NS2 Protein of Hepatitis C Virus Interacts with Structural and Non-Structural Proteins towards Virus Assembly

Abstract

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.

Author Summary

Hepatitis C virus (HCV) causes major health problems worldwide. Understanding the major steps of the life cycle of this virus is essential to developing new and more efficient antiviral molecules. Virus assembly is the least understood step of the HCV life cycle. Growing experimental evidence indicates that, in addition to the physical virion components, the HCV non-structural proteins are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. Molecular interactions between NS2 and other HCV proteins were demonstrated. Furthermore, NS2 was shown to accumulate over time in endoplasmic reticulum-derived structures and to colocalize with the viral envelope glycoproteins and viral components of the replication complex in close proximity to the HCV core protein and lipid droplets. Importantly, specific mutations within NS2 that affected HCV infectivity could also alter the subcellular localization of NS2 protein and its interactions, suggesting that this subcellular localization and its interactions are essential for HCV particle assembly. Altogether, these observations indicate that NS2 protein plays an important role in connecting different viral components that are essential for virus assembly.