Dengue is caused by a taxonomic group of four viruses, dengue virus types 1-4 (DENV1-DENV4). A molecular understanding of the antibody-mediated protection against this disease is critical to design safe vaccines and therapeutics. Here, the energetic epitope of antibody mAb4E11, which neutralizes the four serotypes of DENV but no other flavivirus, and binds domain 3 (ED3) of their envelope glycoprotein, was characterized. Alanine-scanning mutagenesis of the ED3 domain from serotype DENV1 was performed and the affinities between the mutant domains and the Fab fragment of mAb4E11 were measured. The epitope residues (307-312, 387, 389 and 391) were at the edges of two distinct b-sheets. Four residues constituted hot spots of binding energy. They were aliphatic and contributed to form a hydrophobic pocket (Leu308, Leu389), or were positively charged (Lys307, Lys310). They may bind the diversity residues of mAb4E11, H-Trp96-Glu97. Remarkably, cyclic residues occupy and block the hydrophobic pocket in all unrelated flaviviruses. Transplanting the epitope from the ED3 domain of DENV into those of other flaviviruses restored affinity. The epitope straddles residues of ED3 that are involved in virulence, e.g. Asn/Asp390. These results define the epitope of mAb4E11 as an antigenic signature of the DENV group and suggest mechanisms for its neutralization potency. INTRODUCTIONDengue is a re-emerging viral disease. It is caused by four types of virus, dengue virus types 1-4 (DENV1-DENV4), which belong to the species Dengue virus in the genus Flavivirus (Heinz et al., 2000). They are transmitted by Aedes mosquitoes and infect between 50 million and 100 million persons each year. The disease generally takes a mild form, dengue fever, but severe forms, dengue haemorragic fever and dengue shock syndrome, have recently become epidemic (Gubler, 2002).The immune response against an infection by DENV involves both a humoral component, in the form of neutralizing antibodies, and a cell-mediated component (Guzman & Kouri, 2002). The preferential reactivation of the memory B cells that correspond to a primary infection, and an antibody-dependent enhancement of infection, might constitute triggering mechanisms of the severe forms during a secondary infection by a different viral serotype (Halstead, 2003; Mongkolsapaya et al., 2003). A molecular understanding of the events that lead to antibody neutralization, enhancement or escape is critical to the development of efficient and secure vaccines and therapeutics. DENV1-DENV4 are enveloped RNA viruses, like all flaviviruses. The structures of the whole virus and of the envelope glycoprotein E (gpE) have been solved by electron cryomicroscopy and X-ray crystallography (Modis et al., 2003(Modis et al., , 2005 Zhang et al., 2003). Ninety dimers of gpE cover the surface of the virus. Each monomer comprises three ectodomains, ED1-ED3, and a transmembrane segment. ED2 includes the dimerization interface, a glycosylation site and the peptide of fusion with the cellular membrane. ED3 is continuou...
Dengue is a disease which is re-emerging, viral and transmitted by the Aedes mosquitoes. Approximately 100 million individuals are affected by the disease annually and one billion are at risk, mainly in the subtropical regions. Severe forms of the disease can lead to death within hours. There is an urgent need for preventive or curative tools to fight against the dengue virus, because no such specific treatment exists to date.The virus has four serotypes, DEN1 to DEN4. Several tetravalent vaccines are under development but they will not be available for at least a decade, and comprehensive vaccinal coverage might be difficult to achieve [1,2].The dengue virus is an enveloped RNA virus. The structures of the whole virus and of its envelope glycoprotein E have been elucidated by a combination of Dengue is a re-emerging viral disease, affecting approx. 100 million individuals annually. The monoclonal antibody mAb4E11 neutralizes the four serotypes of the dengue virus, but not other flaviviruses. Its epitope is included within the highly immunogenic domain 3 of the envelope glycoprotein E. To understand the favorable properties of recognition between mAb4E11 and the virus, we recreated the genetic events that led to mAb4E11 during an immune response and performed an alanine scanning mutagenesis of its third hypervariable loops (H-CDR3 and L-CDR3). The affinities between 16 mutant Fab fragments and the viral antigen (serotype 1) were measured by a competition ELISA in solution and their kinetics of interaction by surface plasmon resonance. The diversity and junction residues of mAb4E11 (D segment; V H -D, D-J H and V L -J L junctions) constituted major hotspots of interaction energy. Two residues from the D segment (H-Trp96 and H-Glu97) provided > 85% of the free energy of interaction and were highly accessible to the solvent in a three-dimensional model of mAb4E11. Changes of residues (L-Arg90 and L-Pro95) that statistically do not participate in the contacts between antibodies and antigens but determine the structure of L-CDR3, decreased the affinity between mAb4E11 and its antigen. Changes of L-Pro95 and other neutral residues strongly decreased the rate of association, possibly by perturbing the topology of the electrostatic field of the antibody. These data will help to improve the properties of mAb4E11 for therapeutic applications and map its epitope precisely.Abbreviations -, covalent bond; ::, noncovalent bond; CDR, complementary determining region; E3, domain 3 of gpE; gpE, glycoprotein E; H-Trp96, a tryptophan residue in position 96 of the heavy chain; H-W96A, mutation of residue H-Trp96 into Ala; RU, resonance unit; SDR, structure determining residue.
Coordinating homeostasis of multiple metabolites is a major task for living organisms, and complex interconversion pathways contribute to achieving the proper balance of metabolites. AMP deaminase (AMPD) is such an interconversion enzyme that allows IMP synthesis from AMP. In this article, we show that, under specific conditions, lack of AMPD activity impairs growth. Under these conditions, we found that the intracellular guanylic nucleotide pool was severely affected. In vivo studies of two AMPD homologs, Yjl070p and Ybr284p, indicate that these proteins have no detectable AMP, adenosine, or adenine deaminase activity; we show that overexpression of YJL070c instead mimics a loss of AMPD function. Expression of the yeast transcriptome was monitored in a AMPD-deficient mutant in a strain overexpressing YJL070c and in cells treated with the immunosuppressive drug mycophenolic acid, three conditions that lead to severe depletion of the guanylic nucleotide pool. These three conditions resulted in the up-or downregulation of multiple transcripts, 244 of which are common to at least two conditions and 71 to all three conditions. These transcriptome results, combined with specific mutant analysis, point to threonine metabolism as exquisitely sensitive to the purine nucleotide balance.T HE purine nucleotides, ATP and GTP, are involved in almost all aspects of cellular life. In addition to their role as building blocks of nucleic acids, adenylic and guanylic nucleotides have specific roles. For example, GTP is critical for translation and for signaling through GTPases, while ATP is the major energyproviding molecule in the cell. In yeast, intracellular concentrations of ATP and GTP are clearly different ($5 and 1.5 mm, respectively; Breton et al. 2008;Gauthier et al. 2008), most probably as the result of regulatory processes that maintain homeostasis. In most eukaryotic cells, including yeast, adenylic and guanylic nucleotides either are synthesized from a common precursor (IMP) or are recycled from preformed bases or nucleosides (Figure 1). While most enzymes involved in these processes have been identified, the physiological consequences of purine nucleotide imbalance are far from being understood. Interestingly, drugs specifically inhibiting GTP synthesis, such as mycophenolic acid (MPA), have a strong immunosuppressive effect and are now widely used to limit allograft rejection. We have previously established the effect of MPA on the yeast proteome and have identified numerous yeast mutants hypersensitive to this drug (Desmoucelles et al. 2002). MPA effects are due to GTP shortage, since they are reversed by exogenous guanine, allowing replenishment of the GTP pool. However, drugs often have secondary effects and can be detoxified and/or diluted during cell growth. As an alternative, consequences of purine nucleotide imbalance can be investigated using yeast mutants. In two previous studies, we have used specific mutants to increase the GTP pool or decrease the ATP pool (Breton et al. 2008;Gauthier et al. 20...
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