The NS 5 A Protein of Hepatitis C Virus Transcriptionally Upregulates the AGR 3 Gene Expression

The non-structural protein 5A (NS5A) of hepatitis C virus (HCV) is a multifunctional protein involved in the HCV lifecycle and pathogenesis. The precise molecular mechanisms of NS5A-mediated pathogenesis still remain to be clarified. In this study, we performed cDNA microarray analysis on NS5A-expressing HEK293 cells and the non-expressing control to screen the possible cellular genes dysregulated by NS5A. Subsequent quantitative real time PCR (qRT-PCR) analysis on NS5A-expressing cells and the control confirmed that NS5A upregulated the anterior gradient homolog 3 (AGR3) mRNA expression. The domain III of NS5A was responsible for the activation of AGR3 gene expression. AGR3 mRNA expression levels were upregulated also in Huh7.5 cells harboring a full-genome HCV-1b RNA replicon (FGR) and in those infected with HCV-2a. Moreover, AGR3 promoter activity was activated in NS5A-expressing cells, FGR-harboring cells and HCV-infected cells. Taken together, our present results suggest that HCV NS5A transcriptionally activates the cancer-associated AGR3 gene. This may be a novel mechanism of HCV-mediated pathogenesis, especially hepatocarcinogenesis. INTRODUCTION Hepatitis C virus (HCV) infection is associated with severe liver disease, including chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC) (1). HCV is an enveloped virus with single-stranded, positive-sense RNA that belongs to the Hepacivirus genus of the Flaviviridae family. The HCV genome is approximately 9.6 kb in length and encodes a single polyprotein with ~3000 amino acids (aa), which is coand post-translationally processed by cellular and viral proteases into 10 individual viral proteins: the structural proteins, core, E1 and E2, and p7, the non-structural proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B (2). HCV NS5A is a pleiotropic protein involved in not only viral replication and assembly, but also HCV-induced pathogenesis. NS5A inhibits interferon (IFN) antiviral activity, dysregulates glucose and lipid metabolism, and induces HCC through interactions with numerous host proteins (3). However, the exact mechanism by which NS5A induces HCC remains to be fully clarified. The anterior gradient 3 (AGR3) gene encodes a protein, AGR3, also known as breast cancer membrane protein 11 (BCMP11) (4). It belongs to the protein disulfide isomerase (PDI) family involved in relieving endoplasmic reticulum (ER) stress by regulating protein disulfide bond formation, protein folding and maturation (5). AGR3 has been implicated in breast, prostate and ovary tumorigenesis (4, 6-8). In this study, we adopted microarray assay to screen the possible genes dysregulated by NS5A. Subsequent quantitative real time PCR (qRT-PCR) analysis confirmed that NS5A upregulated the AGR3 gene expression. Also, HCV replication and HCV infection induced AGR3 gene expression and promoter activity. Our results thus provide a clue to clarify a novel molecular mechanism of HCV-induced pathogenesis. MATERIALS AND METHODS Plasmids Myc-tagged NS5A expression plasmid, HA-tagged full-length NS5A and NS5A deletion mutant expression plasmids were generated as described previously (9). The expression plasmids of the other HCV proteins were as described elsewhere (10).