c High-risk human papillomavirus type 16 (HPV16) is the primary causative agent of cervical cancer and therefore is responsible for significant morbidity and mortality worldwide. Cellular transformation is mediated directly by the expression of viral oncogenes, the least characterized of which, E5, subverts cellular proliferation and immune recognition processes. Despite a growing catalogue of E5-specific host interactions, little is understood regarding the molecular basis of its function. Here we describe a novel function for HPV16 E5 as an oligomeric channel-forming protein, placing it within the virus-encoded "viroporin" family. The development of a novel recombinant E5 expression system showed that E5 formed oligomeric assemblies of a defined luminal diameter and stoichiometry in membranous environments and that such channels mediated fluorescent dye release from liposomes. Hexameric E5 channel stoichiometry was suggested by native PAGE studies. In lieu of high-resolution structural information, established de novo molecular modeling and design methods permitted the development of the first specific smallmolecule E5 inhibitor, capable of both abrogating channel activity in vitro and reducing E5-mediated effects on cell signaling pathways. The identification of channel activity should enhance the future understanding of the physiological function of E5 and could represent an important target for antiviral intervention.H uman papillomaviruses (HPVs) are small, double-stranded DNA viruses that infect squamous epithelial cells and produce a range of clinical lesions, including common warts, genital warts, and cancers of the anogenital tract and oropharynx. A subset of HPVs are carcinogenic, and among these high-risk types, HPV16 is detected in approximately 60% of all cervical cancer cases worldwide (4). The virus encodes three oncoproteins: E5, E6, and E7. The roles of E6 and E7 in cervical carcinogenesis have been extensively studied, and the contributions of both proteins to HPV pathogenesis are well accepted. The least characterized of the three oncoproteins is the highly hydrophobic, 83-amino-acid E5 protein, which associates with internal membranes, most notably those of the endoplasmic reticulum, Golgi apparatus, and perinuclear region (24). HPV16 E5 is classified as an oncoprotein due to its ability to induce anchorage-independent growth in murine fibroblasts and human keratinocytes (34). Transgenic mouse model systems demonstrate that high levels of E5 expression in the skin induce epithelial hyperproliferation, resulting in spontaneous tumor formation (15, 30). These mice also display increased dysplastic disease in the cervical epithelium (29). E5 mRNA is highly abundant in HPV lesions (37), and the protein is expressed in the early stages of malignant transformation, where the episomal viral genome is present (2). Therefore, E5 represents a target for early-stage intervention, prior to the progression of premalignant lesions to cervical cancer.E5 hyperactivates ligand-dependent epidermal growth fact...
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 transmembrane 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-␣) 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 prot...
The hepatitis C virus (HCV) p7 ion channel plays a critical role during infectious virus production and represents an important new therapeutic target. Its activity is blocked by structurally distinct classes of small molecules, with sensitivity varying between isolate p7 sequences. Although this is indicative of specific protein-drug interactions, a lack of highresolution structural information has precluded the identification of inhibitor binding sites, and their modes of action remain undefined. Furthermore, a lack of clinical efficacy for existing p7 inhibitors has cast doubt over their specific antiviral effects. We identified specific resistance mutations that define the mode of action for two classes of p7 inhibitor: adamantanes and alkylated imino sugars (IS). Adamantane resistance was mediated by an L20F mutation, which has been documented in clinical trials. Molecular modeling revealed that L20 resided within a membrane-exposed binding pocket, where drug binding prevented low pH-mediated channel opening. The peripheral binding pocket was further validated by a panel of adamantane derivatives as well as a bespoke molecule designed to bind the region with high affinity. By contrast, an F25A polymorphism found in genotype 3a HCV conferred IS resistance and confirmed that these compounds intercalate between p7 protomers, preventing channel oligomerization. Neither resistance mutation significantly reduced viral fitness in culture, consistent with a low genetic barrier to resistance occurring in vivo. Furthermore, no cross-resistance was observed for the mutant phenotypes, and the two inhibitor classes showed additive effects against wild-type HCV. Conclusion: These observations support the notion that p7 inhibitor combinations could be a useful addition to future HCV-specific therapies. (HEPATOLOGY 2011;54:79-90) H epatitis C virus (HCV) infects over 3% of the population, causing severe liver disease. Current therapy comprising pegylated interferon (IFN) and ribavirin (Rib) is inadequate, which, combined with high cost and poor patient compliance, has driven the demand for new virus-specific drugs.1 Future standard of care will replace IFN/Rib with combinations of specific inhibitors, such as seen for human immunodeficiency virus (HIV) therapy. However, extensive HCV variability raises concerns over the ability of relatively few compounds to suppress resistance. Thus, great effort focuses on expanding the repertoire of HCV drug targets, expedited by the availability of the Japanese fulminant hepatitis clone 1 (JFH-1) infectious isolate.
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