected by MTT assay. HBsAg and HBeAg in HepG 2.2.15 cell supernatant were evaluated by enzyme linked immunosorbent assay (ELISA). Results showed that water extract from Radix Trichosanthis had a stronger inhibitive effect on expression of HBsAg and HBeAg in HepG2.2.15 cells than the alcohol extract from the same plant. Considering that the most active component of Radix Trichosanthis was in its aqueous extract and this might be related to the active component Trichosanthin. Trichosanthin was further used for related experiments to confirm this hypothesis. The results showed that Trichosanthin, in the aqueous extract from Radix Trichosanthis, is likely the main component responsible for the anti-hepatitis B viral effect.

Construction of a eukaryotic vector expressing human miR-155 and inhibitory effect of miR-155 on HBeAg in HepG2.2.15 cells

Construction of a eukaryotic vector expressing human miR-155 and inhibitory effect of miR-155 on HBeAg in HepG2.2.15 cells

Anti-Hepatitis B Virus Effects of Dehydrocheilanthifoline fromCorydalis saxicola

In this report, the anti-hepatitis B virus (HBV) activity of dehydrocheilanthifoline (DHCH), a quaternary ammonium alkaloid isolated from the traditional Chinese medicine Corydalis saxicola Bunting (Papaveraceae), was determined in vitro. Following six days of treatment, DHCH efficiently suppressed the secretions of HBsAg and HBeAg in HepG2.2.15 cell cultures, with a half-maximal inhibitory concentration (IC(50)) of 15.84 and 17.12 μM, and with a therapeutic index (TI) of 7.32 and 6.77, respectively. Further studies revealed that DHCH reduced the levels of extracellular DNA, intracellular DNA and covalently closed circular DNA (cccDNA) of HBV in a dose-dependent and time-dependent manner, with IC(50) values of 15.08, 7.62 and 8.25 μM, respectively after six days of treatment. In contrast, the level of viral pre-genomic RNA (pgRNA) increased 6.13-fold after treatment with DHCH. Together, it was demonstrated for the first time that DHCH could significantly inhibit the replication of HBV, which warrants further studies on the antiviral mechanisms of DHCH, and suggests that it may be a promising candidate in the therapy of HBV infection.

New amide alkaloids from Piper longum

Bioassay-guided phytochemical investigation on Piper longum resulted in the isolation of two new compounds. By analyses of the MS, IR, UV, 1D and 2D NMR data, the two new compounds were identified as 3β, 4α-dihydroxy-1-(3-phenylpropanoyl)-piperidine-2-one (1) and (2E, 4E, 14Z)-6-hydroxyl-N-isobutyleicosa-2,4,14-trienamide (2). In vitro anti-HBV bioassay demonstrated compound 1 possessed remarkable activity suppressing the secretion of HBeAg in Hep G2.2.15 cell line, with an IC50 value of 0.21mM and SI value of 16.4.

Inhibition on hepatitis B virus in vitro of lectin from Musca domestica pupa via the activation of NF-κB

The present study reported that the secretions of HBsAg and HBeAg in HepG2.2.15 cells were significantly decreased under the treatment of lectin from Musca domestica pupa (MPL). Both the replication of hepatitis B virus (HBV) DNA and HBV cccDNA in cells, and the copies of extracellular HBV DNA were inhibited by MPL. The mRNA expressions of interleukin-2 (IL-2), gamma interferon (INF-γ) and MxA were up-regulated by MPL treatments, but down-regulated when nuclear factor-κB (NF-κB) signal pathway was blocked by pyrrolidine dithiocarbamate (PDTC). Subsequent investigation revealed that nuclear factor-κB inhibitory κB (IκB) in endochylema was inhibited and NF-κB was translocated into the nucleus. These findings indicate that MPL could inhibit HBV replication via the induction of the expression of IL-2, INF-γ and MxA through the activation of NF-κB.

PreC/C gene-targeting RNA interference suppresses hepatitis B virus replication and expression in human hepatoma cells

To explore the antiviral efficacy of small interfering RNAs (siRNAs)/shRNA targeting preC/C of HBV in human hepatoma cells Huh-7 and HepG2.2.15 cells. Three 21 nucleotide(nt) siRNAs for treating HBV preC/C gene were designed and synthesized according to the HBV genome in GenBank accession numbers (U95551); simultaneously, one 21-nt-long non-homologous siRNA was also designed randomly for negative control. They were cloned into vector pU6 for constructing shRNA-expressing plasmids pU6-C1, pU6-C2, pU6-C3 and control pU6-C4. To assess the function of siRNAs, a reporter gene system was constructed. The HBV preC/C gene was synthesized by PCR with pT-HBV1.3 as the template. The preC/C gene was then inserted into the enhanced green fluorescent protein expression vector (EGFP-N1) in order to construct the recombinant plasmid pEGFP-preC/C (E-C), which carries the EGFP reporter gene. The three shRNA-expressing plasmids-pU6-C1, pU6-C2, or pU6-C3-was each then cotransfected into Huh-7 cells along with either reporter gene expression vector E-C or the controls; or these three plasmids-pU6-C1, pU6-C2, or pU6-C3-was each cotransfected into HepG2.2.15 cells along with the controls. First, upon determination of the number of cells exhibiting EGFP expression in Huh-7cells as detected by an BH-2 fluorescence microscope and FACS-440 flow cytometry at different times after cotransfection, the investigators evaluated the inhibitory efficiency of the three shRNA-expressing plasmids by an EGFP reporter system in cultured cells. Subsequently, the expression amount of HBsAg and HBeAg in HepG2.2.15 cell supernatant at 24, 48, 72 and 96 h post-cotransfection was detected by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence was used to detect the expression of HBsAg and HBcAg at 72 h post-cotransfection in HepG2.2.15 cells. The copy level of HBV mRNA transcripts cDNA in HepG2.2.15 cells was further investigated through quantitative real-time polymerase chain reaction (real-time PCR). In comparison with single plasmid transfection pEGFP-N1 or E-C, fluorescence microscope examination and flow cytometry detection at 48 hours after cotransfection indicated that the expression of the reporter gene EGFP in cotransfected group Huh-7 cell involving pU6-C1, pU6-C2 or pU6-C3 resulted in an 80% reduction in EGFP signal relative to the controls (P < 0.01). It was also found through immunofluorescence that the expression of HBsAg and HBcAg in HepG2.2.15 cells was reduced markedly (P < 0.01), that the copy level of HBV mRNA transcripts cDNA as detected at 48 hours after cotransfection by quantitative real-time PCR was reduced respectively by 73.9% ± 1.2% (P = 0.029), 48.2% ± 1.8% and 35.8% ± 1.4% (P = 0.037, 0.040) relative to the control, that it conformed with that detected by fluorescence microscope/flow cytometry, ELISA, and immunofluorescence (P < 0.01). Thereby further corroborating the antiviral efficacy of RNAi. The efficacy was obvious at 48 h, reaching a peak at 72 h. For the first time it has been found that RNAi induced by siRNA/shRNA targeting HBV preC/C gene is effective and specific in inhibiting HBV replication and expression in human hepatoma cells Huh-7 and HepG2.2.15 cells. Our data suggest that RNAi may provide an effective, viable approach in gene therapy to treating major infectious diseases such as HBV/HCV/HIV infection.

Based on the low-density cDNA Macroarray for screening of antiviral proteins of IFNa tissues

To screen the gene expression profiles of IFN-alpha antiviral proteins based on a low-density cDNA Macroarray, and to explore the relationship between the expression of antiviral protein and the HBV replication. The HepG2 and HepG2.2.15 cells were treated with various concentrations of IFN-alpha (0 IU/ml, 100 IU/ml, 1000 IU/ml) of IFN-alpha for 6 h, and then the low-density cDNA Macroarray was used for analysing the expression profiles of antiviral genes and screening differential expressions of antiviral proteins. Meanwhile, the HepG2 cells were transiently transfected with HBV core protein-expressed plasmid pHBc-EGFP, and the expressions of antiviral proteins were analysed by RT-PCR assay. Moreover, the HepG2.2.15 cells were also transfected with the antiviral protein-expressed plasmid pcDNA3.1-Flag-MxA. ELISA was used for analysing the secreted HBV antigens, while dot blot and Southern blot were applied for analysing the extracellular HBV DNA and intracellular replicative intermediate HBV DNA in HepG2.2.15 cells. All data were presented as mean+/-SD and analyzed using the t-test and one-way analysis of variance (ANOVA) in the experiments. The Macroarray results suggested that the expression of IFN-alpha antiviral genes like 6-16, IFITM1, IFITM2, IFITM3 and RING4 in HepG2.2.15 cells were partially inhibited. More importantly, it was found, in this research, the expression of antiviral protein MxA in HepG2.2.15 cells was completely suppressed. RT-PCR analysis indicated that the expression of MxA was also significantly decreased in HepG2 cells transfected with pHBc-EGFP plasmid. Although HepG2.2.15 cells transfected with pcDNA3.1-Flag-MxA plasmid could not inhibit extracellular HBV DNA and intracellular replicative intermediate HBV DNA, the MxA exerted some antiviral activities as it effectively suppressed the secretion of HBsAg and HBeAg in HepG2.2.15 cells. HBV and its antigen components probably influence the expression of antiviral proteins. IFN- resistance may be related to the down-regulation of antiviral proteins expression.

Effects of 1,2,4,6-tetra-O-galloyl-β-D-glucose from P. emblica on HBsAg and HBeAg secretion in HepG2.2.15 cell culture

A polyphenolic compound, 1,2,4,6-tetra-O-galloyl-β-D-glucose (1246TGG), was isolated from the traditional Chinese medicine Phyllanthus emblica L. (Euphorbiaceae) and assayed for its potential as an anti-hepatitis B virus (HBV) agent. The cytotoxicity of 1246TGG on HepG2.2.15 as well as HepG2 cells was determined by observing cytopathic effects, and the effects of 1246TGG on secretion of HBsAg and HBeAg in HepG2.2.15 cells were assayed by enzyme immunoassay. Results indicates that treatment with 1246TGG (6.25 μg/mL, 3.13 μg/mL), reduced both HBsAg and HBeAg levels in culture supernatant, yet the inhibitory effects tend to decline with the assay time. This study provides a basis for further investigation of the anti-HBV activity and possible mechanism of action of 1246TGG.

Adeno-associated virus Rep78 protein inhibits Hepatitis B virus replication through regulation of the HBV core promoter

Rep78, the rep gene product of adeno-associated virus (AAV), has been shown to inhibit the replication of several DNA viruses. This study investigated the effects of Rep78 on replication of Hepatitis B virus (HBV) and possible mechanisms of inhibition. We have shown that HBV DNA replication and secretion of HBsAg and HBeAg in HepG2 2.2.15 cells were inhibited by Rep78. We have also demonstrated, using in vitro transcription and luciferase assay, that Rep78 binds to the HBV core promoter (HBV CP) and inhibits HBV CP activity. Furthermore, after Rep78 and HBV core protein expression plasmids were co-transfected into HepG2 cells, the expression of HBV core protein was inhibited significantly. These results suggest that Rep78 can inhibit the replication of HBV, correlating strongly with suppression of HBV CP activity.

An earthworm protease cleaving serum fibronectin and decreasing HBeAg in HepG2.2.15 cells

BackgroundVirus-binding activity is one of the important functions of fibronectin (FN). It has been reported that a high concentration of FN in blood improves the transmission frequency of hepatitis viruses. Therefore, to investigate a protease that hydrolyzes FN rapidly is useful to decrease the FN concentration in blood and HBV infection. So far, however, no specific protease digesting FN in serum has been reported.MethodsWe employed a purified earthworm protease to digest serum proteins. The rapidly cleaved protein (FN) was identified by MALDI-TOF MS and western blotting. The cleavage sites were determined by N-terminus amino acid residues sequencing. The protease was orally administrated to rats to investigate whether serum FN in vivo became decreased. The serum FN was determined by western blotting and ELISA. In cytological studies, the protease was added to the medium in the culture of HepG2.2.15 cells and then HBsAg and HBeAg were determined by ELISA.ResultsThe protease purified from earthworm Eisenia fetida was found to function as a fibronectinase (FNase). The cleavage sites on FN by the FNase were at R and K, exhibiting a trypsin alkaline serine-like function. The earthworm fibronectinase (EFNase) cleaved FN at four sites, R259, R1005, K1557 and R2039, among which the digested fragments at R259, K1557 and R2039 were related to the virus-binding activity as reported. The serum FN was significantly decreased when the earthworm fibronectinase was orally administrated to rats. The ELISA results showed that the secretion of HBeAg from HepG2.2.15 cells was significantly inhibited in the presence of the FNase.ConclusionThe earthworm fibronectinase (EFNase) cleaves FN much faster than the other proteins in serum, showing a potential to inhibit HBV infection through its suppressing the level of HBeAg. This suggests that EFNase is probably used as one of the candidates for the therapeutic agents to treat hepatitis virus infection.