Impact of directly sequenced core and non-structrural protein 5B regions on hepatitis C virus genotyping / 中华传染病杂志

Objective To explore the sensitivity and accuracy of directly sequenced core and non-structrural protein (NS)5B regions for hepatitis C virus (HCV)genotyping.Methods Fifty-one serum samples from chronic hepatitis C patients were collected in the study.Reverse transcription-polymerase chain reaction was used to amplify core and NS5B regions.Genotypes or subtypes were determined by the phylogenetic analysis of directly sequenced core and NS5B regions.Results Among the 51 samples,49 (96.1 %)were successfully typed by phylogenetic analysis of directly sequenced core region.There were overall five genotypes determined in the area,including 1b (61 .2%,30/49 ),2a (20.4%,10/49 ),2b (2.0%,1/49),3a (4.1 %,2/49 )and 6a (12.2%,6/49 ).The positive rate of HCV genotying was 88.2% (45/51 )on the basis of NS5B region.HCV genotypes 1b,2a,2b,3a and 6a were found in 62.2% (28/45),20.0% (9/45 ),2.2% (1/45 ),4.4% (2/45 )and 11 .1 % (5/45 )of the patients, respectively.Conclusion The HCV genotyping based on core regions,compared with that based on NS5B,shows the advantages of primer design,amplification efficiency and accuracy,suggesting that it has the priority to be used in the epidemiological and clinical study of HCV genotyping.

Hepatitis C virus core protein induces energy metabolism disorders of hepatocytes by down-regulation of silent mating type information regulation 2 homolog-1 and adenosine monophosphate-activated protein kinase signaling pathway / 中华传染病杂志

Objective To study the role of silent mating type information regulation 2 homolog1 (SIRT1)-adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway in hepatitis C virus core protein (HCV-core) induced energy metabolism disorders of hepatocytes.Methods HepG2 cells were transfected with recombined expressed plasmid pcDNA3.1-core.The level of reactive oxygen species (ROS),value of ATP/ADP and activity of AMPK α-2,and nicotinamide adenine dinucleotide (NAD)+/NADH in HepG2 cells expressing HCV-core were detected by flow cytometry,liquid scintillation counter and chromatometry,respectively.The activity of SIRT1 was detected with a fluorometric assay kit.Reverse transcription polymerase chain reaction (RT-PCR) and Western blot assay were performed to examine the expression of SIRT1 and AMPK α-2.Quantitative data were analyzed by t-test.Results It was confirmed by Western blot assay that HepG2 cells expressed HCV-core with relative molecular weight of 22 000.Compared to HepG2 cells,the level of ROS in HepG2 cells expressing HCV-core was significantly increased (1.0 ±0.1 vs 4.0±0.5,t=14.411,P＜0.01),the values of ATP/ADP were similar (8.2±2.2 vs 9.3±2.8,t=0.757,P＞0.05),AMPK α-2 (0.8±0.2 vs 0.2±0,t=7.345,P＜0.01),the values of NAD+/NADH (0.08±0.02 vs 0.02±0,t=7.348,P＜0.01),the activity of SIRT1 [(0.30±0.05) pmol· μg-1 · min-1 vs (0.15±0.04) pmol · μg 1 · min 1,t=5.738,P＜0.01] and the mRNA levels of SIRT1 (0.8±0.2 vs 0.4±0.1,t=4.382,P＜0.01) and AMPK α-2 mRNA (0.9±0.3 vs 0.2±0,t=5.715,P＜0.01),and the expression of SIRT1 (0.8±0.2 vs 0.3±0,t=5.941,P＜0.01) and phosphorylated AMPK protein (0.5±0.1 vs 0.1±0,t=9.608,P＜0.01) were all significantly decreased.Conclusion HCV core protein induces energy metabolism disorders of hepatocytes by down-regulation of SIRT1-AMPK signaling pathway.

Clinical relevance of serum hepatitis C virus core protein level and antiviral therapy response / 中华传染病杂志

Objective To evaluate the relationship between serum level of hepatitis C virus (HCV) core protein and antiviral therapy response in patients with hepatitis C. MethodsOne hundred and sixty-two consecutive patients with chronic hepatitis C (CHC) were retrospectively analyzed. Serum level of HCV core protein and HCV RNA were measured. Among them, 35 CHC patients treated with pegylated interferon (PEG-IFN)+ ribavirin (RBV) were followed up before and at week 4, 24, 48 of treatment. The correlations between serum HCV core protein level, HCV RNA and antiviral therapy were evaluated. Comparison between groups was done by t test and comparison of paired data among groups was done by analysis of variance. Results Total 162 patients were divided into six groups according to the HCV virus load: 56 with HCV virus load≤1×103 IU/mL and HCV core protein absorbance (A)=0. 100±0. 029; 9 with 1×103 IU/mL＜ HCV virus load≤1 × 104 IU/mLand A=0. 246±0. 213; 11 with 1 × 104 IU/mL＜ HCV virus load≤1 × 105 IU/mL and A=0. 235±0.179; 28 with 1×105 IU/mL＜ HCVvirusload≤1×106 IU/mL and A=0. 422±0. 319; 51 with 1 × 106 IU/mL＜ HCV virus load≤ 1 × 107 IU/mL and A = 0. 603 ± 0. 330 ; 7 with 1 × 107 IU/mL＜HCV virus load≤ 1 × 108 IU/mL and A = 0. 900± 0. 379. The HCV core protein absorbance was positively correlated with HCV RNA level (r= 0.36, P＜0.05). The HCV core protein absorbance values of 35 CHC patients before therapy, at week 4, 24, and 48 of therapy were 0. 564 ±0. 296,0. 144±0. 062, 0. 091 ±0. 035 and 0. 112±0. 103, respectively. The HCV core protein absorbance values at week 4, 24, 48 all decreased significantly compared with that before therapy (t= 8. 563,9. 195, 9. 250; all P＜0.05), and there was significant difference between those at week 4 and week 24 (t=4. 301, P＜0.05). Conclusion Serum HCV core protein level is closely correlated with HCV viral load during HCV infection process and antiviral treatment.

Expression of Hepatitis C Virus Core Protein in Hepatocytes Does Not Modulate Proliferation or Apoptosis of CD8+ T Cells

Hepatocytes are the primary targets of the hepatitis C virus (HCV). While immunosuppressive roles of HCV core protein have been found in several studies, it remains uncertain whether core protein expressed in hepatocytes rather than in immune cells affects the CD8+ T cell response. In order to transduce genes selectively into hepatocytes, we developed a baculoviral vector system that enabled primary hepatocytes to express a target epitope for CD8+ T cells, derived from ovalbumin (OVA), with or without HCV core protein. Culture of OVA-specific CD8+ T cells with hepatocytes infected with these baculoviral vectors revealed that core protein has no effect on proliferation or apoptosis of CD8+ T cells. Our results suggest that HCV core protein does not exert its suppressive role on the CD8+ T cell immune response through expression in hepatocytes.

The influence of HCV core protein on cell apoptosis,cell cycles and cell telomerase activities of HepG2 cells / 中华传染病杂志

Objective To investigate the influence of HCV core protein on cell apoptosis, cell cycles and cell telomerase activities of HepG2 cells. Methods A eukaryotic expression plasmid containing HCV C gene was constructed by DNA recombinant technique and the recombinant plasmid was transfected into HepG2. Thereafter, HepG2 cells transfected with recombinant eukaryotic expression plasmid were obtained. The HCV C mRNA and protein in HepG2 cells transfected with recombinant plasmid were verified by RT PCR and indirect immunofluorescence assay. The HepG2 cells were studied as follows: (1) The cell proliferation ratio of three groups cells(HepG2 cells transfected with the recombinant plasmid, HepG2 cells transfected with blank plasmid and HepG2 cells without transfection) was evaluated by MTT assay; the cell cycles were also examined by FACS. (2) The apoptotic ratio of three groups cells were examined by FACS. (3) The cell telomerase activities of all three group cells were examined by TRAP ELISA assay. Results (1) The cell proliferation ratio in the group of HepG2 cells transfected with recombinant plasmid was higher than that of the group of HepG2 cells transfected with blank plasmid or the group of HepG2 cells without transfection; The proportion of phase S in the group of HepG2 transfected with the recombinant plasmid was significantly higher than that of the group of HepG2 without transfection. (2) The apoptotic ratio in the group of HepG2 cells transfected with recombinant plasmid was significantly lower than that of the group of HepG2 cells transfected with blank plasmid or the group of HepG2 cells without transfection. (3) There were no significant differences among the three group cell telomerase activities. Conclusions (1) HCV C protein had the potential role in inhibiting cell apoptosis. (2)HCV C protein could induce HepG2 cells from phase G 0/1 to phase S, and might promote cell proliferation, inhibit cell apoptosis. (3) HCV C protein had no influence on cell telomerase activities of HepG2 cells, thus HCV C protein regulated cell cycle, promoted cell proliferation and inhibited cell apoptosis not by enhancing cell telomerase activities.

SUPPRESSION SUBTRACTIVE HYBRIDIZATION FOR CLONING OF GENES TRANSACTIVATED BY HCV CORE PROTEIN / 解放军医学杂志

To construct a cDNA subtractive library of genes transactivated by hepatitis C virus core protein with suppression subtractive hybridization technique. mRNA was isolated from HepG2 cells transfected with pcDNA3 1(-)-core and pcDNA3 1(-) empty vector,respectively, then cDNA was synthesized. After restriction enzyme RsaI digestion, small sized cDNA were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, and then it was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain JM109. The cDNA were sequenced and analyzed in GenBank with Blast search after PCR. The subtractive library of genes transactivated by HCV core protein was constructed successfully. The amplified library contained 233 positive clones. Colony PCR product showed that 213 clones contained 100~ 1 000 bp inserts. Sequence analysis was performed in 63 clones. Six of the sequences were unknown genes before. The full length sequences were obtained with bioinformatics method,which had been accepted by GenBank. It suggested that six novel cDNA sequences might be target genes transactivated by HCV core protein.

Cloning and identification of human gene transactivated by hepatitis C virus core protein 1 / 解放军医学杂志

Objective To screen and clone the target genes transactivated by hepatitis C virus (HCV) core protein. Methods The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-core and pcDNA3.1(-) empty vector, respectively, and suppression subtractive hybridization (SSH) method was employed to analyze the differentially expressed DNA sequences of the two groups. The obtained sequences were searched for homologous DNA sequence from GenBank. One of them was confirmed to be a new gene without homology with known genes in this database.Then, electric polymerase chain reaction was conducted for the cloning of the full-length DNA of the new gene, in conjunction with Kozak rule and the existence of polyadenyl signal sequence. The reverse transcription PCR (RT-PCR) was used to amplify the new gene with mRNA from HepG2 cell as the template. The coding sequence for the new gene was deduced according to the nucleotide sequence. Results A new gene with unknown function was named TAHCCP1.The nucleotide sequence of the new gene and its corresponding protein-encoding amino acid, which was 2 001nt and composing 667aa, have been determined. The sequence of the TAHCCP1 gene has been registered in GenBank with its accession number AY038359. Conclusion TAHCCP1 gene transactivated by HCV core protein was cloned and identified successfully by a combination of molecular biological technology and bioinformatics technique. The results are expected to pave the way for the study of the molecular mechanism of the transactivating effects of HCV core protein and the development of new therapies for chronic hepatitis C.

Preparation and immunohistochemical study of human single chain Fv antibody against hepatitis C virus core antigen / 解放军医学杂志

Objective To detect hepatitis C virus core antigen in 7721 cells transfected with HCV cDNA by immunohistochemistry method with human single chain Fv antibody(scFv). Methods The recombinant phages were panned by core antigen which was coated in a microtiter plate. After three rounds of biopanning, 48 clones were identified specific to core antigen. The affinity and specificity of scFv were evaluated by ELISA and immunohistochemistry. Results ScFv-core DNA digestion and sequence data showed that the scFv gene was composed of 774bp. Conclusion Human single chain Fv antibody against HCV core antigen has a specific combining capacity with hepatitis C virus core antigen.

Expression of soluble human anti-idiotypic single chain Fv antibody to hepatitis C core protein in E. coli / 解放军医学杂志

Objective To express soluble human anti-idiotypic single chain Fv to hepatitis C core protein in E.coli. Methods Using phage display technique, the semisynthetic phage library was panned by HCV core monoclonal antibody which was coated in a microtiter plate. After three rounds of biopanning, 53 clones were identified specific to HCV core antibody. The specificity of anti-idiotypic scFv was determined by ELISA. After digested with Sfi/Not, the selected HCV core anti-idiotypic scFv positive clone was subcloned into the vector pCANTAB5E for the expression of E-tagged soluble anti-idiotypic scFv. The E.coli XL1-Blue was transformed and induced by IPTG. The specificity of anti-Id scFv was evaluated with ELISA. Results HCV core anti-Id scFv DNA digestion and sequence data showed that the scFv gene was composed of 774bp. ELISA results demonstrated that the soluble human HCV core anti-idiotypic scFv to HCV core monoclonal antibody had a specific combination character. The molecular weight of expressed HCV core anti-idiotypic scFv was 28kD as shown by SDS-PAGE. Conclusion HCV core anti-Id scFv has been successfully expressed in E.coli.