Jean-Louis Romette

A Structural Basis for the Inhibition of the NS5 Dengue Virus mRNA 2′-O-Methyltransferase Domain by Ribavirin 5′-Triphosphate

Ribavirin is one of the few nucleoside analogues currently used in the clinic to treat RNA virus infections, but its mechanism of action remains poorly understood at the molecular level. Here, we show that ribavirin 5′-triphosphate inhibits the activity of the dengue virus 2′-O-methyltransferase NS5 domain (NS5MTaseDV). Along with several other guanosine 5′-triphosphate analogues such as acyclovir, 5-ethynyl-1-β-d-ribofuranosylimidazole-4-carboxamide (EICAR), and a series of ribose-modified ribavirin analogues, ribavirin 5′-triphosphate competes with GTP to bind to NS5MTaseDV. A structural view of the binding of ribavirin 5′-triphosphate to this enzyme was obtained by determining the crystal structure of a ternary complex consisting of NS5MTaseDV, ribavirin 5′-triphosphate, and S-adenosyl-l-homocysteine at a resolution of 2.6 Å. These detailed atomic interactions provide the first structural insights into the inhibition of a viral enzyme by ribavirin 5′-triphosphate, as well as the basis for rational drug design of antiviral agents with improved specificity against the emerging flaviviruses.

The flavivirus polymerase as a target for drug discovery

Flaviviruses are emerging pathogens of increasingly important public health concern in the world. For most flaviviruses such as dengue virus (DENV) and West Nile virus (WNV) neither vaccine nor antiviral treatment is available. The viral RNA-dependent RNA polymerase (RdRp) non-structural protein 5 (NS5) has no equivalent in the host cell and is essential for viral replication. Here, we give an overview of the current knowledge regarding Flavivirus RdRp function and structure as it represents an attractive target for drug design. Flavivirus RdRp exhibits primer-independent activity, thus initiating RNA synthesis de novo. Following initiation, a conformational change must occur to allow the elongation process. Structure-function studies of Flavivirus RdRp are now facilitated by the crystal structures of DENV (serotype 3) and WNV RdRp domains. Both adopt a classic viral RdRp fold and present a closed pre-initiation conformation. The so-called priming loop is thought to provide the initiation platform stabilizing the de novo initiation complex. A zinc-ion binding site at the hinge between two subdomains might be involved in opening up the RdRp structure towards a conformation for elongation. Using two different programs we predicted common potential allosteric inhibitor binding sites on both structures. We also review ongoing approaches of in vitro and cell-based screening programs aiming at the discovery of nucleosidic and non-nucleosidic inhibitors targeting Flavivirus RdRps.

Glucose-oxidase electrode. Measurements of glucose in samples exhibiting high variability in oxygen content

Measurements of glucose in samples exhibiting high variability in oxygen content present an important problem to solve for pO2 detection methods. Different solutions exist, all based on the stabilization of the pO2 sample before or during the measurement. The electrode described in this paper itself contains enough O2 to compensate for the variability of the sample oxygen content. The enzyme is cross-linked with gelatin using the bifunctional agent, glutaraldehyde. Amperometric enzyme electrodes have been constructed using these membranes. Measurements have been done by collecting the derivative in time of the signal given by the electrode for blood or plasma samples.

The VIZIER project: preparedness against pathogenic RNA viruses.

Abstract Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules.

Dengue virus replicons: production of an interserotypic chimera and cell lines from different species, and establishment of a cell-based fluorescent assay to screen inhibitors, validated by the evaluation of ribavirin's activity.

The prevention and treatment of flavivirus infections are public health priorities. Dengue fever is the most prevalent mosquito-borne viral disease of humans, affecting more than 50 million people annually. Despite the urgent need to control dengue infections, neither specific antiviral therapies nor licensed vaccines exist and the molecular basis of dengue pathogenesis is not well understood. In this study we produced a novel dengue virus type 2 (DV2) subgenomic replicon that expresses a fusion protein comprised of Enhanced Green Fluorescent Protein (EGFP) and Puromycin N-Acetyltransferase (PAC). We successfully established BHK, COS and Huh7 cell lines that stably expressed the DV2 replicon. Using EGFP as a reporter of DV replication complex activity, we set up a new HTS assay. The assay was validated using the inhibitor ribavirin, confirmed by flow cytometry analysis and the analysis of NS5 expression by Western-blot analysis. In order to develop a system to test antivirals against the NS5 proteins of all four DV serotypes in a similar cellular environment, the replicon was further modified, to allow easy exchange of the NS5 gene between DV serotypes. As proof of principle, a chimeric replicon in which the DV2 NS5 gene was substituted with that of DV type 3 was stably expressed in BHK cells and used in ribavirin inhibition studies. The assays described in this study will greatly facilitate DV drug discovery by serving as primary or complementary screening. The approach should be applicable to the development of fluorescent cell-based HTS assays for other flaviviruses, and useful for the study of many aspects of DV, including viral replication and pathogenesis.

Purification and properties of two laccase isoenzymes produced byBotrytis cinerea

Laccases produced by five strains ofBotrytis cinerea were studied. Extraction and purification have been performed in order to compare the enzymatic characteristics both, physicochemically and kinetically.Two strains produced isoenzymes of laccase. These two molecular forms of laccase had different isoelectric points (2.6 and 2.8) and sugar content (86 and 91%). The optimum reactional pH was found to be very similar for both enzymes, in contrast to the temperature sensitivity, which is very different.

Immobilized respiratory chain activities from Escherichia coli utilized to measure D- and L-lactate, succinate, L-malate, 3-glycerophosphate, pyruvate, or NAD(P)H

The respiratory chain (membranous, multienzymatic system) fromEscherichia coli, was coimmobilized with gelatin and insolubilized in film form by tanning with glutaraldehyde. The film was fixed onto an oxygen sensor. The enzyme electrode can be used for measuring NAD(P)H, d and L-lactate, succinate, l-malate, 3-glycerophosphate, or pyruvate. The range of metabolites concentrations was from 1 to 50 mM.It was possible to dicriminate between the different metabolites (if mixed):(1)By inducing during bacterial growth the specific flavoproteins necessary for l-lactate, succinate, l-malate, and 3-glycerophosphate respirations. The constitutive activities are unaltered on glucose or glycerol, namely D-lactate, NAD(P)H, and pyruvate respiration.2.When intact bacteria were immobilized (with or without induction), D and L-lactate, succinate, 3-glycerophosphate, and L-malate respiration were measured, no activities of pyruvate and NAD(P)H respiration were obtained. For these last activities, French press breakage (see section on Membrane Preparations) of bacteria prior to immobilization was necessary.3.Products of reactions can be used as enzyme inhibitors: Pyruvate inhibits d and l-lactate; fumarate inhibits succinate, and oxaloacetate inhibits l-malate respirations.4.Heat denaturation of the bacteria at 55°C for 1 h maintains full activity of succinate and pyruvate respiration. On the other hand, no activity of d and l-lactate, l-malate, or NAD(P)H respiration was measurable.These enzyme electrodes have many applications in basic and applied research.

The European Virus Archive: A new resource for virology research

Abstract The European Virus Archive (EVA) was conceived as a direct response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry, initially within Europe, but ultimately throughout the world. Although scientists worldwide have accumulated virus collections since the early twentieth century, the quality of the collections and the viruses collected may vary according to the personal interests and agenda of the scientists. Moreover, when laboratories are re-organised or closed, collections are no longer maintained and gradually cease to exist. The tragedy of 9/11 and other disruptive activities have also meant that some previously available biological reagents are no longer openly exchanged between countries. In 2008, funding under the FP7–EU infrastructure programme enabled the initiation of the EVA. Within three years, it has developed from a consortium of nine European laboratories to encompass associated partners in Africa, Russia, China, Turkey, Germany and Italy. There is every reason to believe that EVA will continue to expand and ultimately exist as a globally networked, quality-controlled non-profit archive for the benefit of science. Organizations or individuals who would like to be considered as contributors are invited to contact the EVA coordinator, Jean–Louis Romette, at jean-louis.romette@univmed.fr.

Production of Sm37‐GAPDH, a major therapeutical target in human schistosomiasis

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic metabolism and the production of energy. This probably explains why GAPDH was evidenced as a major therapeutical target in several parasitic diseases; either as a vaccine candidate or as a target for chemotherapeutic treatments. Schistosoma mansoni GAPDH (Sm37-GAPDH) is one of the main schistosome vaccine candidates. The production of recombinant Sm37-GAPDH is essential to evaluate the ability of this molecule to induce protective immunity in animals and possibly in humans. The cDNA encoding Sm37-GAPDH has been cloned and sequenced. In addition, five B cell (including the major B-cell epitope Sm35-5) and two T cell epitopes have been localized on the molecule. Different expression systems have been evaluated in respect with the production yield and the GAPDH enzymatic activity. Some of them have led to either a high production of insoluble material (E. coli) or to an inactive enzyme (Pischia pastoris). The present article describes the production setting of rSm37-GAPDH using the baculovirus-insect cell system. Large amounts of soluble rSm37-GAPDH with enzymatic activity were obtained. Most sera from individuals living in an area endemic for S. mansoni recognised the rSm37 molecule and inhibited its catalytic activity.

Expression in Insect Cells of the Major Parasite Antigen Associated with Human Resistance to Schistosomiasis

We have produced and purified a functional and active recombinant S.mansoni G3PDH. To the best of our knowledge, this report is the first to describe the quantitative production of biologically active rS.mansoni G3PDH. Given that IgG antibodies to the S.mansoni G3PDH are associated with resistance to infection in human, the biologically active rSm37-G3PDH may prove important as a component of an anti S.mansoni vaccine. Recombinant antigens isolated under nondenaturating conditions which retain biological activity as in this case should resemble the natural parasite antigen on the opposite to inactive or denaturated forms.