Possible future Hep C vaccination

The study is to be published by the Journal of General Virology and is available in the journal's March 4 pre-print online edition.www.upi.com
Copyright 2009 by United Press International
Investigation of a role for lysine residues in non-structural proteins 2 and 2/3 of the hepatitis C virus for their degradation and virus assemblySarah Welbourn1, Vlastimil Jirasko2, Valérie Breton1, Simon Reiss2, Francois Penin3, Ralf Bartenschlager2 and Arnim Pause1,4
1 McGill University;2 University of Heidelberg;3 Université de Lyon
4 E-mail: arnim.pause@mcgill.ca

It has been demonstrated that both uncleaved, enzymatically 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 lysine-mutagenesis 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 NS2-lysine mutagenesis of protease-inactive NS2/3 results in a partial stabilisation of this protein, the increased 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.
Received 24 December 2008; accepted 29 January 2009.