Studies On The Mechanisms Of The Interaction Between Nonstructural Protein 4 Of Hepatitis C Virus And The Hepatocyte Proteins

Hepatitis C virus (HCV) is the main cause of acute and chronic hepatitis with a significant risk of end-stage liver cirrhosis and hepatocellular carcinoma in our country. The interaction between the virus nucleotides, proteins and the hepatocyte nucleotides, proteins may contribute to one of the major molecular pathogenesis of the diseases caused by HCV. The HCV genome consists of a positive-stranded RNA molecule of about 9.6 kilobases and has only one ORF which encodes a large polyprotein precursor (about 3010 to 3033 amino acids). This precursor protein is proteolytically processed by the host cellular signal peptidase and viral proteinase to generate at least 10 proteins: the core, envelope 1 (El), E2, p7, nonstructural (NS) 2, NS3, NS4A, NS4B, NS5A, and NS5B. In addition, studies indicate the ORF of HCV core could move to an novel one, named F protein, which weigh 17 kD. These HCV proteins not only play important role in viral replication and propagation but also affect a variety of cellular functions, but the specific mechanisms are poorly understood. In this study, we aim at two of the nonstructural proteins, NS4A and NS4B, to explore their regulation effects on hepatocellular gene expression profiles and the interaction with hepatocellular proteins. These studies will pave the way for better understanding the pathogenesis of NS4A and NS4B and further development the therapeutic strategies of viral hepatitis and related liver diseases.First, the recombined expression plasmids pcDNA3. 1(-)-NS4A and pcDNA3. l(-)-NS4B were constructed, respectively, and were cotransfected into the hepatocellular carcinoma cell lines HepG2 cells with pCAT3-Promoter, which contains chloramphenicol acetyltransferase (CAT) gene regulated by SV40 immediate early promoter. After 48 h, cells werecollected and detected for the expression of the CAT gene by an enzyme-linked immunosorbent assay (ELISA). Moreover, HepG2 cells were transiently transfected with pcDNA3.1(-)-NS4A and pcDNA3. 1(-)-NS4B, respectively, and pcDNA3. 1 (-) empty vector was used as control. The mRNA was isolated from HepG2 cells transfected by the vectors and suppression subtractive hybridization (SSH) method was employed to analyze the differentially expressed DNA sequence between the two groups. The obtained subtractive library was sequenced and analyzed in GenBank with Blast search after PCR. Results indicate that the activity of CAT in HepG2 cells transfected by the pcDNA3. 1(-)-NS4A and pcDNA3. 1(-)-NS4B were higher than that in HepG2 cells transfected by control plasmid. NS4A and NS4B have the transactivation effect on SV40 promoter in some extent. From the SSH analysis, we concluded that NS4A and NS4B could upregulate the expression of many genes in hepatocytes, among which some genes coding proteins involved in cell cycle regulation, signal transduction, translation and synthesis of protein, tumor development, and metabolism. In addition, we have discovered two novel genes with unknown function transactivted by NS4A, named as NS4ATP1 and NS4ATP2. The two novel genes were deposited into GenBank, with the number AY740521 and AY846876, respectively.Second, the yeast expression plasmids pGBKT7-NS4A and pGBKT7-NS4B were constructed and transformed into yeast AH109, respectively. The yeast proteins were isolated and detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis. Then, we adopted yeast two-hybrid system to seek the proteins in hepatocytes interacting with HCV NS4A and NS4B. Results showed that HCV NS4A and NS4B gene were successfully expressed within yeast cells AH109. The relative molecular weight of the expressed fusion products were approximately 27kD and 48kD, respectively. The results of the yeast two-hybrid screening indicated there were seven kinds of hepatocytes proteins interacting with NS4B: metallothionein 2A, eukaryotic translation elongation factor 1 alpha 1, albumin, RNA binding motif protein 21, myomesin, cytochrome c oxidase II, and Na+K+ ATPase. There are five kinds of hepatocytes proteins interacting with NS4B: NADH dehydrogenase...