Determinants of hepatitis C virus p7 ion channel function and drug sensitivity identified in vitro.
Hepatitis C virus (HCV) chronically infects 170 million individuals, causing severe liver disease. Although antiviral chemotherapy exists, the current regimen is ineffective in 50% of cases due to high levels of innate virus resistance. New, virus-specific therapies are forthcoming although their development has been slow and they are few in number, driving the search for new drug targets. The HCV p7 protein forms an ion channel in vitro and is critical for the secretion of infectious virus. p7 displays sensitivity to several classes of compounds, making it an attractive drug target. We recently demonstrated that p7 compound sensitivity varies according to viral genotype, yet little is known of the residues within p7 responsible for channel activity or drug interactions. Here, we have employed a liposome-based assay for p7 channel function to investigate the genetic basis for compound sensitivity. We demonstrate using chimeric p7 proteins that neither the two trans-membrane helices nor the p7 basic loop individually determines compound sensitivity. Using point mutation analysis, we identify amino acids important for channel function and demonstrate that null mutants exert a dominant negative effect over wild-type protein. We show that, of the three hydrophilic regions within the amino-terminal trans-membrane helix, only the conserved histidine at position 17 is important for genotype 1b p7 channel activity. Mutations predicted to play a structural role affect both channel function and oligomerization kinetics. Lastly, we identify a region at the p7 carboxy terminus which may act as a specific sensitivity determinant for the drug amantadine.
StGelais
Corine
C
Institute of Molecular and Cellular Biology, Astbury Centre for Structural Molecular Biology, Leeds, West Yorkshire, United Kingdom.
Foster
Toshana L
TL
Verow
Mark
M
Atkins
Elizabeth
E
Fishwick
Colin W G
CW
Rowlands
David
D
Harris
Mark
M
Griffin
Stephen
S
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology; D016174:Hepacivirus / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D019698:Hepatitis C, Chronic / Q000188:drug therapy / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000086082:Viroporin Proteins
D000998:Antiviral Agents; D014764:Viral Proteins; C088369:p7 protein, Hepatitis C virus; D000086082:Viroporin Proteins