It has been demonstrated that both uncleaved, enzymitically inactive NS2/3 and cleaved NS2 proteins are rapidly degraded upon expression in cells, phenomena described to be blocked by the addition of proteasome inhibitors. As this degradation and its regulation potentially constitute an important strategy of the hepatitis C virus (HCV) to regulate the levels of its non-structural proteins, we further investigated the turnover of these proteins in relevant RNA replication systems. A

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... nesis approach was used in an effort to prevent protein degradation and determine any effect on various steps of the viral replication cycle. We show that, while NS2lysine mutagenesis of protease-inactive NS2/3 results in a partial stabilization of this protein, the increased NS2/3 levels do not rescue the inability of NS2/3 protease inactive replicons to replicate, suggesting that uncleaved NS2/3 is unable to functionally replace NS3 in RNA replication. Furthermore, we show that the cleaved NS2 protein is rapidly degraded in several transient and stable RNA replicon systems and that NS2 from several different genotypes also has a short half-life, highlighting the potential importance of the regulation of NS2 levels for the viral life cycle. However, in contrast to uncleaved NS2/3, neither ubiquitin nor proteasomal degradation appear to be significantly involved in NS2 degradation. Finally, although NS2 lysine-to-arginine mutagenesis does not affect this protein's levels in a JFH-1 cell culture infection system, several of these residues are identified to be involved in virion assembly, further substantiating the importance of regions of this protein for production of infectious virus. Cellular protein levels are tightly regulated by the balance between transcription/translation and degradation. Interestingly, many virally encoded proteins have also been demonstrated to be quickly degraded, suggesting that control of their levels could regulate their role in the viral life cycle (Mousnier et al., 2007; Schlax et al., 2007) . Furthermore, levels of several HCV proteins such as core, E2 and NS5B have been suggested to be regulated at the Supplementary material is available with the online version of this paper.