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.Hepatitis C virus (HCV) chronically infects 170 million individuals and is a major cause of severe liver disease such as cirrhosis and hepatocellular carcinoma. Acute HCV infection is asymptomatic which, combined with the lack of an available vaccine, means that the majority of carriers are unaware of their positive status. Thus, clinical intervention takes place upon the presentation of symptoms when liver damage is already extensive and when the virus is well established. Current therapy comprises a combination of pegylated alpha interferon (IFN-α) with ribavirin (Rib), which is effective in only 50% of cases and is both expensive and poorly tolerated by patients. This relatively low success rate is due to the highly prevalent, IFN-resistant genotype 1 viruses; other genotypes generally respond well to treatment (27). As IFN-Rib acts primarily via stimulation of the immune system, improving current therapy relies on the development of new, virus-specific drugs. A small number of polymerase and protease inhibitors are at late stages of development, but progress has been hampered by the inability until recently to culture HCV in vitro (21, 40, 45). The highly variable nature of HCV, however, means that new drugs will most likely have to be used in combination, making expansion of available drug targets and the development of new inhibitors a major research focus.HCV is the prototype member of the Hepacivirus genus within the Flaviviridae (3). It is enveloped, and its genome is a 9.6-kb positive-sense RNA. This is translated in a cap-independent fashion from an internal ribosome entry site present within the 5′ untranslated region, yielding a 3,000-amino-acid polyprotein, which is cleaved by both cellular and viral proteases to generate 10 mature virus gene products: the structural proteins core and envelope E1 and E2, the p7 ion channel, and the nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B which replicate the viral genome and regulate host cell metabolism (reviewed in reference 23).The p7 ion channel of HCV is sensitive to several classes of inhibitor compounds in vitro (13, 26, 31) and is necessary for HCV to replicate in chimpanzees (32). Recently, p7 was shown to be critical for the secretion of infectious HCV particles in culture (19, 34), and we along with others have shown that drugs which block its activity significantly reduce virus production (12, 35). p7, therefore, represents an important new target for drug development, and clinical trials combining IFN and Rib with a p7 inhibitor, amantadine, have demonstrated improved response rates in genotype 1-infected individuals (6). Recently, we have shown that the sensitivity of HCV to p7 inhibitors varies according to genotype, implying that p7 sequence determines the binding of inhibitor compounds (12). Little is known about how primary sequence governs p7 channel activity or drug interactions although several mutations have been shown to affect particle secretion in culture (19, 34), including the conserved basic charges on the cytosolic loop, which are known to be required for p7 activity in surrogate cell systems (14).p7 channels are heptameric assemblages (4) with a predicted structure whereby the lumen is formed by the amphipathic amino-terminal trans-membrane helices (13, 25). Carboxy-terminal helices are thought to interact with adjacent p7 protomers, serving to stabilize the channel structure. In addition, the basic loop may form a constriction at one end of the channel, possibly serving to mediate channel gating (13). Accordingly, residues within the loop or the amino-terminal helix would be the most likely to mediate channel opening and/or drug binding.Here, we have investigated determinants of both p7 drug sensitivity and channel activity using a liposome-based fluorescent dye release assay for p7 function (36). Surprisingly, we find that p7 drug sensitivity is not, in fact, determined by either helix nor by the basic loop alone, implying that overall channel structure strongly influences drug binding. Several mutations specifically blocked fluorescent dye release from liposomes without adversely affecting oligomerization or membrane insertion, validating the system as a convenient means of investigating p7 function. Lastly, we identify a region that influences resistance of genotype 1b p7 to amantadine. Developing our understanding of how p7 sequence is linked to drug sensitivity could have important implications for the design of future HCV therapies.