The Hepatitis C virus NS5A and core proteins exert antagonistic effects on HAMP gene expression: the hidden interplay with the MTF-1/MRE pathway.

Hepcidin, a 25-amino acid peptide encoded by the HAMP gene and produced mainly by hepatocytes and macrophages, is a mediator of innate immunity and the central iron-regulatory hormone. Circulating hepcidin controls iron efflux by inducing degradation of the cellular iron exporter ferroportin. HCV infection is associated with hepatic iron overload and elevated serum iron, which correlate with poor antiviral responses. The HCV nonstructural NS5A protein is known to function in multiple aspects of the HCV life cycle, probably exerting its activity in concert with cellular factor(s). In this study, we attempted to delineate the effect of HCV NS5A on HAMP gene expression. We observed that transient transfection of hepatoma cell lines with HCV NS5A resulted in down-regulation of HAMP promoter activity. A similar effect was evident after transduction of Huh7 cells with a recombinant baculovirus vector expressing NS5A protein. We proceeded to construct an NS5A-expressing stable cell line, which also exhibited down-regulation of HAMP gene promoter activity and significant reduction of HAMP mRNA and hepcidin protein levels. Concurrent expression of HCV core protein, a well-characterized hepcidin inducer, revealed antagonism between those two proteins for hepcidin regulation. In attempting to identify the pathways involved in NS5A-driven reduction of hepcidin levels, we ruled out any NS5A-induced alterations in the expression of the well-known hepcidin inducers SMAD4 and STAT3. Further analysis linked the abundance of intracellular zinc ions and the deregulation of the MTF-1/MRE/hepcidin axis with the observed phenomenon. This effect could be associated with distinct phases in HCV life cycle.

Volatile allosteric antagonists of mosquito odorant receptors inhibit human-host attraction.

Odorant-dependent behaviors in insects are triggered by the binding of odorant ligands to the variable subunits of heteromeric olfactory receptors. Previous studies have shown, however, that specific odor binding to ORco, the common subunit of odorant receptor heteromers, may allosterically alter olfactory receptor function and profoundly affect subsequent behavioral responses. Using an insect cell-based screening platform, we identified and characterized several antagonists of the odorant receptor coreceptor of the African malaria vector Anopheles gambiae (AgamORco) in a small collection of natural volatile organic compounds. Because some of the identified antagonists were previously shown to strongly repel Anopheles and Culex mosquitoes, we examined the bioactivities of the identified antagonists against Aedes, the third major genus of the Culicidae family. The tested antagonists inhibited the function of Ae. aegypti ORco ex vivo and repelled adult Asian tiger mosquitoes (Ae. albopictus). Binary mixtures of specific antagonists elicited higher repellency than single antagonists, and binding competition assays suggested that this enhanced repellence is due to antagonist interaction with distinct ORco sites. Our results also suggest that the enhanced mosquito repellency by antagonist mixtures is due to additive rather than synergistic effects of the specific antagonist combinations on ORco function. Taken together, these findings provide novel insights concerning the molecular aspects of odorant receptor function. Moreover, our results demonstrate that a simple screening assay may be used for the identification of allosteric modifiers of olfactory-driven behaviors capable of providing enhanced personal protection against multiple mosquito-borne infectious diseases.

Odorant-binding protein-based identification of natural spatial repellents for the African malaria mosquito Anopheles gambiae.

There is increasing interest in the development of effective mosquito repellents of natural origin to reduce transmission of diseases such as malaria and yellow fever. To achieve this we have employed an in vitro competition assay involving odorant-binding proteins (OBPs) of the malaria mosquito, Anopheles gambiae, with a predominantly female expression bias to identify plant essential oils (EOs) containing bioactive compounds that target mosquito olfactory function. EOs and their fractions capable of binding to such OBPs displayed repellence against female mosquitoes in a laboratory repellent assay. Repellent EOs were subjected to gas chromatographic analysis linked to antennogram (EAG) recordings from female A. gambiae to identify the biologically active constituents. Among these compounds cumin alcohol, carvacrol, ethyl cinnamate and butyl cinnamate proved as effective as DEET at an equivalent dose in the repellent assay, and combinations of carvacrol with either butyl cinnamate or cumin alcohol proved to be significantly more effective than DEET in the assay. When tested as spatial repellents in experimental shelters housing sleeping humans in northern Nigeria a binary mixture of carvacrol plus cumin alcohol caused mosquitoes to leave shelters in significantly higher numbers to those induced by DEET in female Anopheles spp. and in numbers equivalent to that of DEET in Culex spp. mosquitoes. These findings indicate an approach for the identification of biologically active molecules of natural origin serving as repellents for mosquitoes.

Positive Allosteric Modulation of Insect Olfactory Receptor Function by ORco Agonists.

Insect olfactory receptors (ORs) are heteromeric ligand-gated cation channels composed of a common olfactory receptor subunit (ORco) and a variable subunit (ORx) of as yet unknown structures and undetermined stoichiometries. In this study, we examined the allosteric modulation exerted on Anopheles gambiae heteromeric ORx/ORco olfactory receptors in vitro by a specific class of ORco agonists (OAs) comprising ORcoRAM2 and VUAA1. High OA concentrations produced stronger functional responses in cells expressing heteromeric receptor channels relative to cells expressing ORco alone. These OA-induced responses of ORx/ORco channels were also notably much stronger than those obtained upon administration of ORx-specific ligands to the same receptors. Most importantly, small concentrations of OAs were found to act as strong potentiators of ORx/ORco function, increasing dramatically both the efficacy and potency of ORx-specific odorants. These results suggest that insect heteromeric ORs are highly dynamic complexes adopting different conformations that change in a concerted fashion as a result of the interplay between the subunits of the oligomeric assemblies, and that allosteric modulation may constitute an important element in the modulation and fining tuning of olfactory reception function.

Construction, complete sequence, and annotation of a BAC contig covering the silkworm chorion locus.

The silkmoth chorion was studied extensively by F.C. Kafatos' group for almost 40 years. However, the complete structure of the chorion locus was not obtained in the genome sequence of Bombyx mori published in 2008 due to repetitive sequences, resulting in gaps and an incomplete view of the locus. To obtain the complete sequence of the chorion locus, expressed sequence tags (ESTs) derived from follicular epithelium cells were used as probes to screen a bacterial artificial chromosome (BAC) library. Seven BACs were selected to construct a contig which covered the whole chorion locus. By Sanger sequencing, we successfully obtained complete sequences of the chorion locus spanning 871,711 base pairs on chromosome 2, where we annotated 127 chorion genes. The dataset reported here will recruit more researchers to revisit one of the oldest model systems which has been used to study developmentally regulated gene expression. It also provides insights into egg development and fertilization mechanisms and is relevant to applications related to improvements in breeding procedures and transgenesis.

A comprehensive analysis of the chorion locus in silkmoth.

Despite more than 40 years of intense study, essential features of the silkmoth chorion (eggshell) are still not fully understood. To determine the precise structure of the chorion locus, we performed extensive EST analysis, constructed a bacterial artificial chromosome (BAC) contig, and obtained a continuous genomic sequence of 871,711 base pairs. We annotated 127 chorion genes in two segments interrupted by a 164 kb region with 5 non-chorion genes, orthologs of which were on chorion bearing scaffolds in 4 ditrysian families. Detailed transcriptome analysis revealed expression throughout choriogenesis of most chorion genes originally categorized as "middle", and evidence for diverse regulatory mechanisms including cis-elements, alternative splicing and promoter utilization, and antisense RNA. Phylogenetic analysis revealed multigene family associations and faster evolution of early chorion genes and transcriptionally active pseudogenes. Proteomics analysis identified 99 chorion proteins in the eggshell and micropyle localization of 1 early and 6 Hc chorion proteins.

Inhibition of Anopheles gambiae odorant receptor function by mosquito repellents.

The identification of molecular targets of insect repellents has been a challenging task, with their effects on odorant receptors (ORs) remaining a debatable issue. Here, we describe a study on the effects of selected mosquito repellents, including the widely used repellent N,N-diethyl-meta-toluamide (DEET), on the function of specific ORs of the African malaria vector Anopheles gambiae. This study, which has been based on quantitative measurements of a Ca(2+)-activated photoprotein biosensor of recombinant OR function in an insect cell-based expression platform and a sequential compound addition protocol, revealed that heteromeric OR (ORx/Orco) function was susceptible to strong inhibition by all tested mosquito repellents except DEET. Moreover, our results demonstrated that the observed inhibition was due to efficient blocking of Orco (olfactory receptor coreceptor) function. This mechanism of repellent action, which is reported for the first time, is distinct from the mode of action of other characterized insect repellents including DEET.

Poly(ethylene oxide)-b-poly(propylene oxide) amphiphilic block copolymer-mediated growth of silver nanoparticles and their antibacterial behavior.

Silver nanoparticles were grown in self-assembled amphiphilic poly(ethylene oxide)/poly(propylene oxide) (PEO/PPO) triblock copolymers in selective solvents. Ternary systems of block copolymer, water, and p-xylene were used, forming a dispersion of water droplets in oil (reverse micellar) as well as binary water/block copolymer solutions. Besides its stabilizing affect, the role of the copolymer as a reducing agent for the metal salt precursors was examined. It was found that block copolymer-enabled reduction, carried out mainly by the PEO blocks, could take place only under particular conditions mostly related to the metal precursor, the block copolymer concentration, and the self-assembled micellar configuration. The effect of the triblock copolymers on growth and stabilization of gold nanoparticles was also examined. The antibacterial effect of the silver nanoparticles was investigated against Escherichia coli cells, and their performance was evaluated through a series of parametrization experiments, including the effect of the metal concentration, stability, activity over time, and dosage, while particular emphasis was given on the role of ions versus nanoparticles on the antibacterial performance.

MEK5/ERK5/mef2: a novel signaling pathway affected by hepatitis C virus non-enveloped capsid-like particles.

Hepatitis C virus (HCV) is an RNA positive strand virus, member of the Flaviviridae family. The viral particle is composed of a capsid containing the genome, surrounded by E1 and E2 proteins, however different forms of viral particles have been observed including non-enveloped particles. Previous reports have proposed that hepatitis C non-enveloped capsid-like particles (HCVne) enter cells of hepatic origin via clathrin-mediated endocytosis, during which different signaling events occur. In this report we show that HCVne particles are capable of inducing the recently discovered ERK5 pathway, in a dose dependent way. The ERK5 pathway can be activated by growth factors and other extracellular signals. This specific activation occurs through a well characterized upstream kinase, MEK5, and is capable of inducing gene regulation of mef2. In contrast, when HCV core structural and NS5A non-structural proteins were expressed endogenously no activation of this pathway was detected. These cell signaling events could be of critical importance and might give clues for the elucidation of cellular manifestations associated with HCV infection.

Modulation of IL-2 expression after uptake of hepatitis C virus non-enveloped capsid-like particles: the role of p38 kinase.

Hepatitis C virus (HCV) has been shown to actively replicate in cells of the immune system, altering both their function and cytokine expression. Naked nucleocapsids have been reported in the serum of infected patients. We investigated interference of recombinant non-enveloped capsid-like particles with signaling pathways in T cells. HCV non-enveloped particles (HCVne) internalization was verified in Jurkat and Hut 78 T cells, as well as primary human peripheral blood and intrahepatic mononuclear cells. HCVne uptake leads to activation of the MAPKs-p38 signaling pathway. Using specific phosphoantibodies, signaling pathways inhibitors, and chemical agents, it was demonstrated that p38 activation in T cells correlated with IL-2 transcriptional activation and was accompanied by a parallel increase of IL-2 cytokine secretion. c-fos and egr-1, two transcription factors, essential for IL-2 promoter activity, were also found to be elevated. We propose that HCVne uptake by T lymphocytes results in increased MAPKs-p38 activity and IL-2 expression, thus altering the host immune response.

Expression and membrane topology of Anopheles gambiae odorant receptors in lepidopteran insect cells.

A lepidopteran insect cell-based expression system has been employed to express three Anopheles gambiae odorant receptors (ORs), OR1 and OR2, which respond to components of human sweat, and OR7, the ortholog of Drosophila's OR83b, the heteromerization partner of all functional ORs in that system. With the aid of epitope tagging and specific antibodies, efficient expression of all ORs was demonstrated and intrinsic properties of the proteins were revealed. Moreover, analysis of the orientation of OR1 and OR2 on the cellular plasma membrane through the use of a novel 'topology screen' assay and FACS analysis demonstrates that, as was recently reported for the ORs in Drosophila melanogaster, mosquito ORs also have a topology different than their mammalian counterparts with their N-terminal ends located in the cytoplasm and their C-terminal ends facing outside the cell. These results set the stage for the production of mosquito ORs in quantities that should permit their detailed biochemical and structural characterization and the exploration of their functional properties.

Endocytosis of hepatitis C virus non-enveloped capsid-like particles induces MAPK-ERK1/2 signaling events.

Although HCV is an enveloped virus, naked nucleocapsids have been reported in the serum of infected patients. The HCV core particle serves as a protective capsid shell for the viral genome and recombinant in vitro assembled HCV core particles induce strong specific immunity. We investigated the post-binding mechanism of recombinant core particle uptake and its intracellular fate. In hepatic cells, these particles are internalized, most likely in a clathrin-dependent pathway, reaching early to late endosomes and finally lysosomes. The endocytic acidic milieu is implicated in trafficking process. Using specific phosphoantibodies, signaling pathway inhibitors and chemical agents, ERK(1/2) was found to be activated in a sustained way after endocytosis, followed by downstream immediate early genes (c-fos and egr-1) modulation. We propose that the intriguing properties of cellular internalization of HCV non-enveloped particles can induce specific ERK(1/2)-MAPKs events that could be important in HCV life cycle and pathogenesis of HCV infection.

The Anopheles gambiae odorant binding protein 1 (AgamOBP1) mediates indole recognition in the antennae of female mosquitoes.

Haematophagous insects are frequently carriers of parasitic diseases, including malaria. The mosquito Anopheles gambiae is the major vector of malaria in sub-Saharan Africa and is thus responsible for thousands of deaths daily. Although the role of olfaction in A. gambiae host detection has been demonstrated, little is known about the combinations of ligands and odorant binding proteins (OBPs) that can produce specific odor-related responses in vivo. We identified a ligand, indole, for an A. gambiae odorant binding protein, AgamOBP1, modeled the interaction in silico and confirmed the interaction using biochemical assays. RNAi-mediated gene silencing coupled with electrophysiological analyses confirmed that AgamOBP1 binds indole in A. gambiae and that the antennal receptor cells do not respond to indole in the absence of AgamOBP1. This case represents the first documented instance of a specific A. gambiae OBP-ligand pairing combination, demonstrates the significance of OBPs in odor recognition, and can be expanded to the identification of other ligands for OBPs of Anopheles and other medically important insects.

Green fluorescent protein - Tagged HCV non-enveloped capsid like particles: development of a new tool for tracking HCV core uptake.

Circulating 'free' non-enveloped Hepatitis C virus (HCV) core protein has been demonstrated in HCV-infected patients, and HCV subgenomes with deletions of the envelope proteins have been previously identified. Initial studies from our laboratory, previously published, indicated that expression of HCV core in insect cells can direct the formation of capsid-like particles lacking the envelope glycoproteins. These protein nanospheres, morphologically similar to natural capsids, were shown to be taken up by human hepatic cells and to produce cell-signalling events. To follow the intracellular fate of these particles we fused the core protein to eGFP. We demonstrate that the chimeric proteins core(173)-eGFP, eGFP-core(191) and eGFP-core(173) can be efficiently expressed, self-assembled, and form fluorescent non-enveloped capsid-like particles. By using confocal microscopy and FACS analysis, we provide evidence that the fluorescent nanospheres can not only enter human hepatic cells - the main target of HCV - but also human immune cells such as T and B lymphocytes, as well as human myeloid leukaemia cells differentiated along the monocyte/macrophage-like pathway. The fluorescent particles might thus be used to trace the intracellular trafficking of naked HCV capsids as showed by live microscopy and to further understand their biological significance.

Evidence for cellular uptake of recombinant hepatitis C virus non-enveloped capsid-like particles.

Although the hepatitis C virus (HCV) is an enveloped virus, naked nucleocapsids have been reported in the serum of infected patients, and most recently novel HCV subgenomes with deletions of the envelope proteins have been identified. However the significance of these findings remains unclear. In this study, we used the baculovirus expression system to generate recombinant HCV capsid-like particles, and investigated their possible interactions with cells. We show that expression of HCV core in insect cells can sufficiently direct the formation of capsid-like particles in the absence of the HCV envelope glycoproteins and of the 5' untranslated region. By confocal microscopy analysis, we provide evidence that the naked capsid-like particles could be uptaken by human hepatoma cells. Moreover, our findings suggest that they have the potential to produce cell-signaling effects.

The protein phosphatase 2A represents a novel cellular target for hepatitis C virus NS5A protein.

It is well established that HCV NS5A protein when expressed in mammalian cells perturbs the extracellular signal regulated kinase (ERK) pathway. The protein serine/threonine phosphatase 2A controls the phosphorylation of numerous proteins involved in cell signaling and one characterized function is the regulation of Ras-Raf mitogen activated protein (MAP) kinase signaling pathways. Our results showed that expression of HCV NS5A protein stimulates phosphatase 2A (PP2A) activity in cells, indicating the relevance of NS5A as a regulator of PP2A in vivo. We found that transient expression of the full length NS5A protein in different cell lines leads to a significant increase of the PP2A activity and this activity is specifically inhibited by the addition of okadaic acid, a PP2A inhibitor, in living cells. Further investigation showed that NS5A protein interacts in vivo and in vitro with the scaffolding A and the catalytic C subunits of PP2A. We propose that HCV NS5A represents a viral PP2A regulatory protein. This is a novel function for the NS5A protein which may have a key role in the ability of the virus to deregulate cell growth and survival.