Description
Hepatitis C Virus (HCV) is the most common chronic blood- borne infection in the United States and is the main cause for liver cancer worldwide. The non-specific treatment available, together with the two protease inhibitors recently approved by the FDA, are expensive, toxic and only effective for certain patients. Therefore, a better understanding of the HCV life cycle is essential for developing novel drug compounds exhibiting higher efficacy and better tolerance. HCV belongs to the Flaviviridae family which is an enveloped, single stranded (+) sense RNA virus encoding a single polyprotein. The main viral NS3 protease processes the non- structural (NS) region of HCV in conjunction with the NS2 and NS4A cofactors, an essential step for particle assembly. Several studies have been aimed at understanding the substrate requirement for polyprotein processing by NS3, but the role of cofactors and their mechanism of action remain elusive. In our laboratory we are utilizing our Gal4 tetracycline-inducible cell-based assay previously adapted for HIV-1 protease, for the monitoring of NS3 activity in hepatocytes; the natural milieu of HCV infection. To elucidate the role of NS2 and NS4A in the viral life cycle, we exploit the power of flow cytometry, where GFP expression serves as biosensor for NS3 activity. We have produced NS2, NS3, and NS4A with wild type and mutated cleavage sites, to test in cis as well as in trans, the cleavage activity within the assay. The subcellular localization of the various forms of NS3 and NS3/4A expressed in hepatocytes was investigated by indirect immunofluorescence and confocal laser scanning microscopy. We have observed that full length and various forms of NS3 were found in a diffused cytoplasmic and nuclear localization. In contrast, NS3/4A was found to be localized in the ER. These results demonstrate the importance of studying HCV proteins in their biological context. To further elucidate the role of NS2/3/4A we are analyzing their effect on signaling cascades using PhosphoFlow, a technique that allows detection of phosphorylation events through intracellular staining. PhosphoFlow coupled with a cell-based assay can greatly facilitate the study of the viral cofactors on the HCV viral life cycle.
