The non-structural proteins NS3 and NS5A of Hepatitis C Virus (HCV) are degraded by two host proteolysis systems

Abstract

Hepatitis Virus C (HCV) is still a major medical challenge worldwide, with more than 70 million infected individuals. HCV is a 9.6 kb RNA virus that encodes a single polyprotein, which is split into 10 structural and non-structural (NS) proteins. The NS proteins are involved in various steps of the virus life cycle. They are required in much lower concentrations than the structural proteins, which are packaged in the viral particle. Nevertheless, they do not accumulate to very high concentrations, therefore, they must be degraded during an ongoing infection. In this study two non-structural proteins were chosen, NS3 and NS5A, and it was analyzed whether two major protein degradation pathways of the host are involved in their turnover. The half-lives of NS3 and NS5A were quantified in the absence and in the presence of modulators of the autolysosome and the proteasome. Inhibitors of both systems increased the half-life, while inducers decreased the half-life. In addition, polyubiquitination of NS3 and NS5A was observed, in agreement with their degradation via the proteasome. Furthermore, intracellular co-localization of both proteins with marker proteins for the autolysosome (LAMP2) and the proteasome (PSMB5) was observed using fluorescence microscopy, and inhibitors of both systems increased the degree of co-localization. Finally, the HCV replication complex was isolated using the membrane flotation assay, and the two autolysosome marker proteins LAMP2 and P62 were found in the same fractions. Taken together, various biochemical and cell biological assays provided evidence that both host proteolysis systems, the autolysosome and the proteasome, are involved in degrading HCV non-structural proteins, at least NS3 and NS5A. A better understanding of NS protein degradation might help to improve medical interventions during HCV infections in the future.