Eléonore Real

Genetically encoded chloride indicator with improved sensitivity.

Chloride (Cl) is the most abundant physiological anion. Abnormalities in Cl regulation are instrumental in the development of several important diseases including motor disorders and epilepsy. Because of difficulties in the spectroscopic measurement of Cl in live tissues there is little knowledge available regarding the mechanisms of regulation of intracellular Cl concentration. Several years ago, a CFP-YFP based ratiometric Cl indicator (Clomeleon) was introduced [Kuner, T., Augustine, G.J. A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons. Neuron 2000; 27: 447-59]. This construct with relatively low sensitivity to Cl (K(app) approximately 160 mM) allows ratiometric monitoring of Cl using fluorescence emission ratio. Here, we propose a new CFP-YFP-based construct (Cl-sensor) with relatively high sensitivity to Cl (K(app) approximately 30 mM) due to triple YFP mutant. The construct also exhibits good pH sensitivity with pK(alpha) ranging from 7.1 to 8.0 pH units at different Cl concentrations. Using Cl-sensor we determined non-invasively the distribution of [Cl](i) in cultured CHO cells, in neurons of primary hippocampal cultures and in photoreceptors of rat retina. This genetically encoded indicator offers a means for monitoring Cl and pH under different physiological conditions and high-throughput screening of pharmacological agents.

Targeting of incoming retroviral Gag to the centrosome involves a direct interaction with the dynein light chain 8.

The role of cellular proteins in the replication of retroviruses, especially during virus assembly, has been partly unraveled by recent studies. Paradoxically, little is known about the route taken by retroviruses to reach the nucleus at the early stages of infection. To get insight into this stage of virus replication, we have studied the trafficking of foamy retroviruses and have previously shown that incoming viral proteins reach the microtubule organizing center (MTOC) prior to nuclear translocation of the viral genome. Here, we show that incoming viruses concentrate around the MTOC as free and structured capsids. Interestingly, the Gag protein, the scaffold component of viral capsids, targets the pericentrosomal region in transfected cells in the absence of any other viral components but in a microtubule- and dynein/dynactin-dependent manner. Trafficking of Gag towards the centrosome requires a minimal 30 amino acid coiled-coil motif in the N-terminus of the molecule. Finally, we describe a direct interaction between Gag and dynein light chain 8 that probably accounts for the specific routing of the incoming capsids to the centrosome prior to nuclear import of the viral genome.

Molecular basis for the interaction between rabies virus phosphoprotein P and the dynein light chain LC8: dissociation of dynein-binding properties and transcriptional functionality of P

The lyssavirus phosphoprotein P is a co-factor of the viral RNA polymerase and plays a central role in virus transcription and replication. It has been shown previously that P interacts with the dynein light chain LC8, which is involved in minus end-directed movement of organelles along microtubules. Co-immunoprecipitation experiments and the two-hybrid system were used to map the LC8-binding site to the sequence (139)RSSEDKSTQTTGR(151). Site-directed mutagenesis of residues D(143) and Q(147) to an A residue abolished binding to LC8. The P-LC8 association is not required for virus transcription, since the double mutant was not affected in its transcription ability in a minigenome assay. Based on the crystal structure of LC8 bound to a peptide from neuronal nitric oxide synthase, a model for the complex between the peptide spanning residues 140-150 of P and LC8 is proposed. This model suggests that P binds LC8 in a manner similar to other LC8 cellular partners.

Functional interaction map of lyssavirus phosphoprotein : identification of the minimal transcription domains

ABSTRACT Lyssaviruses, the causative agents of rabies encephalitis, are distributed in seven genotypes. The phylogenetically distant rabies virus (PV strain, genotype 1) and Mokola virus (genotype 3) were used to develop a strategy to identify functional homologous interactive domains from two proteins (P and N) which participate in the viral ribonucleoprotein (RNP) transcription-replication complex. This strategy combined two-hybrid and green fluorescent protein–reverse two-hybrid assays in Saccharomyces cerevisiae to analyze protein-protein interactions and a reverse genetic assay in mammalian cells to study the transcriptional activity of the reconstituted RNP complex. Lyssavirus P proteins contain two N-binding domains (N-BDs), a strong one encompassing amino acid (aa) 176 to the C terminus and a weak one in the 189 N-terminal aa. The N-terminal portion of P (aa 52 to 189) also contains a homomultimerization site. Here we demonstrate that N-P interactions, although weaker, are maintained between proteins of the different genotypes. A minimal transcriptional module of the P protein was obtained by fusing the first 60 N-terminal aa containing the L protein binding site to the C-terminal strong N-BD. Random mutation of the strong N-BD on P protein identified three highly conserved K residues crucial for N-P interaction. Their mutagenesis in full-length P induced a transcriptionally defective RNP. The analysis of homologous interactive domains presented here and previously reported dissections of the P protein allowed us to propose a model of the functional interaction network of the lyssavirus P protein. This model underscores the central role of P at the interface between L protein and N-RNA template.

Peptides that mimic the amino-terminal end of the rabies virus phosphoprotein have antiviral activity.

ABSTRACT We wanted to develop a therapeutic approach against rabies disease by targeting the lyssavirus transcription/replication complex. Because this complex (nucleoprotein N-RNA template processed by the L polymerase and its cofactor, the phosphoprotein P) is similar to that of other negative-strand RNA viruses, we aimed to design broad-spectrum antiviral drugs that could be used as a complement to postexposure vaccination and immunotherapy. Recent progress in understanding the structure/function of the rabies virus P, N, and L proteins predicts that the amino-terminal end of P is an excellent target for destabilizing the replication complex because it interacts with both L (for positioning onto the N-RNA template) and N (for keeping N soluble, as needed for viral RNA encapsidation). Thus, peptides mimicking various lengths of the amino-terminal end of P have been evaluated, as follows: (i) for binding properties to the N-P-L partners by the two-hybrid method; (ii) for their capacity to inhibit the transcription/replication of a rabies virus minigenome encoding luciferase in BHK-21-T7 cells; and (iii) for their capacity to inhibit rabies virus infection of BHK-21-T7 cells and of two derivatives of the neuronal SK-N-SH cell line. Peptides P60 and P57 (the first 60 and first 57 NH2 residues of P, respectively) exhibited a rapid, strong, and long-lasting inhibitory potential on luciferase expression (>95% from 24 h to 55 h). P42 was less efficient in its inhibition level (75% for 18 to 30 h) and duration (40% after 48 h). The most promising peptides were synthesized in tandem with the Tat sequence, allowing cell penetration. Their inhibitory effects were observed on BHK-21-T7 cells infected with rabies virus and Lagos bat virus but not with vesicular stomatitis virus. In neuronal cells, a significant inhibition of both nucleocapsid inclusions and rabies virus release was observed.

Antiviral Drug Discovery Strategy Using Combinatorial Libraries of Structurally Constrained Peptides

ABSTRACT We have developed a new strategy for antiviral peptide discovery by using lyssaviruses (rabies virus and rabies-related viruses) as models. Based on the mimicry of natural bioactive peptides, two genetically encoded combinatorial peptide libraries composed of intrinsically constrained peptides (coactamers) were designed. Proteomic knowledge concerning the functional network of interactions in the lyssavirus transcription-replication complex highlights the phosphoprotein (P) as a prime target for inhibitors of viral replication. We present an integrated, sequential drug discovery process for selection of peptides with antiviral activity directed against the P. Our approach combines (i) an exhaustive two-hybrid selection of peptides binding two phylogenetically divergent lyssavirus Ps, (ii) a functional analysis of protein interaction inhibition in a viral reverse genetic assay, coupled with a physical analysis of viral nucleoprotein-P complex by protein chip mass spectrometry, and (iii) an assay for inhibition of lyssavirus infection in mammalian cells. The validity of this strategy was demonstrated by the identification of four peptides exhibiting an efficient antiviral activity. Our work highlights the importance of P as a target in anti-rabies virus drug discovery. Furthermore, the screening strategy and the coactamer libraries presented in this report could be considered, respectively, a general target validation strategy and a potential source of biologically active peptides which could also help to design pharmacologically active peptide-mimicking molecules. The strategy described here is easily applicable to other pathogens.

Mitochondrial Dysfunction in Lyssavirus-Induced Apoptosis

ABSTRACT Lyssaviruses are highly neurotropic viruses associated with neuronal apoptosis. Previous observations have indicated that the matrix proteins (M) of some lyssaviruses induce strong neuronal apoptosis. However, the molecular mechanism(s) involved in this phenomenon is still unknown. We show that for Mokola virus (MOK), a lyssavirus of low pathogenicity, the M (M-MOK) targets mitochondria, disrupts the mitochondrial morphology, and induces apoptosis. Our analysis of truncated M-MOK mutants suggests that the information required for efficient mitochondrial targeting and dysfunction, as well as caspase-9 activation and apoptosis, is held between residues 46 and 110 of M-MOK. We used a yeast two-hybrid approach, a coimmunoprecipitation assay, and confocal microscopy to demonstrate that M-MOK physically associates with the subunit I of the cytochrome c (cyt-c) oxidase (CcO) of the mitochondrial respiratory chain; this is in contrast to the M of the highly pathogenic Thailand lyssavirus (M-THA). M-MOK expression induces a significant decrease in CcO activity, which is not the case with M-THA. M-MOK mutations (K77R and N81E) resulting in a similar sequence to M-THA at positions 77 and 81 annul cyt-c release and apoptosis and restore CcO activity. As expected, the reverse mutations, R77K and E81N, introduced in M-THA induce a phenotype similar to that due to M-MOK. These features indicate a novel mechanism for energy depletion during lyssavirus-induced apoptosis.

Monitoring of chloride and activity of glycine receptor channels using genetically encoded fluorescent sensors

Genetically encoded probes have become powerful tools for non-invasive monitoring of ions, distributions of proteins and the migration and formation of cellular components. We describe the functional expression of two molecular probes for non-invasive fluorescent monitoring of intracellular Cl ([Cl]i) and the functioning of glycine receptor (GlyR) channels. The first probe is a recently developed cyan fluorescent protein–yellow fluorescent protein-based construct, termed Cl-Sensor, with relatively high sensitivity to Cl (Kapp∼30 mM). In this study, we describe its expression in retina cells using in vivo electroporation and analyse changes in [Cl]i at depolarization and during the first three weeks of post-natal development. An application of 40 mM K+ causes an elevation in [Cl]i of approximately 40 mM. In photoreceptors from retina slices of a 6-day-old rat (P6 rat), the mean [Cl]i is approximately 50 mM, and for P16 and P21 rats it is approximately 30–35 mM. The second construct, termed BioSensor-GlyR, is a GlyR channel with Cl-Sensor incorporated into the cytoplasmic domain. This is the first molecular probe for spectroscopic monitoring of the functioning of receptor-operated channels. These types of probes offer a means of screening pharmacological agents and monitoring Cl under different physiological and pathological conditions and permit spectroscopic monitoring of the activity of GlyRs expressed in heterologous systems and neurons.

Calcium-dependent modulation of human glycine receptors expressed in cultivated cell lines

Glycine receptors (GlyRs) provide the main inhibitory neurotransmission in spinal cord and brain-stem synapses of vertebrates. Fucile et al. (2000) discovered that elevation of intracellular Ca2+ caused rapid potentiation of GlyRs. This modulation develops in less than 100 ms. It is characterized by an increase in GlyR apparent affinity for glycine. It has been suggested that the phenomenon of Ca-induced potentiation involves an unknown Ca2+-binding protein (CaBP). Using the yeast two-hybrid system, screening of a human brain cDNA library against the cytoplasmic loop of human alpha 1 subunit (GlyRh1) allowed us to identify five new interactors. One of them belongs to the family of Ca-binding proteins. We analyzed the effect of “short” forms of this protein (CaBP-1) on functional properties of GlyRh1 expressed in HEK-293 and CHO cells. Using whole-cell recordings and rapid agonist application, we constructed concentration dependences of glycine-induced currents. This analysis revealed statistically significant differences in EC50s between control cells (expressing only GlyRh1) and those expressing CaBP-1. In HEK-293 cells recorded under conditions of low intracellular Ca concentration (BAPTA 20 mM in the recording pipette), EC50 for glycine in control cells and expressing GlyRh1+CaBP-1 were, correspondingly, 68 ± 49 μM (n = 29) and 409 ± 421 μM (n = 60). In CHO cells, EC50 were 54 ± 43 μM (n = 25) and 123 ± 104 μM (n = 28). The differences were not statistically significant for recordings made with an intracellular solution containing high Ca concentration (50 μM). Under these conditions, EC50 values were correspondingly 35 ± 28 μM (n = 7) and 64 ± 38 μM (n = 7). These results suggest that CaBP-1 causes a decrease of GlyR sensitivity to agonist interacting with the cytoplasmic domain of GlyR.

A genetically encoded indicator for monitoring intracellular chloride

Inhibitory drives in the nervous system are provided by synapses operating mostly through Cl− ion channels. We describe a novel CFP-YFP-based fluorescence Cl− indicator, Cl-sensor, characterized by a high sensitivity, which can be successfully used as a tool for monitoring intracellular Cl− in various biological preparations.