The Anti-IFN-α Effects Of The Tp Associated Protein Of Hepatitis B Virus

Hepatitis B virus (HBV) is the member of the Hepadnaviridae family, with a narrow host range which are human or gorillas, infection of HBV resulted in a broad spectrum of clinical manifestations, including asymptomatic carrier, acute hepatitis, chronic hepatitis, cirrhosis and hepatocellular carcinoma. Interferon-α(IFN-α) is one of the most commonly used drugs for the treatment of hepatitis B, it can inhibit and ultimately clear the HBV by immunomodulation and establishing intracellular antiviral state. However, it was found that approximately two thirds of IFN-αtreated patients showed IFN-αnon-responseness, i.e., no loss of HBeAg and reduction of serum HBV DNA lever. In addition to the host factors, it is also related to the anti-IFN-αeffects of virus itself. At present,the anti-IFN-αeffects of the HBV DNA polymerase was recognized specificly. Foster et al demonstrated that terminal protein (TP) of HBV DNA polymerase could dramatically inhibit the cellular response to IFN-αby blocking the IFN pathway, yet the related functional region of TP was remained largely obscure.The genotype B and C HBV are the predominant genotype in china. Several studies have shown that patients with genotype B have a higher probability of hepatitis B e antigen (HBeAg) seroconversion and the reduce of virus titer than patients with genotype C during IFN-αtreatment. Our analysis of a large sample of HBV DNA polymerase amino acid sequences ascertains that there are separately 14 and 44 diversity in TP and Spacer regionsre spectively.Whether these diversity were corresponded with the different responds to IFN-αtreatment in patients with genotype B and patients with genotype C was still uncertainty now.In fact , TP,which is located in the N-terminal of HBV DNA polymerase , can only exist in the form of pol,but, a novel method for efficient amplification of whole hepatitis B virus genome maked it possible to isolate a variety of splice variants and deletion mutants from serum of chronic hepatitis patients. these HBV subgenomic DNA which generated by Splicing and deletion beyond 2309nt from the HBV complete genomic DNA can encode TP Associated proteins. It is presently lack of the systematic study about the anti-IFN-αeffects of these TP Associated protein. To address all these issues, our study includes three parts1. Analysis of the functional region for anti-IFN-αeffects by TP+Spacer of HBV DNA polymeraseThe fragments of the serial deletants of TP+Spacer were amplificated from FMU013 which a recombinated vector of HBV complete genome and cloned into the pcDNA3.1/HisC vector. Huh7 hepatocytes were co-transfected with p6-16CAT and the recombinant vectors harboring deleted TP+Spacer,then, treated with IFN-α.The intracellular CAT expression were calculated to evaluate the anti- IFN-αeffects.2. Comparison of the anti-IFN-αeffects of genotype B and C TP+Spacer regionIsolated DNA from IFN pre-treatment serum samples which from chronic hepatitis patients including responsers and non-responsers. Genotypes of all samples were determined by restriction fragment length polymorphism。The Tp257 fragments of all samples were amplificated from isolated DNA and cloned into the pcDNA3.1/HisC vector. Huh7 hepatocytes were co-transfected with p6-16CAT and the recombinant vectors harboring Tp257, then, treated with IFN-α.The intracellular CAT expression were calculated to comparison of the anti-IFN-αeffects of genotype B and C TP associated protein.3. The anti-IFN-αeffects of the Tp associated protein encoded by HBV splice variants and deletion mutantsIsolated DNA from serum samples of chronic hepatitis patients, complete HBV genomes were amplificated from isolated serum DNA and cloned into pUC18 vector and sequenced. Identified all the possible existent Splice variants and deletion mutants which spliced and deleted beyond 2309nt. The PCR fragment of the Tp Associated proteins encoded by these HBV subgenomic DNA were cloned into the pcDNA3.1/HisC vector. Huh7 hepatocytes were co-transfected with p6-16CAT and the recombinant vectors harboring the fragment of the Tp Associated proteins , then, treated with IFN-α.The intracellular CAT expression were calculated to determine the anti-IFN-αeffects of the Tp associated protein encoded by HBV splice variants and deletion mutants . Our study demonstrated that functional region for anti-IFN-αeffects of the Tp associated protein was closely related to the Spacer region, and the complete TP indispensable for this effect. Although,the differences of amino acids sequences from Tp + Spacer region between genotype B and C were significant, it showed that they were not attributed to the different anti-IFN-αeffects. Eight kinds of splice variants and five kinds of deletion mutants were obtained,and two kinds of TP associated proteins with anti-IFN-αeffects were confirmed.