Function Of Hepatitis C Virus Non-structural Protein NS4B

Hepatitis C virus (HCV) infection is an important public health problem worldwide, which is a major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma.HCV-encoded proteins have been identified in vitro and in vivo system in spite of the lack of reliable cell culture. HCV encodes a long polyprotein which products at least ten discrete proteins by host and viral proteinase. These include core protein(C),envelope proteins (El and E2), and other non-structural proteins (NS2 to NS5B).Research efforts are focusing on the properties and functions of individual HCV gene product in the interst of unraveling the mechanisms of its pathogenesis.The function of NS4B is incompletely understood. The aim of the study is to understand its function. Conventional methods to search for genes are usually time-consuming and limited by the view scope. DNA microarray provides a powerful alternation with an unprecedent view scope in monitoring gene expression levels and leads to discoveries of regulatory pathways involved in complicated biological processes.HeLa cells transfected by pCDNA3.1(-) and recombinant pCDNA3.1(-)NS4B served as control group and test group, respectively. Stably expressing NS4B HeLa cell line was established through G418 selection and was confirmed by RT-PCR and western blot analysis. By a cDNA microarray representing 2308 signal transduction-related genes, we studied the expression profiles of HeLa cells impacted by NS4B. The criterion for inclusion of a cDNA in a group as either induced or repressed expression was whetherbalanced differential expression was greater than 2.5 in either direction. If Cy5/Cy3>2.5, the gene was considered to be induced, and if Cy3/Cy5>2.5, the gene was considered to be repressed. Among 90 genes, whose expression has changed by more than 2.5 fold, 34 genes are up-regulated and 56 genes are down-regulated. In those 34 up-regulated genes, the expression of NPTX1 changes up to 14.44 fold. The expression of Dickkopf homolog 1 (DKK1), syndecan 1 (SDC1) and API change over 5 fold. The 10 genes down-regulated most dramatically are AKR1C1, IL10RA, substance K, transforming growth factor, beta(TGF- β ), leukemia inhibitory factor receptor (LIFR), H factor (complement)-like 3 (HFL3), fibronectin 1 (FN1), adducin 3 (gamma) (ADD3), carboxypeptidase E (CPE) and dihydropyrimidinase-like 3 (DPYSL3). The expression of these genes changes more than 8 fold. We classified the differentially expressed genes into seven functional groups. These groups are: 1) tumor-associated 2) cytokines, receptors, adhesions and complement 3) transcription and translation -associated 4) kinase and phosphatase 5) cellular stress 6) cytoskeleton 7) others. To verify the results from DNA microarray data,four genes (two up-regulated genes, NPTX1 and API; two down-regulated genes, AKR1C1 and IL10RA) were chosed to analyse with teal-time quantitative PCR,the results of which were consistent with that of DNA microarray data. Additionally, AKR1C1 enzyme activity was detected in HeLa and Huh-7 cells thansfected by NS4B.The results showed that AKR1C1 activity was inhibited either in stably expressing NS4B HeLa cells or in HeLa and Huh-7 cells transiently transfected by pCDNA3.1(-)NS4B.So DNA microarray data was reliable.Based on DNA microarray data, the genes impacted by NS4B mainly involved in carcinogenesis, the host defense system and cell homeostatic mechanisms.Thus HCV-NS4B could play some important roles in the pathogenesis mechanism of HCV, in particular in chronic hepatitis, even in hepatocellular carcinoma.NS4B is hydrophobic protein, and contains at least four trans-membrane domains (TMD), and interacts with ATF6.HCV subreplicons contained NS4B could induce unfolded protein response (UPR).In mammalian cells, three proteins, ATF6JRE1 and PERK located in ER could sense UPR and were activated, and induce the downstreamgenes. The study aimed to demonstrate whether NS4B can induce UPR. We detected the localization of NS4B; the unspliced and spliced forms of XBP1 in HeLa cells with NS4B; The transcriptional levels of activating transcription factor-6 (ATF6), glucose response protein (Grp78) and caspase-12 in HeLa and Huh-7 cells expressing NS4B, and the luciferase activity in X-box-binding protein (XBP1) and Grp78 promoter. The results showed that HCV NS4B localized in the cytoplasm, the two forms of XBP1 were detected in HeLa cells expressing NS4B, moreover, ATF6 and Grp78 transcription levels and the luciferase activity in XBP1 and Grp78 promoter in cells expressing NS4B increased compared with the controls due to XBP1 binding to their promoter sites. Collectively, our results imply the possibility that NS4B induces UPR through ATF6 or IRE1-XBP1 pathways upon ER stress, and maybe play some roles in HCV pathogenesis.