Potential Role Of Alternate Reading Frame Protein In Chronic Hepatitis C Infection And The Association Of TBX21 Polymorphisms With Virus Persistence From A High-risk Chinese Population

Hepatitis C virus(HCV) infection is one of the major causes of chronic liver disease and has a high rate of chronicity. It has been reported that around 75-85% of newly infected people developed into chronic infection and 20% of infected people developed into chronic liver disease, including cirrhosis and hepatocellular carcinoma(HCC). HCV infection is prevalent worldwidely, and recent data from WHO declared that more than 180 million people get infected with this virus so far.Although the mechanisms underlying chronic HCV infection are not well understood, it has been accepted that newly discovered alternative reading frame protein(ARFP) may play a pivotal role in the progression of HCV infection. ARFP protein was reported to prolong virus survival through activing NF-κB which inhibits the cell apoptosis induced by TNF-α. Moreover, experimental data suggested that the novel ARFP protein certainly possesses various activities attributed to Core protein, such as pro- and anti-apoptotic effects, regulation of cell growth, transcriptional activities and immune-regulatory properties including decreasing Th1 cytokine secretions.Our previous results demonstrated that the rate of ARFP-seropositivity was associated with progression of the disease and it showed higher rate of anti-F antibody(F-Ab) seropositivity in chronic hepatitis patients(CHP) than acute hepatitis patients(AHP). And we have made an intriguing observation that ARFP protein may participate in the progression and/or outcomes of the infection through regulating the secretion of cytokines such as gamma interferon(IFN-γ), interluekin-2, 4, 5, 10(IL-2, IL-4, IL-5 and IL-10) in F-Ab(+)CHP and F-Ab(-)CHP. In addition, we found that decreased IFN-γ and/or IL-2 and increased IL-4 and/or IL-5 levels appeared in cultured PBMCs supernatant after HCV Core/ARFP protein stimulation in vitro. It suggested that ARFP protein may be a novel product for the virus to adapt to the host environment through imbalance of Th1/Th2 cytokines. It has been reported that T-bet is a Th1-specific transcription factor which appears to act as a master switch for Th1 development. ARFP protein may participate in the progress of chronic HCV infection by affecting the expression of the transcription factor. Therefore, the relevant research is of great significance.In order to explore the correlation between ARFP protein and Th1/Th2 biased cytokine responses and the mechanisms which may contribute to chronic inflammation or malignancy, HCV 1b F gene sequences was introduced into the upstream of a expression vector pCold II after a hexahistidine tail, which allowing its overexpression in Escherichia Coli and easy purification. ARFP protein was purified by a Ni-nitrilotriacetic acid agarose column. A total of 104 cases including CHC patients and healthy donors were enrolled. T-bet and GATA3 expression levels were analyzed in peripheral blood mononuclear cells(PBMCs). The levels of signal transducer and activator of transcription-1/-6(STAT1/6) and phosphorylated STAT1/6(pSTAT1/6) in PBMCs were measured by Western blotting. Our results showed that the levels of T-bet in PBMCs, as well as the levels of IFN-γ in sera, were decreased in F-Ab(+) patients when compared with F-Ab(-), whereas the levels of GATA3 didn’t show difference between two groups. Moreover, decreased pSTAT1 and increased pSTAT6 were observed in PBMCs by HCV Core/ARFP protein stimulation, while STAT1/6 expression stayed constant. Taken together, it suggested that T-bet may be involved in Th1/Th2 bias induced by HCV ARFP protein and the disruption of STAT phosphorylation may participate in this mediation.In the study of the relationships of the TBX21 gene polymorphisms and HCV infection, we used logistic regression analysis. Three single nucleotide polymorphisms(SNPs)(rs17250932, rs2074190, rs4794067) in TBX21 gene were genotyped using improved multiple ligase detection reaction(iMLDR).All subjects were recruited from Jurong and Danyang, Jiangsu Province, including 354 healthy controls and 747 CHC patients(190 anti-ARFP protein antibody seronegative patients and 557 anti-ARFP protein antibody seropositive patients). The study was carried out in a case-control and case-case study in a cohort of high-risk group. Results showed that rs4794067 C allele in TBX21 promoter was significantly more common in CHC patients(OR=1.335, 95% CI=1.058-1.684, P=0.015), especially in anti-ARFP protein seropositive patients(OR=1.547, 95% CI=1.140-2.101, P=0.005), compared with healthy controls. In stratified analysis, the risk effect was also significantly high in patients with HCV 1b genotype and mild fibrosis(P=0.021, P=0.010, respectively). Compared with the most frequent haplotype TAT, the distribution of TAC was significantly different between the chronic HCV carrier group and the healthy group, and so was anti-ARFP antibody seronegativity group and anti-ARFP antibody seronegativity group(all P<0.001). Our results indicated the association of SNPs in TBX21 with susceptibility to chronic HCV infection and the ARFP protein generation for the first time. It suggested that TBX21 variants may be involved in the etiology of this disease.Our subject conducted a study that a novel pathogenic antigen(viral gene mutation), human molecular network(Th1/Th2 balance), and TBX21 gene polymorphisms may be implicated in the pathogenesis of chronic HCV infection. These findings may help to clarify the mechanisms and associated factors influencing the pathogenesis of hepatitis C virus infection. And, it means a lot in the development of preventive and therapeutic anti-HCV agents and new strategies of antiviral therapy.