Core promoter: A critical region where the hepatitis B virus makes decisions

The core promoter(CP) of the viral genome plays an important role for hepatitis B virus(HBV) replication as it directs initiation of transcription for the synthesis of both the precore and pregenomic(pg) RNAs. The CP consists of the upper regulatory region and the basa core promoter(BCP). The CP overlaps with the 3'-end of the X open reading frames and the 5'-end of the precore region,and contains cis-acting elements that can independently direct transcription of the precore mRNA and pgRNA. Its transcription regulation is under strict control of viral and cellular factors. Even though this regulatory region exhibits high sequence conservation,when variations appear,they may contribute to the persistence of HBV within the host,leading to chronic infection and cirrhosis,and eventually,hepatocellular carcinoma. Among CP sequence variations,those occurring at BCP may dysregulate viral gene expression with emphasis in the hepatitis B e antigen,and contribute to disease progression. In this review these molecular aspects and pathologic topics of core promoter are deeply evaluated.

Precore/basal core promoter mutants quantification throughout phases of hepatitis B virus infection by Simpleprobe

AIM:To investigate precore/basal core promoter(PC/BCP) mutants throughout hepatitis B virus(HBV) infection and to determine their relationship to hepatitis B early antigen(HBeA g) titers.METHODS:We enrolled 191 patients in various stages of HBV infection at the Huashan Hospital and the Taizhou Municipal Hospital from 2010 to 2012.None of the patients received antiviral therapy.HBV DNA from serum,was quantified by real-time PCR.The HBV genotype was determined by direct sequencing of the S gene.We used the Simpleprobe ultrasensitivequantitative method to detect PC/BCP mutants in each patient.We compared the strain number,percentage,and the changes in PC/BCP mutants in different phases,and analyzed the relationship between PC/BCP mutants and HBe Ag by multiple linear regression and logistic regression.RESULTS:Patients with HBV infection(n = 191) were assigned to groups by phase:Immune tolerance(IT) = 55,Immune clearance(IC) = 67,Low-replicative(LR) = 49,and HBeA g-negative hepatitis(ENH) = 20.Of the patients(male,112; female,79) enrolled,122 were HBe Ag-positive and 69 were HBe Ag-negative.The median age was 33 years(range:18-78 years).PC and BCP mutation detection rates were 84.82%(162/191) and 96.86%(185/191),respectively.In five HBe Ag-negative cases,we detected double mutation G1896A/G1899 A.The logarithm value of PC mutant quantities(log10 PC) significantly differed in IT,IC,and LR phases,as well as in the ENH phase(F = 49.350,P < 0.001).The logarithm value of BCP mutant quantities(log10 BCP) also differed during the four phases(F = 25.530,P < 0.001).Log10 PC and log10 BCP values were high in the IT and IC phases,decreased in the LR phase,and increased in the ENH phase,although the absolute value at this point remained lower than that in the IT and IC phases.PC mutant quantity per total viral load(PC%) and BCP mutant quantity per total viral load(BCP%) differed between phases(F = 20.040,P < 0.001; F = 10.830,P < 0.001),with PC% and BCP% gradually increasing in successive phases.HBeA g titers negatively correlated with PC%(Spearman's rho =-0.354,P < 0.001) and BCP%(Spearman's rho =-0.395,P < 0.001).The negative correlation between PC% and HBeA g status was significant(B =-5.281,P = 0.001),but there was no such correlation between BCP% and HBeA g status(B =-0.523,P = 0.552).CONCLUSION:PC/BCP mutants become predominant in a dynamic and continuous process.Log10 PC,log10 BCP,PC% and BCP% might be combined to evaluate disease progression.PC% determines HBeA g status.

Review on hepatitis B virus precore/core promoter mutations and their correlation with genotypes and liver disease severity

Of 350 million people worldwide are chronically infected with hepatitis B virus(HBV)and are at risk of developing cirrhosis and hepatocellular carcinoma(HCC)later in life.HBV is the most diverse DNA virus,and its genome is composed of four open reading frames:Presurface antigen/surface antigen gene(preS/S),precore/core gene(preC/C),polymerase gene(P),and theχgene(χ).HBV produces quasispecies naturally or in response to antiviral agents because of the absence of proofreading activity amid reverse transcription and a high replication rate.The virus has 10 genotypes(A to J)with different geographical distributions.There are various HBV mutations in the HBV genome,including preC/C mutations,preS/S mutations,P gene mutations,andχgene mutations.The core promoter region plays a vital part in the replication,morphogenesis and pathogenesis of the virus.The precore region also plays a crucial role in viral replication.Both core promoter and precore mutations rescue the virus from host immune surveillance and result in the formation of mutated strains that may have altered pathogenicity.preC/C mutations are associated with liver disease progression.Precore mutations stop hepatitis B e antigen(HBeAg)production and basal core promoter mutations downregulate HBeAg production.Mutations in the basal core promoter are also associated with increased HBV replication and an increased incidence of advanced liver diseases such as cirrhosis and HCC.The emergence of antiviral-resistant mutations is the main reason for treatment failure.This review focuses mainly on preC/C promoter mutations and their correlation with genotypes and liver disease severity.Thorough perception and knowledge of HBV genetic variety and mutants could be vital to discover techniques for the prognosis and control of HBV infection.

Association of core promoter mutations of hepatitis B virus and viral load is different in HBeAg(+) and HBeAg(-) patients

AIM:To identify the prevalence of hepatitis B e antigen (HBeAg) and to assess the association of hepatitis B virus (HBV) core promoter mutations and viral load in Indonesian patients.METHODS:Sixty-four patients with chronic hepatitis,65 with liver cirrhosis and 50 with hepatocellular carcinoma were included in this study.HBeAg and hepatitis B e antibody (HBeAb) tests were performed using enzyme-linked immunosorbent assay and the mutations were analyzed by sequencing.Viral load was measured by real-time polymerase chain reaction.RESULTS:Of 179 patients,108 (60.3%) were HBeAg(-) and 86 (79.6%) of these HBeAg(-) patients had been seroconverted.The A1896 mutation was not found in HBeAg(+) patients,however,this mutation was detected in 70.7% of HBeAg(-) patients.This mutation was frequently found when HBeAg was not expressed (87.7%),compared to that found in HBeAg seroconverted patients (65.1%).The A1899 mutation was also more prevalent in HBeAg(-) than in HBeAg(+) patients (P=0.004).The T1762/A1764 mutation was frequently found in both HBeAg(+) and HBeAg(-) patients,however,the prevalence of this mutation did not significantly differ among the two groups (P=0.054).In HBeAg(+) patients,the T1762/A1764 mutation was correlated with lower HBV DNA (P < 0.001).The A1899 mutation did not correlate with HBV DNA (P=0.609).In HBeAg(-) patients,the T1762/A1764 mutation alone was not correlated with HBV DNA (P=0.095),however,the presence of either the T1762/A1764 or A1896 mutations was associated with increased HBV DNA (P < 0.001).CONCLUSION:The percentage of HBeAg(-) patients is high in Indonesia,and most of the HBeAg(-) patients had been seroconverted.The A1896 mutation was most likely the major cause of HBeAg loss.The T1762/A1764 mutation alone was associated with lower viral loads in HBeAg(+) patients,but not in HBeAg(-) patients.

Quasispecies groups in the core promoter region of hepatitis B virus

Objectives: To investigate the mutation of the basic core promoter (BCP) of hepatitis B virus (HBV) and clarify the significance of HBV quasispecies groups in patients with chronic HBV infection. Methods: A set of specific primers was synthesized according to the HBV DNA sequence of a Chinese strain. The BCP was amplified by PCR method from the serum of 40 patients with chronic HBV infection, and the PCR products of 2 patients were subcloned into pGEM Teasy vectors. Polyacrylamide gel elec- trophoresis (PAGE) was employed to display the de- letion mutations, and clones with differential length were selected to be sequenced. Sequence comparison was made to find the difference. Results: Two or three bands were displayed by PAGE in 60% patients. The results of sequence anal- ysis showed that there are some kinds of mutations in the BCP region. The substitution always occurs in TATA-like boxes, especially from T to C on 140 site. The deletion mutations were detected in TA1, TA2 and TA3. The 8bp, 20bp deletion mutations fre- quently happened. Conclusions: There is a hot deletion region in the BCP. The deletion and the substitution in the TATA- like box may influence the expression of preC/C pro- tein. The sequencing results indicate that there are HBV quasispecies groups in patients with chronic HBV infection.

Specific activation of 2'-5'oligoadenylate synthetase gene promoter by hepatitis C virus-core protein:A potential for developing hepatitis C virus targeting gene therapy

AIM： TO examine whether 2＇-5＇oligoadenylate synthetase （OAS） gene promoter can be specifically activated by hepatitis C virus （HCV）-core protein. METHODS： Human embryo hepatic cell line L02 was transfected with pcDNA3.1-core plasmid and selected by G418. Expression of HCV-core was detected by reverse transcription polymerase chain reaction and Western blotting. The OAS promoter sequence was amplified from the genomic DNA and inserted into pGL3-basic vector. The resultant pGL3-OAS-Luci plasmid was transiently transfected into L02/core cells and luciferase activity was assayed. I^ESULTS： L02/core cell line stably expressing HCV- core protein was established. The pGL3-OAS-Luci construct exhibited significant transcriptional activity in the L02/core cells but not in the L02 cells. CONCLUSION： HCV-core protein activates the OAS gene promoter specifically and effectively. Utilization of OAS gene promoter would be an ideal strategy for developing HCV-specific gene therapy.

Advances in discovery of novel investigational agents for functional cure of chronic hepatitis B:A comprehensive review of phases II and III therapeutic agents

Chronic hepatitis B virus(HBV)infection affects over 295 million people globally and an estimated 1.6 million people in the United States.It is associated with significant morbidity and mortality due to cirrhosis,liver failure,and liver cancer.Antiviral therapy with oral nucleos(t)ide analogues is associated with high rates of virologic suppression,which in turn has been associated with a decreased risk of liver complications.However,current antiviral regimens are limited by concerns with adverse effects,adherence,resistance,long-term treatment,and ongoing risk for liver events.Novel investigational agents are currently in development and are targeted at achieving functional cure with sustained hepatitis B surface antigen(HBsAg)loss and suppression of HBV DNA.Herein we review key evidence from phases II and III trials defining the efficacy and safety profiles for key investigational agents for functional cure of chronic hepatitis B,including core/capsid inhibitors,entry inhibitors,RNA interference(siRNA/ASO),HBsAg inhibitors,Toll-like receptor agonists,checkpoint inhibitors,and therapeutic vaccines.

Precore/core region mutations of hepatitis B virus related to clinical severity

Despite the availability of an effective vaccine, hepatitis B virus (HBV) infection remains a major health problem, with more than 350 million chronically infected people worldwide and over 1 million annual deaths due to cirrhosis and liver cancer. HBV mutations are primarily generated due both to a lack of proofreading capacity by HBV polymerase and to host immune pressure, which is a very important factor for predicting disease progression and therapeutic outcomes. Several types of HBV precore/core (preC/C) mutations have been described to date. The host immune response against T cells drives mutation in the preC/C region. Specifically, preC/C mutations in the MHC class II restricted region are more common than in other regions and are significantly related to hepatocellular carcinoma. Certain mutations, including preC G1896A, are also significantly related to HBeAg-negative chronic infection. This review article mainly focuses on the HBV preC/C mutations that are related to disease severity and on the HBeAg serostatus of chronically infected patients.

Precore/basal core promoter mutants and hepatitis B viral DNA levels as predictors for liver deaths and hepatocellular carcinoma

AIM： To conduct a retrospective study in 400 chronic hepatitis B patients in order to identify hepatitis B viral factors associated with complications of liver disease or development of hepatocellular carcinoma. METHODS： The mean follow-up time was 83.6 ± 39.6 mo. Alpha-fetoprotein test and abdominal ultrasound were used for cancer surveillance. Hepatitis B basal core promoter mutants, precore mutants, genotypes, hepatitis B viral DNA （HBV DNA） level and hepatitis B e antigen （HBeAg） were measured. Univariate analysis and logistic regression were used to assess odds ratios for viral factors related to liver deaths and hepatocellular carcinoma development. RESULTS： During follow-up, 38 patients had liver deaths not related to hepatocellular carcinoma. On multivariate analysis, older age [odds ratio： 95.74 （12.13-891.31）, P 〈 0.0001], male sex [odds ratio： 7.61 （2.20-47.95）; P = 0.006], and higher Iogzo HBV DNA [odds ratio： 4.69 （1.16-20.43）; P 〈 0.0001] were independently predictive for these liver related deaths. Also, 31 patients developed hepatocellular carcinoma. Multivariate analysis showed that older age [odds ratio： 26.51 （2.36-381.47）; P = 0.007], presence of precore mutants [odds ratio： 4.23 （1.53-19.58）, P = 0.02] and presence of basal core promoter mutants [odds ratio： 2.93 （1.24-7.57）; P = 0.02] were independent predictors for progression to hepatocellular carcinoma. CONCLUSION： Our results show that high levels of baseline serum HBV DNA are associated with non- hepatocellular carcinoma-related deaths of liver failure, while genetic mutations in the basal core promoter and precore regions are predictive for development of hepatocellular carcinoma.

Safety of hepatitis B virus core antibody-positive grafts in liver transplantation: A single-center experience in China

BACKGROUND Given the shortage of suitable liver grafts for liver transplantation, proper use of hepatitis B core antibody-positive livers might be a possible way to enlarge the donor pool and to save patients with end-stage liver diseases. However, the safety of hepatitis B virus core antibody positive(HBcAb+) donors has been controversial. Initial studies were mainly conducted overseas with relatively small numbers of HBcAb+ liver recipients, and there are few relevant reports in the population of China's Mainland. We hypothesized that the safety of HBcAb+ liver grafts is not suboptimal.AIM To evaluate the safety of using hepatitis B virus(HBV) core antibody-positive donors for liver transplantation in Chinese patients.METHODS We conducted a retrospective study enrolling 1071 patients who underwent liver transplantation consecutively from 2005 to 2016 at West China Hospital Liver Transplantation Center. Given the imbalance in several baseline variables, propensity score matching was used, and the outcomes of all recipients were reviewed in this study.RESULTS In the whole population, 230 patients received HBcAb+ and 841 patients received HBcAb negative(HBcAb-) liver grafts. The 1-, 3-and 5-year survival rates in patients and grafts between the two groups were similar(patient survival: 85.8% vs 87.2%, 77.4% vs 81.1%, 72.4% vs 76.7%, log-rank test, P = 0.16; graft survival: 83.2% vs 83.6%, 73.8% vs 75.9%, 70.8% vs 74.4%, log-rank test, P = 0.19). After propensity score matching, 210 pairs of patients were generated. The corresponding 1-, 3-and 5-year patient and graft survival rates showed no significant differences. Further studies illustrated that the post-transplant major complication rates and liver function recovery after surgery were also similar. In addition, multivariate regression analysis in the original cohort and propensity score-matched Cox analysis demonstrated that receiving HBcA b+ liver grafts was not a significant risk factor for long-term survival. These findings were consistent in both HBV surface antigen-positive(HBsAg+) and HBsA g negative(HBsAg-) patients.Newly diagnosed HBV infection had a relatively higher incidence in HBsAg-patients with HBcAb+ liver grafts(13.23%), in which HBV naive recipients suffered most(31.82%), although this difference did not affect patient and graft survival(P = 0.50 and P = 0.49, respectively). Recipients with a high HBV surface antibody(anti-HBs) titer(more than 100 IU/L) before transplantation and antiviral prophylaxis with nucleos(t)ide antiviral agents post-operation, such as nucleos(t)ide antiviral agents, had lower de novo HBV infection risks. CONCLUSION HBcA b+ liver grafts do not affect the long-term outcome of the recipients. Combined with proper postoperative antiviral prophylaxis, utilization of HBcAb+ grafts is rational and feasible.

Interleukin-10 promoter polymorphisms in patients with hepatitis B virus infection or hepatocellular carcinoma in Chinese Han ethnic population

BACKGROUND: Since single nucleotide polymorphisms (SNPs) can serve as gene markers, polymorphism profiles may help scientists to identify the full collection of genes that contribute to the development of complex diseases such as cancer. The distribution of interleukin-10 (IL-10) promoter polymorphisms in Chinese Han ethnic patients with hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC) was investigated in this study. METHODS: The polymorphisms of IL-10 promoter region were detected by pulymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing. Sixty-six health controls, 42 patients with HBV infection, 30 HCC patients, and cell line SMMC-7721 were examined this way. RESULTS: Polyrnorphisms of T/C or T/N on-872 site occurred frequently in Han ethnic population. Pulyrnorphisms were detected in HBV and HCC patients and cell line SMMC-7721. The hotspot among the pulymorphisms was inserting base A between-1058 and-1057. CONCLUSION: Polymorphisms of IL-10 promoter in HBV and HCC patients may be associated with HBV infection and HCC development.

Enhancement of CTLs induced by DCs loaded with ubiquitinated hepatitis B virus core antigen

AIM： To investigate whether hepatitis B virus （HBV） could induce a hepatitis B virus core antigen （HBcAg）specific cytotoxic T lymphocyte （CTL） response in vitro by dendritic cells （DCs） transduced with lentiviral vector-encoding ubiquitinated hepatitis B virus core antigen （LV-Ub-HBcAg）.METHODS： Recombinant LV-Ub-HBcAg were transfected into highly susceptible 293 T cells to obtain high virus titres, Bone marrow-derived DCs isolated from BALB/c mice were cultured with recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleukin （IL）-4. LV-Ub-HBcAg, lentiviral vector-encoding hepatitis B virus core antigen （LV-HBcAg）, lentiviral vector （LV） or lipopolysaccharide were added to induce DC maturation, and the DC phenotypes were analyzed by flow cytometry. The level of IL-12 in the supernatant was detected by enzyme-linked immunosorbent assay （ELISA）. T lymphocytes were proliferated using Cell Counting Kit-8. DCs were cultured and induced to mature using different LVs, and co-cultured with allogeneic T cells to detect the secretion levels of IL-2, IL-4, IL-10and interferon-γ in the supernatants of T cells by ELISA. Intracellular cytokines of proliferative T cells were analyzed by flow cytometry, and specific CTL activity was measured by a lactate dehydrogenase release assay.RESULTS： LV-Ub-HBcAg-induced DCs secreted more IL-12 and upregulated the expression of CD80, CD86 and major histocompatibility class ]I, DCs sensitised by different LVs effectively promoted cytokine secretion; the levels of IL-2 and interferon-y induced by LV-Ub- HBcAg were higher than those induced by LV-HBcAg, Compared with LV-HBcAg-transduced DCs, LV-Ub- HBcAg-transduced DCs more efficiently stimulated the proliferation of T lymphocytes and generated HBcAgspecific cytotoxic T lymphocytes.CONCLUSION： LV-Ub-HBcAg effectively induced DC maturation. The mature DCs efficiently induced T cell polarisation to Thl and generated HBcAg-specific CTLs.

Biological impact of hepatitis B virus X-hepatitis C virus core fusion gene on human hepatocytes

AIM： To investigate the biological impact of hepatitis B virus X- hepatitis C virus core （HBV X-HCV C） fusion gene on hepatoma cells.METHODS： The recombinant adenoviruses Ad- XC, Ad-X and Ad-C expressing HBV X-HCV C fusion gene, HBVX gene and HCV C gene were constructed, respectively. Hepatoma cells were infected with different recombinant adenoviruses. MTT, colony- forming experiment, FCM, TUNEL assay were performed to observe the biological impact of the HBV X-HCV C fusion aene on liver cells.RESULTS： MTT showed that the Ad-XC group cells grew faster than the other group cells. Colony-forming experiment showed that the colony-forming rate for the Ad-XC group cells was significantly higher than that for the other group cells. FCM analysis showed that Ad-XC/Ad-X/Ad-C infection enhanced the progression of G1→S phase in the HepG2 cell cycle. The apoptosis index of the Ad-XC, Ad-X, Ad-C group cells was significantly lower than that of the AdO and control group cells. Semi-quantitative RT-PCR showed that the expression level of c-myc was the highest in Ad- XC infected cells. Tumor formation was found at the injected site of mice inoculated with Ad-XC-infected LO2 cells, but not in control mice.CONCLUSION： Ad-XC, Ad-X and Ad-C facilitate the proliferation activity of HepG2 cells and inhibit their apoptosis in vitro. The effect of Ad-XC is significantly stronger than that of Ad-X and Ad-C. Up-regulation of c-myc may be one of the mechanisms underlying the synergism of HBVX and HCV C genes on hepatocarcinogenesis in athymic nude mice.

Novel DNA vaccine based on hepatitis B virus core gene induces specific immune responses in Balb/c mice

AIM： To investigate the immunogenidty of a novel DNA vacoine, pSW3891/HBc, based on HBV core gene in Balb/c mice. METHODS： A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plasmid pSW3891. Balb/c mice were immunized with either pSW3891/HBc or empty vector DNA via gene gun. IgG anti-HBc responses in mouse sera were demonstrated by ELISA. Specific cytotoxicity of cytotoxic T lymphocytes （CTLs） of mice was quantitatively measured by lactate dehydrogenase release assay. RESULTS： HBcAg was expressed effectively in 293T cell line transiently transfected with pSW3891/HBc. Strong IgG anti-HBc responses were elicited in mice immunized with pSW3891/HBc. The end-point titers of anti-HBc reached the highest 1：97 200, 4 wk after the third immunization. The specific CTL killing with the highest specific lysis reached 73.25% at effector：target ratio of 20：1 in mice that received pSW3891/HBc DNA vaccine. CONCLUSION： pSW3891/HBc vaccination elicits specific anti-HBc response and induces HBc-specific CTL response in immunized Balb/c mice.

Mutations in hepatitis B virus core regions correlate with hepatocellular injury in Chinese patients with chronic hepatitis B

AIM: To elucidate the relationship between the frequency of core mutations and the clinical activity of hepatitis B virus (HBV)-related liver disease and to characterize the amino acid changes in the core region of HBV.METHODS: We studied 17 Chinese patients with chronic hepatitis B according to their clinical courses and patterns of the entire core region of HBV.RESULTS: Amino acid changes often appeared in the HBV core region of the HBV gene in patients with high values of alanine aminotransferase (ALT) or with the seroconversion from HbeAg to anti-HBe. The HBV core region with amino acid changes had high frequency sites that corresponded to HLA Ⅰ/Ⅱ restricted recognition epitopes reported by some investigators.CONCLUSION: The core amino acid changes of this study occur due to influence of host immune system. The presence of mutations in the HBV core region seems to be important for predicting the clinical activity of hepatitis B in Chinese patients.

Effects of two novel nucleoside analogues on different hepatitis B virus promoters

AIM： To explore the effects of the nucleoside analogues β-L-D4A and β-LPA on hepatitis B virus （HBV） promoters. METHODS： Four HBV promoters were amplified by polymerase chain reaction （PCR） and subcloned into the expression vector pEGFP-1. The four recombinants controlled by HBV promoters were confirmed by restriction analysis and sequencing. Human hepatoma HepG2 cells transfected with the recombinant plasmids were treated with various concentrations of β-L-D4A and β-LPA. Then, enhanced green fluorescent protein （EGFP）-positive cells were detected by fluorescence microscopy and using a fluorescence activated cell sorter RESULTS： Four HBV promoters were separately obtained and successfully cloned into pEGFP-1, Expression of EGFP under the control of the surface promoter （Sp） and the X promoter （Xp） was inhibited by β-L-D4A in a dosedependent manner, while expression of EGFP under the control of the core promoter （Cp） and Xp was inhibited by β-LPA in a dose-dependent manner. CONCLUSION： The two novel nucleoside analogues investigated here can inhibit the activities of HBV promoters in a dose-dependent manner. These findings may explain the mechanisms of action by which these two novel compounds inhibit HBV DNA replication.

Screening and identification of interacting proteins with hepatitis B virus core protein in leukocytes and cloning of new gene C1

AIM： To investigate the biological function of HBcAg in pathogenesis of HBV replication in peripheral blood mononuclear cells （PBMCs）.METHODS： HBcAg region was amplified by polymerase chain reaction （PCR） and HBV HBcAg bait plasmid pGBKT7-HBcAg was constructed by routine molecular biological methods. Then the recombinant plasmid DNA was transformed into yeast AH109. After the HBV core protein was expressed in AH109 yeast strains （Western blot analysis）, yeast-two hybrid screening was performed by mating AH109 with Y187 containing leukocyte cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium （SD/-Trp-Leu-His- Ade） （QDO） and synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade） （TDO）. The second screening was performed with the LacZ report gene （ yeast cells were grown in QDO medium containing X-a-gal）. The interaction between HBV core protein and the protein obtained from positive colonies was further confirmed by repeating yeast-two hybrid. After plasmid DNA was extracted from blue colonies and sequenced, the results were analyzed by bioinformatic methods.RESULTS： Eighteen colonies were obtained and sequenced, including hypermethylated in cancer 2 （3 colones）, eukaryotic translation elongation factor 2 （2 colones）, acetyl-coenzyme A synthetase 3 （1 colone）, DNA polymerase gamma （1 colone）, putative translation initiation factor （1 colone）, chemokine （C-C motif） receptor 5 （1 colone）, mitochondrial ribosomal protein L41 （1 colone）, kyot binding protein genes （1 colone）, RanBPM （1 colone）, HBeAg-binding protein 3 （1 colone）, programmed cell death 2 （1 colone）. Four new genes with unknown function were identified.CONCLUSION： Successful cloning of genes of HBV core protein interacting proteins in leukocytes may provide some new clues for studying the biological functions of HBV core protein.

Hepatitis B virus X protein regulates the mEZH2 promoter via the E2F1-binding site in AML12 cells

Histone lysine methyltransferase EZH2 has been reported to be frequently overexpressed in hepatocellular carcinoma(HCC) tissues and associated with hepatocarcinogenesis.However,the exact mechanism of EZH2 up-regulation in HCC has not been determined.In this study,we used murine hepatocyte AML12 cells to investigate the role of hepatitis B virus X protein(HBx) in regulating the expression of mEZH2.Western blot analysis demonstrated that the expression level of mEZH2 protein in AML12 cells was up-regulated by HBx in a dose-dependent manner.To further investigate the mechanism of mEZH2 overexpression,the 2500 bp regulatory sequence upstream from the first exon of the mEZH2 gene was amplified from AML12 genomic DNA and constructed into a luciferase reporter plasmid.The luciferase activity of the mEZH2 promoter significantly increased in AML12 cells co-transfected with HBx plasmid,and deleting the-486/-214 promoter region decreased HBx-induced mEZH2 promoter activation by nearly 50%.The-486/-214 region was then analyzed in the TRANSFAC 6.0 database and a typical E2F1-binding site was found.Mutation of this E2F1-binding site or knockdown of E2F1 expression by RNAi led to a dramatic decrease in HBx-induced activation of the mEZH2 promoter and mEZH2 overexpression in AML12 cells.These results provide evidence that HBx up-regulates mEZH2 expression by transactivating the mEZH2 promoter through E2F1 transcription factor,thereby providing new epigenetic evidence for the carcinogenic effect of HBx.

Cloning and characterization of a novel hepatitis B virus core binding protein C12

AIM： To elucidate the biological function of HBV core antigen （HBcAg） on pathogenesis of hepatitis B, a novel gene C12 coding for protein with unknown function interacting with HBcAg in hepatocytes was identified and characterized. METHODS： HBcAg bait plasmid pGBKT7-HBcAg was constructed and transformed into yeast AH109, then the transformed yeast was mated with yeast Y187 containing liver complementary DNA （cDNA） library plasmid in 2xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade） and synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade） containing X-α-gal for screening twice. After extracting and sequencing of plasmid from blue colonies, we isolated a cDNA clone encoding a novel protein designated as C12 that directly interacted with HBcAg. The interaction between HBcAg and C12 was verified again by re-mating. pEGFP-N1-C12 fluorescent protein fusion gene was transfected in 293 and L02 cell, and observed by fluorescent microscope. M-FI reduction assay was used to study the action of C12 protein effect on metabolism of mammal cell. Yeast two-hybrid and cDNA microarray were performed to search binding protein and differential expression genes regulated by C12 protein. RESULTS： C12 gene was screened and identified by yeast two-hybrid system 3. The interaction between HBcAg and the novel protein coded by the new gene C12 was further confirmed by re-mating. After 48 h, fluorescence of fusion protein could be observed steadily in the 293 and L02 cell plasma. Under MTT assay, we found that the expression of C12 did not influence the growth of liver cells. Seventeen differential expression genes in HepG2 cells transfected with C12 protein expression plasmid by cDNA microarray, of which 16 genes were upregulated and 1 gene was downregulated by C12 protein. Twenty-one colonies containing 16 different genes coding for C12 protein binding proteins were isolated by yeast two-hybrid, there were 2 new genes with unknown function. CONCLUSION： The novel protein C12 is located in cell plasma, and its overexpression has no significant effect on the metabolism of liver cell. It interacts with many proteins in hepatocytes and may be involved in many processes of gene expression. 2005 The W.IG Press and Elsevier Inc. All rights reserved

Hepatitis B Virus S Promoter Deletion in Hepatocellular Carcinoma

Objective To identify the difference and significance of dominant types of hepatitis B virus(HBV) pre-S mutation between liver tumor tissues and paired adjacent non-tumor tissues and to test if the mutations were tumor tissue specific.Methods HBV DNA isolated from 34 paired intratumoral and peritumoral tissues of Hepatocellular Carcinoma(HCC) patients were screened by PCR and direct sequencing. All patients carried HBV with genotype C, except for one B/C heterozygote. The expression, localization and excretion of LHBs mutant carrying pre-S deletions were characterized in vitro. The expression of endoplasmic reticulum(ER) GRP78 m RNA was assayed.Results Four patterns of pre-S mutations were identified: pre-S1 in-frame deletion involving the first start codon; pre-S2 in-frame deletion; pre-S2 start codon mutation with or without in-frame deletion; and S promoter in-frame deletion(ΔSP). The first two types were evenly found in both tumor and non-tumor tissues. They were rarely present as dominant strains. The last two types were frequently found in the dominant strains in tumor tissues. The overall prevalence of HBV carrying ΔSP was 17.64%(6/34) in tumor tissues, but none were dominant in non-tumor tissues. HBV carrying ΔSP was unable to produce S protein in vitro. Immunocytofluorescence assay showed that the ΔSP LHBs mutant aggregated in the cytoplasm, accumulating mainly in the ER. Transient transfection and expression of ΔSP mutant caused GRP78 up-regulation in vitro.Conclusion HBV S promoter deletion was found dominantly in HCC tumor tissue. The aggregation of mutant large surface proteins in the ER possibly involved in HBV-related HCC.