Structural and functional studies on Hepatitis C virus non-structural proteins.

Mustapha Aoubala, Katherine Crowder, John Holt, Andrew Macdonald, Mick Miller

and Mark Harris

Hepatitis C virus (HCV) is an increasingly important cause of liver disease. The virus has a single stranded positive sense RNA genome of 9.5Kb that contains a long open reading frame encoding a single polyprotein of 3000 amino acids. This is cleaved into 10 individual polypeptides by a combination of host cell and virus specific proteases (see Figure 1). The molecular mechanisms of pathogenesis remain to be elucidated. To this end the laboratory is interested in the potential for the non-structural proteins (expressed from the 3' end of the genome and designated non-structural as they do not form part of the viral particle) to interfere with host cell metabolism and signal transduction pathways.


The non-structural protein NS3 has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). We have used recombinant baculoviruses to express full-length, catalytically-active NS3 and shown that it is also able to inhibit PKA C-subunit in vitro. However both mutational analysis and experiments in which a constant ATP concentration was maintained by the addition of an ATP regeneration system, demonstrated that the ability to inhibit PKA was due to ATPase activity. We are currently pursuing the functional consequences of NS3 expression in mammalian cell lines to determine whether ATPase activity might play a role in pathogenesis of this virus.

NS5A contains a poly-proline motif reported to interact with the SH3 domain of the intracelllular adaptor protein Grb2. Further to this study we have shown that NS5A is also able to interact with the SH3 domains derived from members of the Src family of protein tyrosine kinases. We are currently using phage display, surface plasmon resonance and in vitro binding assays to more precisely understand these interactions. Additionally we are expressing NS5A transiently in mammalian cells to identify the functional consequences of these interactions for signal transduction pathways within the cell.

Collaborators

Roger Clegg, Hannah Research Institute
Derek Mann, University of Southampton
Kalle Saksela, University of Tampere, Finland

Funding: This work is funded by the BBSRC and the MRC.