Genotype-dependent sensitivity of hepatitis C virus to inhibitors of the p7 ion channel

Abstract

The hepatitis C virus (HCV) p7 protein plays a critical role during particle formation in cell culture and is required for virus replication in chimpanzees. The discovery that it displayed cation channel activity in vitro led to its classification within the "viroporin" family of virus-coded ion channel proteins, which includes the influenza A virus (IAV) M2 protein. Like M2, p7 was proposed as a potential target for much needed new HCV therapies, and this was supported by our finding that the M2 inhibitor, amantadine, blocked its activity in vitro. Since then, further compounds have been shown to inhibit p7 function but the relationship between inhibitory effects in vitro and efficacy against infectious virus is controversial. Here, we have sought to validate multiple p7 inhibitor compounds using a parallel approach combining the HCV infectious culture system and a rapid throughput in vitro assay for p7 function. We identify a genotype-dependent and subtype-dependent sensitivity of HCV to p7 inhibitors, in which results in cell culture largely mirror the sensitivity of recombinant protein in vitro; thus building separate sensitivity profiles for different p7 sequences. Inhibition of virus entry also occurred, suggesting that p7 may be a virion component. Second site effects on both cellular and viral processes were identified for several compounds in addition to their efficacy against p7 in vitro. Nevertheless, for some compounds antiviral effects were specific to a block of ion channel function. Conclusion: These data validate p7 inhibitors as prototype therapies for chronic HCV disease. H epatitis C virus (HCV) chronically infects over3% of the population causing severe liver disease. Despite intensive efforts, no vaccine exists and asymptomatic acute infection results in most carriers being unaware of their positive status. Combination antiviral therapy is available based on interferon ␣ (IFN␣) and ribavirin (Rib). This treatment is expensive, poorly tolerated, and its outcome is largely determined by virus genotype 1 ; resistance of many genotype 1 isolates results in a sustained response for only 50% of patients overall, despite good response rates for other virus strains. New, virus-specific, therapies are in production, yet face resistance caused by HCV sequence variation. 2 Future therapies will likely require combinatorial approaches, targeting multiple virus-specific processes.HCV is the prototype member of the Hepacivirus genus within the Flaviviridae. It is enveloped and possesses a positive strand RNA genome of ϳ9.6 kb. An internal ribosome entry site (IRES) present in the 5Ј untranslated region (UTR) drives translation of a single polyprotein that is cleaved to yield 10 mature proteins. The core and envelope (E) glycoproteins, with the RNA genome, comprise the virion, while nonstructural (NS) proteins modulate host metabolism and replication of the viral RNA. 3 Viral insensitivity to IFN/Rib maps to regions within the NS proteins that confer resistance to the innate immune response, 4,5 whereas t...

NMR Structure and Ion Channel Activity of the p7 Protein from Hepatitis C Virus

“…These polypeptides modify the membrane cell permeability to ions and small molecules. Isolated p7 reconstituted in artificial membranes has been reported to form oligomers and to possess cation channel activity, which is inhibited by long alkyl chain imino-sugar derivatives (18), amiloride compounds (19), and amantadine (20), albeit the latter appeared to be genotype-dependent (21,22). Nevertheless, the relationship between the ion channel activity and the role of p7 in assembly and release of infectious HCV particles in cell culture is not known.…”

The 3-dimensional structure of a hepatitis C virus p7 ion channel by electron microscopy

“…Fab fragments were generated from antibodies 2716 and 2717 (a kind gift from Dr. Steve Griffin, University of Leeds, UK), which are directed against the N and C termini of JFH-1 p7, respectively (6). Twenty micrograms of both full-length antibodies were dialyzed in 30 L against Fab digestion buffer (Pierce) and digested individually for 4 h shaking at 800 rpm, 37°C, on an agarose column with immobilized papain (Pierce).…”

“…p7 belongs to the viroporins, small virally encoded proteins with at least 1 membrane-spanning helix that oligomerize to form channels or pores that modify the permeability of the cell membrane to ions and other small molecules (7). In planar lipid bilayers, p7 monomers oligomerize to form cation-selective ion channels that can be specifically inhibited by long alkylchain iminosugars, amiloride, and amantadine derivatives, with varying reported efficacies (6,(8)(9)(10)(11)(12)(13)(14)(15). Each HCV p7 monomer consists of 63 aa, most of which are hydrophobic and possibly contain endoplasmic reticulum (ER) retention signals (16-18).…”

“…p7 is critical for the release of infectious virions in vitro (1,2) and in vivo (3). It is not involved in HCV RNA replication (4,5), but is required for late steps of viral particle assembly (2) and potentially cell entry (6). However, the prerequisite incorporation of p7 into budding virions has not been demonstrated.…”

Development and Validation of a High-Throughput Screening Assay for the Hepatitis C Virus p7 Viroporin

“…however, these inhibitors are active against only certain hCV genotypes, and different groups have reported differing sensitivities. [8][9][10][11] ideally, inhibitors would have similar potency against all genotypes. more potent p7 inhibitors are required to assess the potential to inhibit p7 from different hCV genotypes.…”