Characterization of HCV NS5A cellular interactors: role of MOB1B and LRPPRC in viral replication and assembly

Hepatitis C virus (HCV) is a hepatotropic positive-strand RNA virus which belongs to the Flaviviridae family. With almost 170 million people infected worldwide, HCV is a leading cause of chronic hepatitis, liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC) (Poynard, T., et al., 2003). Major improvements in patient treatment were achieved by the approval of the first direct acting antivirals (DAAs) in 2011 (Welsch, C., et al., 2012). Nonetheless, HCV variants resistant to DAAs may arise during treatment as well as a heavy burden of side effects, thus affecting therapy outcome. Cellular models of HCV replication and infection have recently allowed the characterization of molecular details of HCV life cycle. HCV RNA replication occurs in association with ER-like cellular membranes and requires several viral non-structural (NS) proteins including; NS3, NS4A, NS4B, NS5A, and NS5B, as well as host cell factors (Moriishi, K., and Matsuura, Y., 2003; Gosert R., et al., 2003). The site of virus assembly is yet unknown, but recent data haves proposed the recruitment of HCV RNA from the replicase complex to lipid droplets mediated by the HCV core and the non-structural proteins as an early event in virion assembly (Miyanari, Y., et al., 2007). Since HCV is a relative small virus, in order to accomplish genome replication and formation of new viral particle, it needs to interact with and subvert cellular machineries for its own purpose. A large number proteinprotein interaction has been described between HCV and host cells. However to date, most of them are only descriptive and their functions in HCV life cycle remain to be characterized (Tellinghuisen and Rice, 2002). NS5A is an important component of the viral replication complex and also participates to HCV particle assembly (Kim, S., et al., 2010). While no known enzymatic function has been ascribed to NS5A, it is known to interact with host cell proteins to affect a variety of processes, including innate immunity, host cell growth and proliferation (He, Y., et al., 2006). The molecular mechanism by which NS5A contributes to HCV life cycles remains largely uncharacterized. To get insights on NS5A function, we performed a screening to search for cellular proteins interacting with NS5A in HCV replicon cells, by means of a tandem affinity purification approach coupled to mass spectrometry (TAPMS/MS). NS5A protein complexes were isolated from HCV replicon cells, resolved by monodimensional gel (SDS-PAGE) and revealed by SYPRORuby staining. Protein bands were submitted to trypsin digestion and identified by MALDI-TOF/TOF mass spectrometry. 24 proteins were identified as NS5A binding proteins, including 14 factors not previously reported. Among them, we functionally characterized the interaction of NS5A with the kinase protein Mob1b, an important regulator of the Hippo pathway, and the mitochondrial protein LRPPRC. Here we show that individually knockdown of Mob1b and LRPPRC significantly reduces new HCV particle production and, to lesser extent, intracellular HCV RNA replication. We also found that Mob1b relocalizes to NS5A-positive structures and Mob1b downregulation results in a decrease in NS5A phosphorylation. Finally, we show that the Hippo signaling pathway is perturbed in HCV replicon cells, as indicated by the increased nuclear localization of the Mob1b target transcription factor Yap. Furthermore we observed that NS5A interaction with LRPPRC occurs at mitochondria-ER synapses (MAM) and that LRPPRC downregulation causes a significant increase in different the inflammation-related genes: IFNβ, Mx1, IL 6 and TNFα. Altogether, our results indicate that, through NS5A interactions, HCV hijacks the cellular functions of Mob1b and LRPPRC to ensure its life cycle