Prevalence Of Anti-F In Patients Infected With HCV, And The Modulating Effect Of F Protein On (Anti-)oncogenes

Hepatitis C virus (HCV) is a positive, single-stranded, enveloped RNA virus belonging to the Flaviviridae family, genus Hepacivirus. HCV causes acute and chronic liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). The HCV genome consists of about 9,600 nucleotides and contains a single open reading frame (ORF) encoding a polyprotein of about 3,010 amino acids. The ORF is flanked by 5'- and 3'- untranslated regions and directs the polyprotein precursor translation, which is cotranslationally and posttranslationally processed by both cellular and viral proteases at the level of the endoplasmic reticulum membrane to yield 10 mature proteins including the structural proteins core (C), the envelope glycoproteins El and E2, P7, and the nonstructural (NS) proteins NS2 to NS5b. F protein is a newly identified HCV genomic product synthesized by an alternative open reading frame(ARF) that +1 overlaps core-encoding gene. The functional significance of this viral F protein remains unknown. Using recombinant F protein as coated antigen and enzyme-linked immunosorbent assay(ELISA), we assessed the prevalence of anti-F antibodies in patients infected with HCV to see whether the F protein could be translated in human body naturally. The possiblities of F protein as an alternative laboratory detection antigen against HCV infection and as a vaccinal component inducing protective neutralized antibody are evaluated through analyzing the correlation of anti-F with the clinical data from hepatitis C cases. On the other hand, the effect of F protein on HCV related HCC occurrence was observed by determinations of oncogene(c-myc) and anti-oncogenes(p21 and p53) in HepG₂ cells transfected with pcDNA3.1+-F alone or in combination with C containing plasmids, using Western blot or real-time quantitive PCR. Part I Anti-F prevalence in patients infected with HCVTruncated F protein(codon 75^codon 134) was used as coated antigen to assess the prevalence of anti-F in sera of 35 patients infected with hepatitis C virus, 8 patients with other liver diseases, and 10 healthy controls with ELISA after being expressed in Escherichia coli using the vector pET32a and Ni-NTA resin purification. The cut-off value of anti-F antibodies was defined as mean OD_450nm plus 0.1, and the association betweenanti-F and HCV infection was evaluated statistically. Fifteen (43%) of 35 HCV serum samples reacted with the recombinant F protein, whereas no reactivity against F protein was detected in any of the controls (p<0.005), which demonstrated the F protein was able to be synthesized naturally in patients infected with HCV. Chi-square test was used to show that there were insignificant correlations between anti-F and the clinical status including age, sex, stage of liver disease or HCV RNA serum level in patients infected with HCV. Three(50%) of 6, 7(50%) of 14, 3(27%) of 11, and 2(50%) of 4 showed positive reactions in stages of acute, chronic mild, chronic moderate, and chronic severe, respectively( p>0.1). F protein has a potential usage in laboratory detection as an alternative antigen against HCV infection when used in ELISA, due to the reliable immunogenicity and immunoreactivity in 41% to 50% individuals infected with HCV observed in acute and chronic cases in our study. Anti-F has little posibility in immune injury to host due to no correlation with stage of liver disease, which suggests a possible usage in vaccine development for inducing protective neutralizing antibodies against HCV infection.Part II Effects of F protein on expressions of c-myc, p21, and p53 in HepG2 cellspGEM-HCJ4 containing the whole HCV lb genome was used as template for amplification and cloning of the desired F and C regions by proper primers. The amplified DNAs were cloned in-frame into pcDNA3.1+ to construct pcDNA3.1+-F, pcDNA3.1+-C, and pcDNA3.1+-CF. The F gene containing plasmids were transfected into HepG2 cells alone or in combination with C gene containing plasmids in varied ratios (10:1, 1:1, 1:10) for transient expression. After 48h transfection, Western blot analysis confirmed the expression of F protein and C protein, using a rabbit antiserum and a pooled anti-HCV positive serum as the first antibodiy to F protein and C protein respectively. Western blot and real-time quantitive PCR were used to determine the expression changes of c-myc, p21, and p53 in order to examine whether F protein has regulatory properties against these celluar genes. At 48h post-transfection, F protein enhanced the expression of c-myc and p21 by 3.8 times and 41% respectively, decreased that of p53 by 67%, compared with the empty vector DNA(defined as 100%). while, C protein had the similar tendency ininfluencing the (anti-)oncogenes by enhancing c-myc and p21 by 60% and 21%, decreasing p53 by 59%. The changes of p21 and p53 by qPCR were parallelled with those by Western blot, which demonstrated the effects exerted by F protein took place in the transcription level. The co-transfection tests failed to support the interaction between F and C proteins in terms of modulating the transcriptions of these three (anti-)oncogenes. F protein has cellular transformation activity through up-regulating oncogene c-myc and down-regulating anti-oncogene p53. Whileas, F ptotein could inhibit the transformation activity by up-regulating anti-oncogene p21. The transformational function of F protein merits further investigation. Summary1. EIA demonstrated that 15 (43%) of 35 HCV serum samples had anti-F, and F proteins could synthesized in patients infected with HCV naturally. Our study suggests that F protein has a potential usage in laboratory detection as an alternative antigen against HCV infection, because of the steady anti-F positive rates in acute or chronic cases, it also implies the newly identified protein as a possible protective vaccine against HCV infection.2. HCV F and C proteins could be synthesized transiently alone or in combination when transfected into HepG2 cells, after cloned into pcDNA3.1+. Western blot or qPCR analysis confirmed that the F protein, similar to C protein, could increase the transcrptions of c-myc and p21, with decrease in p53. Co-transfection tests failed to support the interaction between F and C proteins on modulating the three (anti-)oncogene transcriptions. F protein has bi-directional cellular transformation activities through up-regulating oncogene c-myc, down-regulating anti-oncogene p53, and up-regulating anti-oncogene p21.