Toshana L. Foster

All Three Domains of the Hepatitis C Virus Nonstructural NS5A Protein Contribute to RNA Binding

ABSTRACT The hepatitis C virus (HCV) nonstructural protein NS5A is critical for viral genome replication and is thought to interact directly with both the RNA-dependent RNA polymerase, NS5B, and viral RNA. NS5A consists of three domains which have, as yet, undefined roles in viral replication and assembly. In order to define the regions that mediate the interaction with RNA, specifically the HCV 3′ untranslated region (UTR) positive-strand RNA, constructs of different domain combinations were cloned, bacterially expressed, and purified to homogeneity. Each of these purified proteins was probed for its ability to interact with the 3′ UTR RNA using filter binding and gel electrophoretic mobility shift assays, revealing differences in their RNA binding efficiencies and affinities. A specific interaction between domains I and II of NS5A and the 3′ UTR RNA was identified, suggesting that these are the RNA binding domains of NS5A. Domain III showed low in vitro RNA binding capacity. Filter binding and competition analyses identified differences between NS5A and NS5B in their specificities for defined regions of the 3′ UTR. The preference of NS5A, in contrast to NS5B, for the polypyrimidine tract highlights an aspect of 3′ UTR RNA recognition by NS5A which may play a role in the control or enhancement of HCV genome replication.

Morphological characteristics of the limbal epithelial crypt

Aim: In 2005 we reported the discovery of a novel anatomical structure at the limbus, which we termed the limbal epithelial crypt (LEC). The purpose of this study was to further evaluate the distribution, immunophenotypical, and ultra structural characteristics of the LEC as a putative niche of stem cells. Methods: Sequential histological sections of human corneo-scleral limbal rims were examined for the presence and distribution of the LEC. Immunophenotypical characterisation of the LEC cells using a panel of antibodies of interest was undertaken. Transmission electron microscopy of the LEC was used to examine the ultra structural and morphometric features of cells within the LEC and adjacent limbus. Results: A total of 74 LECs were identified in eight corneo-scleral rims. These varied in number, size and distribution within rims. Cells within the crypt demonstrated the following phenotype: CK3−/CK19+/CD 34−/Vimentin+/p63+/Connexin 43+/MIB1 (Ki67)−. Presence of Cx43 was also demonstrated in the rete pegs adjacent to the LEC. Basal cells of the LEC were significantly smaller than basal cells found in adjacent rete pegs and also smaller than suprabasal limbal and central corneal epithelial cells (p<0.05). Morphologically they had a high nuclear:cytoplasmic ratio and were adherent to the underlying basement membrane by means of complex convolutions of cytoplasmic processes. Conclusions: LECs are sparse but a consistent finding in the human corneo-scleral limbus. The LEC contains a unique sub-population of cells expressing several characteristics that are consistent with it representing a putative stem cell niche.

Cyclophilin A Interacts with Domain II of Hepatitis C Virus NS5A and Stimulates RNA Binding in an Isomerase-Dependent Manner

ABSTRACT NS5A plays a critical, yet poorly defined, role in hepatitis C virus genome replication. The protein consists of three domains, each of which is able to bind independently to the 3′ untranslated region (UTR) of the viral positive strand genomic RNA. The peptidyl-prolyl isomerase cyclophilin A (CypA) binds to domain II, catalyzing cis-trans isomerization. CypA inhibitors such as cyclosporine (CsA) have been shown to inhibit hepatitis C virus (HCV) replication. We show here that CypA stimulated domain II RNA binding activity, and this stimulation was abrogated by CsA. An isomerase mutant of CypA (H126Q) failed to bind to domain II and did not stimulate RNA binding. Finally, we demonstrate that the RNA binding of two domain II mutants, the D316E and D316E/Y317N mutants, previously shown to exhibit CypA independence for RNA replication, was unaffected by CypA. This study provides an insight into the molecular mechanism of CypA activity during HCV replication and further validates the use of CypA inhibitors in HCV therapy.

Determinants of Hepatitis C Virus p7 Ion Channel Function and Drug Sensitivity Identified In Vitro

ABSTRACT 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.

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

MINT‐7890784, MINT‐7890776: SH (uniprotkb:P04852) and SH (uniprotkb:P04852) bind (MI:0407) by electron microscopy (MI:0040)

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

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. (HEPATOLOGY 2011;)

Resistance of Transmitted Founder HIV-1 to IFITM-Mediated Restriction

Summary Interferon-induced transmembrane proteins (IFITMs) restrict the entry of diverse enveloped viruses through incompletely understood mechanisms. While IFITMs are reported to inhibit HIV-1, their in vivo relevance is unclear. We show that IFITM sensitivity of HIV-1 strains is determined by the co-receptor usage of the viral envelope glycoproteins as well as IFITM subcellular localization within the target cell. Importantly, we find that transmitted founder HIV-1, which establishes de novo infections, is uniquely resistant to the antiviral activity of IFITMs. However, viral sensitivity to IFITMs, particularly IFITM2 and IFITM3, increases over the first 6 months of infection, primarily as a result of neutralizing antibody escape mutations. Additionally, the ability to evade IFITM restriction contributes to the different interferon sensitivities of transmitted founder and chronic viruses. Together, these data indicate that IFITMs constitute an important barrier to HIV-1 transmission and that escape from adaptive immune responses exposes the virus to antiviral restriction.

Structure‐guided design affirms inhibitors of hepatitis C virus p7 as a viable class of antivirals targeting virion release

Current interferon‐based therapy for hepatitis C virus (HCV) infection is inadequate, prompting a shift toward combinations of direct‐acting antivirals (DAA) with the first protease‐targeted drugs licensed in 2012. Many compounds are in the pipeline yet primarily target only three viral proteins, namely, NS3/4A protease, NS5B polymerase, and NS5A. With concerns growing over resistance, broadening the repertoire for DAA targets is a major priority. Here we describe the complete structure of the HCV p7 protein as a monomeric hairpin, solved using a novel combination of chemical shift and nuclear Overhauser effect (NOE)‐based methods. This represents atomic resolution information for a full‐length virus‐coded ion channel, or “viroporin,” whose essential functions represent a clinically proven class of antiviral target exploited previously for influenza A virus therapy. Specific drug‐protein interactions validate an allosteric site on the channel periphery and its relevance is demonstrated by the selection of novel, structurally diverse inhibitory small molecules with nanomolar potency in culture. Hit compounds represent a 10,000‐fold improvement over prototypes, suppress rimantadine resistance polymorphisms at submicromolar concentrations, and show activity against other HCV genotypes. Conclusion: This proof‐of‐principle that structure‐guided design can lead to drug‐like molecules affirms p7 as a much‐needed new target in the burgeoning era of HCV DAA. (Hepatology 2014;59:408–422)

Serine Phosphorylation of HIV-1 Vpu and Its Binding to Tetherin Regulates Interaction with Clathrin Adaptors

HIV-1 Vpu prevents incorporation of tetherin (BST2/ CD317) into budding virions and targets it for ESCRT-dependent endosomal degradation via a clathrin-dependent process. This requires a variant acidic dileucine-sorting motif (ExxxLV) in Vpu. Structural studies demonstrate that recombinant Vpu/tetherin fusions can form a ternary complex with the clathrin adaptor AP-1. However, open questions still exist about Vpu’s mechanism of action. Particularly, whether endosomal degradation and the recruitment of the E3 ubiquitin ligase SCFβTRCP1/2 to a conserved phosphorylated binding site, DSGNES, are required for antagonism. Re-evaluation of the phenotype of Vpu phosphorylation mutants and naturally occurring allelic variants reveals that the requirement for the Vpu phosphoserine motif in tetherin antagonism is dissociable from SCFβTRCP1/2 and ESCRT-dependent tetherin degradation. Vpu phospho-mutants phenocopy ExxxLV mutants, and can be rescued by direct clathrin interaction in the absence of SCFβTRCP1/2 recruitment. Moreover, we demonstrate physical interaction between Vpu and AP-1 or AP-2 in cells. This requires Vpu/tetherin transmembrane domain interactions as well as the ExxxLV motif. Importantly, it also requires the Vpu phosphoserine motif and adjacent acidic residues. Taken together these data explain the discordance between the role of SCFβTRCP1/2 and Vpu phosphorylation in tetherin antagonism, and indicate that phosphorylation of Vpu in Vpu/tetherin complexes regulates promiscuous recruitment of adaptors, implicating clathrin-dependent sorting as an essential first step in tetherin antagonism.

Mutations in hepatitis C virus p7 reduce both the egress and infectivity of assembled particles via impaired proton channel function.

Hepatitis C virus (HCV) p7 protein is critical for the efficient production of infectious virions in culture. p7 undergoes genotype-specific protein-protein interactions as well as displaying channel-forming activity, making it unclear whether the phenotypes of deleterious p7 mutations result from the disruption of one or both of these functions. Here, we showed that proton channel activity alone, provided in trans by either influenza virus M2 or genotype 1b HCV p7, was both necessary and sufficient to restore infectious particle production to genotype 2a HCV (JFH-1 isolate) carrying deleterious p7 alanine substitutions within the p7 dibasic loop (R33A, R35A), and the N-terminal trans-membrane region (N15 : C16 : H17/AAA). Both mutations markedly reduced mature p7 abundance, with those in the dibasic loop also significantly reducing levels of mature E2 and NS2. Interestingly, whilst M2 and genotype 1b p7 restored the same level of intracellular infectivity as JFH-1 p7, supplementing with the isogenic protein led to a further increase in secreted infectivity, suggesting a late-acting role for genotype-specific p7 protein interactions. Finally, cells infected by viruses carrying p7 mutations contained non-infectious core-containing particles with densities equivalent to WT HCV, indicating a requirement for p7 proton channel activity in conferring an infectious phenotype to virions.