Study Of Hepatitis C Virus Anti-F Protein Distribution In Serum And The Function Of F Protein

Hepatitis C virus is a serious and growing threat to human public health and is estimated to infect over 123 million people worldwide. Hepatitis C, it had become clear that the vast majority of persons who develop acute hepatitis C, perhaps over 80 percent-remain infected. A high proportion of persons with chronic HCV infection will develope cirrhosis and progress eventually to terminal chronic liver disease, in some instances after developing HCC.HCV is an enveloped positive-strand RNA virus, belonging to the genus Hepacivirus of the family Flaviviridae. The single-stranded positive-sense RNA genome of HCV (~9.6 kb) is flanked by conserved, highly structured nontranslated regions (NTRs) and encodes a polyprotein precursor of about 3 000 amino acids (aa). The polyprotein is processed by cellular and viral proteases to yield 10 structural (C, E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins. The core protein is located at the N terminus of the polyprotein and is predicted to have a length of 191 amino acids and a molecular mass of 23 kDa (p23). HCV core has been implicated in immune-mediated mechanisms associated with the development of chronic hepatic diseases. Recently, a new protein, named F protein, has been described to be expressed through a ribosomal frameshift of core gene. Now its characteristics and function are still unclear. F protein is found in HCV infected patients, and is conserved in different HCV genotype, indicate that it can expression spontaneously in HCV infected patients and may play an important role in the viral life cycle. The double-shift F protein (ARFP/DF) was found in HCV genotype 1b by Boulant et al in 2003, then several study showed that DF protein might participate in the process of liver diseases. However, there is no research on the function of DF in China now. In order to understand the biological function, and whether it has shared the same funtion with core protein. We use renal dialysis patients for study population, explore relationship between F and core protein expression, and anti-F distribution features among HCV infected patients. On the other hand, the effect of double-shift F protein was observed by determinations of anti-oncogenes (p16, p21) in HepG2 cells by using Westem blot and semi-quantitative RT-PCR.Section I The relationship between expression of hepatitis C virus F protein and core protein and distribution of anti-FObjective: To find correlates between distribution relationship of F and core protein antibodies, and distribution of F protein antibody, in order to provide a basis for process of HCV chronic with F protein expression. Methods: A total of 128 renal dialysis patients with HCV antibody positive and 100 with HCV antibody negative were selected from Jiangsu province hospital for this study. Sera were taken for HCV antibody test with ELASA and virus genotyping was performed by using HCV Genotype Primer Kit. The purified recombinant protein, HCV-F /GST, were coated onto microtiter plates as antigen. The secondary antibodies were horse radish peroxidase (HRP)-conjugated goat anti-human IgG. Sera of 128 patients were tested by indirect ELISA for detecting anti-HCV-F(C) antibodies. Base on clinical data of objects, analysis on the relationship between expression of HCV F and core protein, and the distribution fetures of F protein. Results: Renal dialysis patients with HCV infection were mainly infected with HCV 1b genetype (86.1%). Constituent ratio between anti-F group and anti-C group were found no statistics difference (P>0.05). F antibody titer showed that F antibody had positive correlation with age and clinical stage of hepatits C, also infection with HBV was higher than HCV single infection over antibody titer. Multivariate analysis identified the statistics differences between anti-F positive rate and several factors (age, time of renal dialysis, coinfection with HBV, clinical stage of hepatitis C). Conclusion: There is no reason to show that F and C protein has the correlation of expression. F protein may participate in the progess of liver disease, and to study the HCV chronic mechanism and correlate factors will benificial to HCV prevention and treatment.Section II Regulation of anti-oncogene of the double-shift F protein of Hepatitis C virus subtype 1bObjective: To investigate the effects of the double-shift F protein of Hepatitis C virus subtype 1b on the expression of anti-oncegene (p16, p21) in HepG2 cells. In order to give basis on the function study of DF protein. Methods: DF gene was amplificated from the whole HCV 1b genome, and cloned into pcDNA3.0 vecter. The recombinant plasmid (pcDNA3.0/HCV-DF) and empty vector were transfected into HepG2 cells. Flow cytometry detection was used to study the effect of DF protein on HepG2 cells cycle. Screening was performed with G418. p16, p21 mRNA were detected by semi-quantitative RT-PCR, and protein was detected by Western blot. Results: Stable expression of the recombinant plasmid was found in HCV DF protein. DF protein could promote proliferation and suppress apoptosis of HepG2 cells. The expression of p16 and p21 in HepG2 cells transfected with pcDNA3.0/HCV-DF were lower than those with blank plasmid. Conclusion: HCV-DF protein could suppress HepG2 cell apoptosis and inhibits expression of p16 and p21 in HepG2 cells. This suggested that HCV-DF protein may participate in the progress of hepatocellular carcinoma.