<b>OBJECTIVEb>To explore the possibility of transacting hepatitis D virus (HDV) ribozyme cleaving in vitro the hepatitis B virus (HBV) mRNA fragments.

<b>METHODSb>According to the established pseudoknot-like structure, its' H1 domain was changed to design the transacting HDV ribozyme Rc1 and Rc2, which targeted the 701-713 site and 776-788 site of HBV C domain. After the chemically synthesised cDNA of the ribozyme was cloned into the vector PGEM-4Z, the transacting HDV ribozyme was transcriped using in vitro transcription technology. The in vitro cleavage characteristics of the ribozyme were studied and the kinetic parameters (Kcat and Km) were determined by Eadie Hofstee plotting.

<b>RESULTSb>Both the two ribozymes had the ability to cleave the substrate, the cleavage percentage at 37 degrees for 90 minutes were 50% and 51%. According to the Eadie Hofstee plot, the Km of the Rc1 and Rc2 were 0.61 micromol and 0.58 micromol, the Kcat were 0.64 x min(-1) and 0.60 x min(-1),respectively.

<b>CONCLUSIONSb>The cleaving ability of trans-acting HDV ribozyme on non-HDV RNA fragment was tested. The results showed a new potential of the antisense antisense regent for HBV gene therapy.

DNA, Antisense ; genetics ; Genome, Viral ; Hepatitis B virus ; genetics ; Hepatitis Delta Virus ; enzymology ; genetics ; Humans ; RNA, Catalytic ; genetics ; metabolism ; RNA, Messenger ; genetics ; RNA, Viral ; genetics ; Transcription, Genetic

HBV infections leads to severe public health problems around the world, especially in China. Improved understanding of the molecular mechanisms of HBV reverse transcription is fundamental for optimization of treatment and solution to drug-resistance. Recently, the main structural basis involved in the process of HBV reverse transcription and the cis-elements were revealed by means of biochemistry and genetics. The entire process of reverse transcription is completed mainly through the first template switch mediated by the P- epsilon structure; the second template switch mediated by 5E/3E and M structure; and the third template switch mediated by 5' r / 3' r structure. The important structure and the cis-elements involved in this process are the focus of this review, at the same time, an overview of the progress in relevent studies is demonstrated to show the whole picture of the HBV reverse process.
Animals ; Hepatitis B ; virology ; Hepatitis B virus ; enzymology ; genetics ; metabolism ; Humans ; RNA, Viral ; genetics ; RNA-Directed DNA Polymerase ; genetics ; metabolism ; Reverse Transcription ; Viral Proteins ; genetics ; metabolism

<b>OBJECTIVEb>To investigate the correlation between spontaneous clearance of HBV DNA and the levels of Alanine Aminotransferase (ALT) in chronic hepatitis B (CHB) patients.

<b>METHODSb>Retrospective review analysis was used in this research. A total of 177 CHB patients with HBV DNA>1x10(4) copies/ml and ALT>800 U/L were recruited in this study and were divided randomly into two groups, 84 patients in control group (received lamivudine therapy) while 96 cases in study group (without anti-viral therapy), the dynamic changes of HBV DNA and HBV markers in these two groups were compared.

<b>RESULTSb>The clinical data of CHB patients were retrospected and followed up in 24 weeks. The negative conversion cases of HBV DNA are 62 (87.3%) in study group and 56 cases (78.87%) in control group at week 24, the negative conversion cases of HBV DNA are 56 (78.9%) in study group and 60 (92.3%) cases in control at week 8. No significant difference (x2=0.058, P>0.05) existed between these two groups. Among 43 patients with HBV DNA is less than or equal to 6 log10 copies/ml, 41 (95.3%) patients converted negatively, while in 28 patients with HBV DNA is more than 6 log10 copies/ml, 21 (75.0%) patients converted negatively. The negative conversion rate of HBV DNA is more than 6 log10 copies/ml was lower than the other group at week 24. The difference between the two groups was significant (x2=0.024, P<0.05). 41 patients with hepatitis B e antigen (HBeAg) negative and 30 patients with HBeAg positive were included in antiviral group. The negative conversion cases of HBV DNA of the former are 36 (87.8%) and the latter are 26 (86.7%). No significant difference found between them (x2=1, P>0.05). HBeAg loss found in 10 patients of 30 HBeAg positive patients with 4 patients occurred as early as at the fourth week. A total of 62 patients HBV DNA converted negatively in antiviral group, but 5 patients were found HBV DNA rebounded (occurred in 24 to 72 weeks) with ALT rebound (47 to 140 U/L).

<b>CONCLUSIONSb>The tendency of spontaneous clearance of HBV DNA when ALT is more than 800 U/L is obvious, so anti-viral therapy should be administrated strictly. The negative conversion rate of HBV DNA has no relation with HBeAg but with the copies of HBV DNA replication.

Adult ; Alanine Transaminase ; metabolism ; Antiviral Agents ; therapeutic use ; DNA, Viral ; blood ; Female ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; drug therapy ; enzymology ; Humans ; Male ; Retrospective Studies ; Young Adult

Clinically being applied treatment against chronic hepatitis has three limitations: low response rates, severe adverse effects and a high rate of drug resistance. Hence, novel targets for antiviral therapy need to be developed so as to provide an armory of different strategies. During the replication of hepatitis B virus, the interaction of viral polymerase (P protein, also called P) and epsilonRNA is indispensable for the initiation of reverse transcription via protein priming and the pregenome RNA (pgRNA) packaging. Three strategies are currently developed for blocking P-epsilon interaction: heat shock protein inhibitors, epsilonaptamers and chemical compounds for blocking formation of P-epsilon complex. Previously, our group has for the first time worldwide in vitro screened several aptamers, which are able to interfere with the P-epsilon interaction. A strong inhibition against HBV was observed in vitro and in vivo experiments, respectively. In conclusion, the so far developed chemicals suppressing the P-epsilon interaction may bypass or overcome the viral resistance problems during clinic treatment and represent a highly attractive option for therapeutic intervention.
Animals ; Gene Expression Regulation, Viral ; Gene Products, pol ; antagonists & inhibitors ; genetics ; metabolism ; Hepatitis B ; therapy ; virology ; Hepatitis B virus ; enzymology ; genetics ; physiology ; Humans ; RNA, Viral ; genetics ; metabolism ; Virus Replication

<b>OBJECTIVEb>To investigate the cleavage activities of 10-23 DNA enzymes targeting at HBV C gene mRNA in vitro.

<b>METHODSb>10-23 DNA enzymes named DrzBC-7, DrzBC-8 and DrzBC-9 specific to HBV C gene ORF A1816UG were designed and synthesized. HBV C gene mRNA was obtained by in vitro transcription method. Cleavage activities were observed in vitro. The influence of MgCl2 concentration on RNA cleaving activity was examined with DrzBC-9. Values of kinetic parameters including Km, Kcat and Kcat/Km were calculated accordingly.

<b>RESULTb>Targeted substrate mRNA with the size of 300 nt was obtained by transcription in vitro. Under the certain cleavage conditions, DrzBC-7, DrzBC-8 and DrzBC-9 all efficiently cleaved target mRNA at specific sites in vitro. Cleavage products of 109 nt and 191 nt were obtained. No cleavage occurred without MgCl2. The most efficient cleavage was obtained at 150 mmol x L(-1) MgCl2. The efficiency of cleavage did not increase when the MgCl2 concentration was more than 200 mmol x L(-1). The kinetic parameters, Km, Kcat and Kcat/Km for DrzBC-9 were 1.4x10(-9) mol x L(-1), 1.6 min(-1) and 1.1x10(9) mol x L(-1) x min(-1), respectively.

<b>CONCLUSIONb>10-23 DNA enzymes targeting at HBV C gene mRNA possess the specific cleavage activities in vitro.

DNA, Catalytic ; metabolism ; DNA, Single-Stranded ; metabolism ; Hepatitis B virus ; enzymology ; genetics ; Open Reading Frames ; RNA, Messenger ; metabolism ; RNA, Viral ; genetics ; metabolism ; Transcription, Genetic

Hepatitis B virus (HBV) is a major cause of chronic liver disease, and frequently results in hepatitis, cirrhosis, and ultimately hepatocellular carcinoma. HBV polymerase (Pol) is an essential viral protein that is important for HBV replication and might be involved in the development of hepatocellular carcinoma. Protein-protein interactions appears to be crucial for its role. The aim of this study was to screen and identify the proteins that interact with Pol using a co-immunoprecipitation-based LC-MS/MS identification technique. The HBV Pol gene was amplified by polymerase chain reaction (PCR) and cloned into pCDNA3.1(+). The recombinant plasmid pCDNA3. 1(+)-Pol-flag was transfected into HeLa cells. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) identified 45 proteins that co-immunoprecipitated with flag-tagged HBV Pol. Eleven of these have previously been reported as proteins that interact with HBV Pol. A proof-of-concept-based Ingenuity Pathway Analysis (IPA, www.ingenuity.com) was used to characterize the functions and pathways of these 45 identified proteins and HBV Pol. Among these proteins, four proteins may play a role in three major molecular cellular networks, and are therefore worthy of further investigation.
Cell Line, Tumor ; Chromatography, Liquid ; methods ; Gene Products, pol ; chemistry ; genetics ; metabolism ; Hepatitis B ; genetics ; metabolism ; virology ; Hepatitis B virus ; chemistry ; enzymology ; genetics ; Humans ; Immunoprecipitation ; methods ; Protein Interaction Maps ; Software ; Tandem Mass Spectrometry ; methods

<b>OBJECTIVEb>To establish a simple and accurate method for rapid detection of lamivudine related mutations in hepatitis B virus (HBV) polymerase gene.

<b>METHODSb>HBV polymerase gene fragments of covering B and C active region were amplified by nested polymerase chain reaction (nPCR) or nested mismatched PCR. The PCR products were digested with Nde I or Nia III and subjected to electrophoresis on agarose gel, respectively. The patterns of restriction fragment length polymorphism (RFLP) were distinguished. Using this method, thirty patients with chronic hepatitis B and treated with lamivudine for at least one year were analysed for the lamivudine related mutations in polymerase gene. Sixteen cases without lamivudine therapy were used as controls. Some of the patients were also analysed by clone sequencing.

<b>RESULTSb>The nested mismatched PCR-RFLP method was simple, accurate and rapid. The whole experiments could be finished in eleven hours. The least titers of HBV DNA which could be detected was 10.3 copies/ml. The wild or mutant strains judged by RFLP were identified by clone sequencing. Mutation in the tyrosine methionine aspartic aspartic acid (YMDD) motif of HBV polymerase gene was found in eight patients and mutations of YMDD motif associated with L526M were found in another three patients. However, there were no such mutations in the control cases.

<b>CONCLUSIONb>The nested PCR-RFLP is considered as a simple and accurate method for rapid detection of lamivudine related mutations in HBV polymerase gene. It is suitable for larger number of sample detection.

Antiviral Agents ; pharmacology ; DNA-Directed DNA Polymerase ; genetics ; Drug Resistance, Viral ; genetics ; Hepatitis B virus ; drug effects ; enzymology ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Lamivudine ; pharmacology ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length

<b>OBJECTIVEb>To screen and clone the genes of protein interacting with the N-terminal protein (TP) of hepatitis B virus DNA polymerase.

<b>METHODSb>TP was amplified by polymerase chain reaction (PCR) and TP bait plasmid was constructed by using yeast two-hybrid system 3, then transformed into yeast AH 109. The transformed yeast was mated with yeast Y187 containing liver cDNA library plasmid in 2 x YPDA medium. Diploid yeast was plated on synthetic dropout medium (SD/-Trp-Leu-His-Ade) and that containing X-alpha-GAL for selecting two times and screening. Plasmids were extracted from blue colonies, and sequence analysis was performed by bioinformatics.

<b>RESULTSb>Forty-seven clones were obtained, these clones included human P36956 sterol regulatory element binding protein-1, RNA polymerase II subunit hsRPB7 mRNA, asialoglycoprotein receptor 2, transcript variant 3, ceruloplasmin (ferroxidase), transmembrane 4 superfamily member 2 and 19 of the hypothetical proteins and so on.

<b>CONCLUSIONb>Genes encoding TP interacting proteins in hepatocytes were successfully cloned and the results suggest that TP has a wide variety of biological functions.

Cloning, Molecular ; DNA-Directed DNA Polymerase ; chemistry ; genetics ; metabolism ; Gene Library ; Hepatitis B virus ; enzymology ; genetics ; Humans ; Liver ; metabolism ; Plasmids ; genetics ; Protein Binding ; Receptors, Virus ; genetics ; isolation & purification ; metabolism ; Transformation, Genetic ; Two-Hybrid System Techniques ; Viral Proteins ; chemistry ; genetics ; metabolism

<b>BACKGROUNDb>To investigate the mutation of HBV polymerase gene in chronic hepatitis B patients treated with lamivudine.

<b>METHODSb>The restriction-fragment-length-polymorphism (RFLP) assay for HBV DNA sequence determination at the codon 528 and 552 in the HBV polymerase gene associated with lamivudine resistance in vitro. HBV DNA samples extracted from sera of 240 patients were subjected to PCR amplification with primer pairs F2/R2 (552), F3/R2 (528). Each PCR product was digested with Nde I or Nla III.

<b>RESULTSb>Serum HBV DNA mutation was found in 51/240 patients (38/51M552V, 26/38L528M, 13/51M552I) after therapy for 52 weeks. DNA sequence analysis was performed on samples of 3 patients, and the results were consistent with those of RFLP assay.

<b>CONCLUSIONb>The RFLP assay was able to detect the mutation of HBV DNA at codon 552 and 528 which are the principal site of HBV DNA resistant to lamivudine. The specific PCR method for HBV DNA mutation is rapid, simple and specific.

Drug Resistance, Viral ; Gene Products, pol ; genetics ; Hepatitis B virus ; enzymology ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Lamivudine ; therapeutic use ; Mutation ; Polymerase Chain Reaction ; methods ; Polymorphism, Restriction Fragment Length ; Reverse Transcriptase Inhibitors ; therapeutic use

In order to investigate the mutation of HBV polymerase gene reverse transcription conserved region (P region) in chronic hepatitis B (CHB) patients, 212 CHB patients who took antiretroviral treatment with nucleotide analogues were chosen. The drug resistance mutations of HBV P region and HBV genotype were detected by Pyrosequencing. Sequence analysis showed that the drug resistance sites of HBV P region located at sites 173; 180; 181; 184; 204; 236 and 250. The main site of HBV P region drug resistance was 204 and 180, accounting for 35.8% and 23.5%, respectively. There were significant differences in the mutation rate of site 180 among different age groups. There were also significant differences in the mutation rate of site 204 among younger than 30 age group, 41 to 50 age group and 51 to 60 age group. (P < 0.05, P < 0.01). The mutation rate of site 180 combined with site 204 was 66.6%. The mutation rate of site 181 combined with site 236 was 23.3%. The age of C genotype infected patients was significantly older than B genotype infected patients (P < 0.01). M204V/I mutation mostly existed in the form of joint L180M mutation, the mutation rate was age-related. The detection of HBV genotypes and drug resistance sites of HBV P region have important clinical implications for the treatment and prognosis of patients with CHB.
Adult ; Aged ; Antiviral Agents ; pharmacology ; China ; Drug Resistance, Viral ; Female ; Gene Products, pol ; genetics ; Genotype ; Hepatitis B virus ; classification ; drug effects ; enzymology ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Male ; Middle Aged ; Mutation, Missense ; Young Adult