Microsoft word - 35rowlands.targeting the hepa.doc

Targeting the Hepatitis C virus ion channel p7 for anti-viral therapy
Stephen Griffin, Dean Clarke, Steve Evans, Alastair Smith, Joachim Jäger,
Introduction
Hepatitis C virus (HCV) currently infects over 3 % of the world population and is the major
indicator for liver transplant surgery in the west. Acute infection is usually asymptomatic but
leads to persistence in the majority of cases causing chronic liver disease. Treatment of the
virus is currently limited to the use of type 1 interferon either alone, or in combination with
the guanosine analogue ribavirin, and this therapeutic regime is expensive, poorly tolerated,
and effective in only 40 % of cases world-wide. Furthermore, resistance
to this treatment is common in the viral genotypes found in the west.
The search for a vaccine and alternative therapies has been hampered by
the current inability to successfully culture the virus in vitro, making the
identification of new anti-viral drug targets paramount.
The HCV p7 protein is a small hydrophobic protein of 63 amino acids
comprised of two trans-membrane alpha helices separated by a short
positively charged cytoplasmic loop (Fig. 1). It is predicted to belong to
a family of proteins known as viroporins, which homo-oligomerise to
form aqueous pores in cellular membranes. Perhaps the best
characterised of these proteins is the M2 channel of Influenza A virus: Fig. 1. Computer
the target of the first anti-viral drug, Amantadine.

Biophysical studies of p7 oligomers
High level expression of a p7 was achieved by fusion with glutathione-S-transferase in E. coli
separated by a 6-histidine linker: GST-HIS-p7. Oligomeric complexes were visualised by Transmission Electron Microscopy (TEM) in collaboration with Dr Lucy Beales (University of Leeds). Both GST-HIS-p7 and cleaved near-native HIS-p7 (Fig. 2) were shown to form ring-like structures with dimensions Fig. 2. 1-3; TEM of HISp7 oligomers. 4 & 5;
modelling performed by Dr Joachim Jaeger collaborating with the laboratories of Dr Alastair Smith and Professor Jennifer Kirkham
(University of Leeds) in order to visualise these complexes by Atomic Force Microscopy
(AFM) in lipid membranes under aqueous conditions.
In vitro ion channel formation by p7 is inhibited by
Amantadine
In collaboration with the laboratory of Professor Stephen
Evans (University of Leeds), purified HIS-p7 was shown
to form ion channels in artificial lipid bilayers using a
Black Lipid Membrane (BLM) system. p7 ion channels
showed a preference for calcium ions over potassium,
consistent with the proteins’ reticular localisation in living
Fig. 3. p7 ion channel activity
cells. Furthermore, this activity was abrogated by the before (top) and after (bottom) addition of Amantadine (Fig. 3). Recent clinical trials that adding 1 µM Amantadine include this drug with existing therapy show efficacy in many cases, particularly in those
patients who were previously non-responsive. We propose that p7 is the target of
Amantadine and are working with GlaxoSmithKline to develop new anti-HCV compounds
based on this finding.
p7 modulates cellular membrane permeability
Recently, we have gone on to assess the effect of expressing p7 in the context of living cells.
Inducible expression of native p7 in E. coli was shown to cause a growth-inhibitory effect
due to membrane leakage, which was reversed by the addition of Amantadine. Furthermore,
mutation of the charged loop also negated this effect consistent with studies in the related
Pestivirus: Bovine Viral Diarrhoea Virus.
We have also demonstrated p7-induced membrane permeability in mammalian cells causing
enhanced susceptibility to Hygromycin B. In addition, we have shown in collaboration with
Dr Wendy Barclay (University of Reading) that p7 can functionally replace Influenza A M2
protein in a bioassay and is again inhibited by the addition of Amantadine.
Further studies of p7 and other viroporins
We are currently looking at chimeric viral systems in order to assess the function of p7 in the
context of virus replication and it’s ability to functionally
complement similar proteins from other viruses. The p7 of the
closely related GB Virus B, BVDV p7, Picornavirus 2B (Fig. 4),
and Flavivirus NS1 proteins are all candidate systems for such
studies. Work has already begun on characterising these proteins
and construction of viral chimeras. We are also attempting to
crystallise the p7 of HCV and of BVDV, as well as pursuing other
structural investigations such as NMR and mass spectroscopy in
order to determine the precise composition of the ion channel
structure for rational drug design.
Fig. 4. GFP-tagged
Publication
Griffin, S.D., Beales, L.P., Clarke, D.S., Worsfold, O., Evans, S.D., Jaeger, J., Harris, M.P. &
Rowlands, D.J. (2003) The p7 protein of hepatitis C virus forms an ion channel that is
blocked by the antiviral drug, Amantadine. (2003) FEBS Lett 535, 34-8
Funding
This work was funded by the MRC “Enabling Technologies” Co-operative, University of
Leeds.

Source: http://www.astbury.leeds.ac.uk/Report/2002/Report/35Rowlands.pdf