Objective To construct the yeast expression vector of a new gene transactivated by hepatitis B virus X protein (XTP11), and to explore the feasibility of cloning the hepatocyte proteins interacting with XTP11 protein by yeast two hybrid system. Methods The XTP11 gene was amplified by polymerase chain reaction (PCR) with specific primers, and the amplified fragment was subcloned into the Nco I/BamH I sites (5′ ends) of yeast expression vector pGBKT7,which was named as pGBKT7(-)-XTP11 encoding fusion proteins of full length of XTP11 and DNA binding domain of yeast protein GAL4. The pGBKT7 (-)-XTP11 plasmid was transformed in the yeast cells AH109 and the expression of XTP11 protein in yeast cells was detected by Western blotting assay. Then the yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp and SD/-Trp-His-Ade) containing X-?-gal, and their autonomous activation was tested. Results The yeast expression vector of XTP11 gene was constructed and the fusion protein in yeast cells was examined. The yeast cells transformed with pGBKT7-XTP11 plasmid could grew well on both of the media, and turned blue on medium containing X-?-gal for the production X-?-galactosidase. This phenomenon suggested that XTP11 protein acted to substitute the functional yeast protein GAL4 activation domain (AD) to activate transcription of reporter genes (ADE2, HIS3, MEL1 and LacZ). Conclusion The XTP11 protein fused to the GAL4 DNA-binding domain functioned as a transcriptional activation domain in yeast, and the transcriptional activation function of XTP11 protein in yeast might restrain researchers to use yeast two hybrid system to clone hepatocyte proteins interacting with XTP11 protein.

Objective MicroRNA(miRNA)has recently been suggested to play a role in certain virus infections.The present study is to investigate if miRNA is associated with hepatitis B virus(HBV)activity by detecting differential expression of miRNA between human hepatoblastoma cell line HepG2.2.15 transfected with full-long HBV genome and its parent cell line HepG2.Methods Total RNA were extracted from HepG2.2.15 cells and control HepG2 cells,respectively.miRNAs were then isolated from the total RNA.Mammalian miRNA microarrays containing 509 miRNA genes were employed to analyze the differentially expressed miRNAs between the two groups.miRNAs were considered to be up-or down-regulated when the fluorescent intensity ratio between the two groups was over 4-fold and global false positive is zero using SAM program.Validation of microarray results was carried out by real-time quantitative RT-PCR(qRT-PCR).Results Compared with those of control HepG2 cells,a total of 27 miRNAs were differentially expressed(5.3% of all probes),among which 7 were up-regulated and 20 were down-regulated in HepG2.2.15 cells.qRT-PCR verified that miR-181d expression was 16-fold up-regulated and miR-15a was 9-fold down-regulated in HepG2.2.15 cells,which was in agreement with the results of the microarray analysis.Conclusion The findings suggest that there are HBV replication-associated miRNAs in HBV genome-transfected HepG2.2.15 cells.These up-and down-regulated miRNAs may be involved in the life cycle of HBV replication.The knowledge is helpful for further study to discover new molecular targets for anti-HBV therapy.

OBJECTIVETo determine the mutational profile and clinical implications of the viral reverse-transcriptase (rt)A 181T mutation in hepatitis B virus (HBV) through population-based analysis of clinical samples.

METHODSSerum samples from 3, 013 patients who visited The 302 Hospital (Beijing, China) were investigated.HBV DNA was extracted and HBV mutations and genotypes were determined by direct sequencing.Recombinant plasmids harboring the rtA181T/sW172* mutant or wild type sequence were constructed and transfected into the HepG2 cell line. The levels of HBsAg in culture supernatants were compared and statistically analyzed.

RESULTSThe incidence of rtA181T across the study population was 4.1% (165/3, 013), and most of the rtAl 81T-positive patients had received adefovir and/or lamivudine.Forty percent (66/165) of the rtA 181T cases were single mutants and treatment responsive, 46.1% (76/165) included the adefovir-resistant mutation rtA 181 V/N236T, 12.1% (20/165) included the lamivudine-resistant mutation rtM204V/rtM2041, and 1.8% (3/165) included multidrug-resistant mutations.Interestingly, 73.9% (122/165) of the rtA181T-positive samples were detected with co-existing wild-type nucleotides at the site. The rates of HBV/C to HBV/B were 92.1% to 7.9% in the rtA181T-positive patients, but 82.1% to 17.9% in the rtA181T-negative paticnts (P less than 0.01).Almost all (98.2%; 129/165) of the rtA181T led to sW172*, while only 1.8% of the rtA181T (3/165) led to sW172L or sW172S.HBsAg secretion in vitro was reduced from the rtA181T/ sW172* strain, but there was no significant difference observed in the average serum HBsAg and HBV DNA levels of patients who carried or did not carry the mutant.

CONCLUSIONThe HBV rtA181T mutation is closely associated with adefovir and lamivudine exposure.rtA181T may led to sW172*, culminating in suppression of HBsAg secretion.However, co-existence of the mutant with wild-type sequences was common among our patient population, suggesting that the mutation had little impact on serum HBsAg and HBV DNA levels across the clinical study population.

Adenine ; analogs & derivatives ; Antiviral Agents ; China ; Genotype ; Hepatitis B Surface Antigens ; Hepatitis B virus ; Humans ; Lamivudine ; Mutation ; Organophosphonates