High-risk human papillomavirus E5 oncoprotein displays channel-forming activity sensitive to small-molecule inhibitors.

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 small-molecule 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.

Mutation of a C-terminal motif affects Kaposi's sarcoma-associated herpesvirus ORF57 RNA binding, nuclear trafficking, and multimerization.

The Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein is essential for virus lytic replication. ORF57 regulates virus gene expression at multiple levels, enhancing transcription, stability, nuclear export, and translation of viral transcripts. To enhance the nuclear export of viral intronless transcripts, ORF57 (i) binds viral intronless mRNAs, (ii) shuttles between the nucleus, nucleolus, and the cytoplasm, and (iii) interacts with multiple cellular nuclear export proteins to access the TAP-mediated nuclear export pathway. We investigated the implications on the subcellular trafficking, cellular nuclear export factor recruitment, and ultimately nuclear mRNA export of an ORF57 protein unable to bind RNA. We observed that mutation of a carboxy-terminal RGG motif, which prevents RNA binding, affects the subcellular localization and nuclear trafficking of the ORF57 protein, suggesting that it forms subnuclear aggregates. Further analysis of the mutant shows that although it still retains the ability to interact with cellular nuclear export proteins, it is unable to export viral intronless mRNAs from the nucleus. Moreover, computational molecular modeling and biochemical studies suggest that, unlike the wild-type protein, this mutant is unable to self-associate. Therefore, these results suggest the mutation of a carboxy-terminal RGG motif affects ORF57 RNA binding, nuclear trafficking, and multimerization.

Resistance mutations define specific antiviral effects for inhibitors of the hepatitis C virus p7 ion channel.

UNLABELLED: 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 high-resolution 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.

Direct visualization of the small hydrophobic protein of human respiratory syncytial virus reveals the structural basis for membrane permeability.

Human respiratory syncytial virus (HRSV) is the leading cause of lower respiratory tract disease in infants. The HRSV small hydrophobic (SH) protein plays an important role in HRSV pathogenesis, although its mode of action is unclear. Analysis of the ability of SH protein to induce membrane permeability and form homo-oligomers suggests it acts as a viroporin. For the first time, we directly observed functional SH protein using electron microscopy, which revealed SH forms multimeric ring-like objects with a prominent central stained region. Based on current and existing functional data, we propose this region represents the channel that mediates membrane permeability.

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.