OBJECTIVETo investigate the effect of hepatitis C virus (HCV) non-structure protein NS5A on the activity of calcium-regulating protein alpha subunit of nascent polypeptide-associated complex (NACA) promoter.

METHODSHepG2 cell plasmid pCAT3-NACA, containing NACA promoter, was transfected alone or cotransfected with pcDNA3.1(-)-NS5A, and chloramphenicol acetyl transferase (CAT) enzyme activity was assayed by enzyme-linked immunoassay (ELISA).

RESULTSThe CAT activity in the pcDNA3.1(-)-NS5A cotransfection group was 20.7% of the CAT activity in the pCAT3-NACA group.

CONCLUSIONHCV non-structural protein NS5A has a down-regulating effect on the promoter of NACA gene.

Base Sequence ; Down-Regulation ; Humans ; Molecular Chaperones ; biosynthesis ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics ; Transcriptional Activation ; Transfection ; Viral Nonstructural Proteins ; genetics ; pharmacology

BACKGROUNDTo investigate the transcriptional inhibitory role of hepatitis B virus X protein on the expression of p53 tumor suppression gene.

METHODSThe promoter sequence of the p53 tumor suppression gene was identified and amplified by bioinformatics and polymerase chain reaction (PCR). The recombinant reporter gene expression vector pCAT3-p53p was constructed and transfected into the hepatoblastoma cell line HepG2 and cotransfected with pcDNA3.1 (-)-X by Fugene 6 transfection reagents. The chloramphenicol acetyl transferase (CAT) activity was detected by enzyme-linked immunosorbent assay (ELISA). The expression of p53 mRNA was further detected by RT-PCR with or without HBV X protein.

RESULTSThe reporter vector pCAT3-p53p has been successfully constructed and identified and the p53 promoter could cis-activate the transcription of the CAT gene. The relative expression level of CAT gene in HepG2 cells cotransfected with pCAT3-p53p and pcDNA3.1 (-)-X was lower than the control, and the inhibitory rate was approximately 78%, which indicate that HBV X protein could transcriptionally inhibit the activity of p53 promoter. After transfected with pcDNA3.1 (-)-X, the expression of p53 mRNA was lower than the control.

CONCLUSIONHBV X protein could transcriptionally inhibit the expression of p53 tumor suppression gene, which might be a possible molecular mechanism responsible for the development of HBV-associated hepatocellular carcinoma.

Base Sequence ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; Molecular Sequence Data ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Trans-Activators ; genetics ; Transcription, Genetic ; Transfection ; methods ; Tumor Suppressor Protein p53 ; genetics

Objective To prove the interaction between hepatitis virus C(HCV)nonstruetural protein 4A(HCV NS4A)and calcium modulating cyclophilin tigand(CAML)with yeast-two hybrid- ization and coimmunoprecipitation.Methods The gene encoding CAML was cloned,and subcloned into the yeast expression vector pGADT7 and eucell expression vector pcDNA3.1/His-A.The back- cross test between HCV NS4A and CAML was performed in yeast cells.After that,the pCMV-Myc/ NS4A plasmid and pcDNA3.1/His-A-CAML plasmid were co transfected into 293 cells and,then, coimmunoprecipitation and Western blot were performed.Results The gene encoding CAML was cloned sucessfully,and then the gene was subcloned into yeast expression vectors,pGADT7.After the interaction between NS4A and CAML was ensured in yeast cells,the eukaryotic expression vec- tors of NS4A and CAML were constructed and their interaction was ensured again by Co-immunopre- cipitation.Conclusions The interaction between HCV NS4A and CAML is proved.CAML is one of the proteins involved in Ca~(2+)signaling,which suggests that the interaction of HCV NS4A and CAML may be a new clue of the chronic mechanism of HCV infection.Future studies will be required to de- fine the physiologic significance of this interaction.

BACKGROUNDThere is still a paucity of data on hepatitis B virus (HBV) subgenotype prevalence in North China based on sequencing of large-size samples. In addition, whether HBV genotypes impact drug-resistance-associated and HBV e antigen (HBeAg)-loss-associated mutations in patients with chronic hepatitis B (CHB) is still under investigation. This study aimed to disclose clinical prevalence of HBV genotypes/subgenotypes in North China and the clinical implications of HBV genotype classification in respect to HBeAg loss and drug-resistant occurrence.

METHODSSera were collected from 1301 nucleos(t)ide analog-experienced CHB patients. Viral DNA was extracted and used as template for HBV genome amplification by nested PCR. DNA sequencing was performed for the analysis of HBV genotypes/subgenotypes, drug-resistance-associated mutations in polymerase gene and HBeAg-loss-associated mutations in precore/basal core promoter (BCP) regions.

RESULTSHBV/B, HBV/C, and HBV/D were detected in 190 (14.6%), 1096 (84.2%), and 15 (1.2%) patients, respectively. HBV/B2 (182/190), HBV/C2 (1069/1096), and HBV/D1 (12/15) were predominant subgenotypes within individual genotypes. By contrast, C2 prevalence is relatively lower in Beijing area (77.2%) than in other north areas (84.9% - 87.4%). HBV/C-infected patients had an older age and a lower serum albumin level but similar HBV DNA and alanine aminotransferase (ALT) levels compared to HBV/B-infected patients. HBV/C infection had a higher incidence of lamivudine-resistant mutations rtM204I/V (44.9% vs. 30.2%, P < 0.01) and BCP mutations A1762T+G1764A (65.8% vs. 40.0%, P < 0.01) compared with HBV/B infection.

CONCLUSIONSC2 is the most prevalent HBV subgenotype followed by B2 in CHB patients in North China; and HBV genotype prevalence is influenced by immigrant population. HBV/C infection is likely to have longer disease duration and severer liver functional impairment and might be more susceptible to develop lamivudine resistance compared to HBV/B infection.

Adult ; Antiviral Agents ; therapeutic use ; China ; DNA, Viral ; genetics ; Drug Resistance, Viral ; genetics ; Female ; Genotype ; Hepatitis B virus ; classification ; drug effects ; genetics ; Hepatitis B, Chronic ; virology ; Humans ; Male ; Middle Aged ; Mutation

OBJECTIVETo investigate the interferon alpha regulation mechanisms by screening binding proteins of interferon alpha promoter by phage display.

METHODSPCR product of interferon-alpha promoter was incubated with a phage display cDNA library that expressed a library of human liver proteins on the surface of bacteriophage T7. Unbound phages were washed off and the phages bound to the interferon alpha promoter were amplified. Positive plaques were amplified by PCR and cloned into a pGEM-Teasy vector in order to perform DNA sequence analysis and subsequent computer blasting analysis.

RESULTSPositive phage-displayed proteins binding with interferon alpha promoter were enriched after five rounds of biopanning. We found that the following proteins were relevant to interferon alpha: mitochondrial ribosomal protein, chromosome clone, fibrinogen A alpha polypeptide, enoyl coenzyme A hydratase short chain, eukaryotic translation elongation factor 1 alpha, PI-3-kinase-related kinase SMG-1-like, xeroderma pigmentosum C group, and Homo sapien activity-dependent neuroprotector (ADNP).

CONCLUSIONMany proteins with different functions could bind with interferon alpha promoter.

DNA, Complementary ; genetics ; DNA-Binding Proteins ; biosynthesis ; genetics ; Gene Library ; Hepatocytes ; cytology ; metabolism ; Humans ; Interferon-alpha ; biosynthesis ; genetics ; Promoter Regions, Genetic ; genetics ; Two-Hybrid System Techniques

OBJECTIVETo screen proteins interacting with HCV NS4A protein in leukocytes by yeast-double hybridization.

METHODSThe bait plasmid pGBKT7-NS4A was transformed into yeast AH109 was transformed, and the expressing of the fusion protein was identified by SDS-page. The transformed yeast was mated with yeast Y187 containing leukocytes 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-alpha-gal for selecting two times and screening. After extracting and sequencing of plasmid from blue colonies, analysis was conducted by bioinformatics. And, the gene encoding the interesting protein was cloned, and back-cross was performed.

RESULTSForty-five colonies were sequenced, among them, 29 colonies were human calcium modulating cyclophilin ligand (CAML). The gene encoding CAML was cloned, and the interaction between NS4A and CAML was ensured.

CONCLUSIONSeven kinds of proteins interacting with NS4A in leukocytes were successfully screened and the results brought some new clues for studying the pathogenesis of HCV.

Adaptor Proteins, Signal Transducing ; genetics ; isolation & purification ; metabolism ; Carrier Proteins ; genetics ; metabolism ; Cloning, Molecular ; Gene Library ; Humans ; Leukocytes ; cytology ; metabolism ; Protein Binding ; Transformation, Genetic ; Two-Hybrid System Techniques ; Viral Nonstructural Proteins ; Viral Proteins ; genetics ; metabolism

OBJECTIVESTo analyze HBV drug-resistant mutations against nucleos(t)ide analogues at 12 reported sites in 340 patients with chronic hepatitis B.

METHODSSerum HBV DNA was extracted and a nested PCR assay was employed for the reverse transcriptase (RT) gene amplification. Direct sequencing of PCR product was performed. The significance of detected mutations was analyzed in view of clinical data of the patients.

RESULTSDrug-resistant mutations were detected in 68 patients taking lamivudine (LAM), 10 taking adefovir (ADV), 8 taking entecavir, and 1 taking telbivudine (LdT). M204V and M204I were the most common LAM-resistant mutations. The former usually emerged with L180M while the latter often emerged alone. N236T +/- A181 substitution was the most frequently seen ADV-resistant mutation. ETV-resistant mutations occurred on the basis of LAM-resistant mutations and T184 change was the most common form. LdT-resistance was observed as M204I. Interestingly, these drug-resistant mutations were detected in a few patients who had not been treated with nucleos(t)ide analogues.

CONCLUSIONDetection of HBV drug-resistant mutations at multiple sites of the viral RT gene is valuable for discovering and verifying drug resistance and thus is very helpful in planning anti-HBV therapy.

Adult ; DNA Mutational Analysis ; DNA, Viral ; genetics ; Drug Resistance, Viral ; genetics ; Female ; Genotype ; Hepatitis B virus ; drug effects ; genetics ; Hepatitis B, Chronic ; virology ; Humans ; Male ; Middle Aged ; Mutation ; Young Adult