Cytomégalovirus humain et antiviraux (supports génétiques des résistances et cibles de nouveaux antiviraux )

Le cytomégalovirus est un herpèsvirus responsable d'infections graves chez les personnes immunodéprimées. Les traitements disponibles, peu nombreux, s appuient sur des inhibiteurs de l ADN polymérase virale pUL54 : le cidofovir, le foscarnet et le ganciclovir. Le ganciclovir, utilisé en première intention pour la prévention ou le traitement des infections à cytomégalovirus, nécessite, pour être actif, d être primo-phosphorylé par la kinase virale pUL97. Du fait de la cytotoxicité de ces agents, de leur faible biodisponibilité et de l émergence de résistances, de nouvelles molécules ne ciblant pas la synthèse de l ADN viral sont en voie de développement. Il s agit de dérivés benzimidazolés dextrogyres, le BDCRB et le TCRB, qui ciblent des protéines impliquées dans l encapsidation de l ADN viral (pUL56, pUL89, et pUL104), et d un dérivé benzimidazolé lévogyre, le maribavir, qui inhibe l exportation extranucléaire des capsides néoformées par un mécanisme impliquant la phosphotransférase pUL97 et la protéine pUL27. Ce travail s articule en deux axes principaux : les antiviraux actuels et les molécules en développement. Concernant les traitements actuels nous avons développé des outils de détection et d étude des résistances. Puis nous avons appliqué ces outils à des études multicentriques, visant à définir la fréquence des résistances et le polymorphisme naturel des cibles des antiviraux actuels (pUL97 et pUL54). Ensuite, nous avons développé des modèles théoriques de la structure tridimensionnelle des domaines fonctionnels de ces enzymes afin de mieux comprendre leur fonction et d évaluer le rôle de certaines mutations dans les résistances. Concernant les molécules antivirales en développement, nous avons utilisé le principe de génotypage développé pour les cibles des antiviraux traditionnels pour étudier le polymorphisme naturel des protéines cibles de ces molécules (pUL27, pUL56, pUL89 et pUL104). Dans un deuxième temps, nous avons analysé la conservation inter-espèces et la structure primaire de ces protéines afin de mieux comprendre leur fonction et les mécanismes moléculaires des résistances.Drugs usually used for treatment or prophylaxis of HCMV disease lean on few inhibitors of viral DNA synthesis targeting the HCMV DNA polymerase pUL54 : ganciclovir, cidofovir and foscarnet. Because of these drugs are associated with multiple side effects and drug resistance emergence, new drugs like benzimidazole derivatives, that target the viral DNA processing, being developed. The benzimidazole d-ribonucleosides inhibit DNA packaging by a process implicating the packaging proteins pUL56 and pUL89 and the portal protein pUL104. The benzimidazole l-ribonucleosides inhibit the virion nuclear egress. Little is known about both the benzimidazole D-ribonucleosides and benzimidazole L-ribonucleosides mechanisms of action. The aim of this study was to develop fast and standardized tools for detection of HCMV drugs resistance and to study the function of the targets of these molecules and their mechanism of action.LIMOGES-BU Sciences (870852109) / SudocSudocFranceF

Ternary polysaccharide complexes: Colloidal drug delivery systems stabilized in physiological media

International audienceChitosan-hyaluronan (HYA) polyelectrolyte complexes (PECs) were designed to maintain their colloidal stabilities in physiological ionic strength and pH, via a new concept of ternary complexes. This strategy relied on the formation of a binary PEC between chitosan and a strong polyacid, dextran sulphate (DS) or heparin (HEP), and further functionalization with HYA. The major parameter leading to stabilized colloids was a high ratio of the degrees of polymerization of chitosan versus the strong polyacid. The process afforded either positive or negative particles when HYA was used in default or in excess (vs. chitosan) for the functionalization of the binary complexes. The most stable formulations were loaded with an antiretroviral drug tenofovir (TF), and could be surface functionalized with targeting IgAs. In vitro, the cationic TF loaded ternary complexes exhibited an inhibition of infection of PBMCs by the HIV-1 virus, superior to the free drug

Zinc-Stabilized Chitosan-Chondroitin Sulfate Nanocomplexes for HIV-1 Infection Inhibition Application

International audiencePolyelectrolyte complexes (PECs) constituted of chitosan and chondroitin sulfate (ChonS) were formed by the one-shot addition of default amounts of polyanion to an excess of polycation. Key variables of the formulation process (e.g., degree of depolymerization, charge mixing ratio, the concentration, and pH of polyelectrolyte solutions) were optimized based on the PECs sizes and polydispersities. The PECs maintained their colloidal stability at physiological salt concentration and pH thanks to the complexation of polyelectrolytes with zinc(II) ion during the nanoPECs formation process. The PECs were capable of encapsulating an antiretroviral drug tenofovir (TF) with a minimal alteration on the colloidal stability of the dispersion. Moreover, the particle interfaces could efficiently be functionalized with anti-OVA or anti-alpha 4 beta 7 antibodies with conservation of the antibody biorecognition properties over 1 week of storage in PBS at 4 degrees C. In vitro cytotoxicity studies showed that zinc(II) stabilized chitosan-ChonS nanoPECs were noncytotoxic to human peripheral blood mononuclear cells (PBMCs), and in vitro antiviral activity test demonstrated that nanoparticles formulations led to a dose-dependent reduction of HIV-1 infection. Using nanoparticles as a drug carrier system decreases the IC50 (50% inhibitory concentration) from an aqueous TF of 4.35 mu mol.L-1 to 1.95 mu mol.L-1. Significantly, zinc ions in this system also exhibited a synergistic effect in the antiviral potency. These data suggest that chitosan-ChonS nanoPECs can be promising drug delivery system to improve the antiviral potency of drugs to the viral reservoirs for the treatment of HIV infection

Zinc-stabilized colloidal polyelectrolyte complexes of chitosan/hyaluronan: a tool for the inhibition of HIV-1 infection

International audienceZinc(II) stabilized polyelectrolyte nano-complexes (PECs) of chitosan and hyaluronan (HYA) were designed as safe and efficient drug delivery systems. HIV-1 reverse transcriptase inhibitor tenofovir (TF) was quantitatively encapsulated and the particle interface could be functionalized in PBS with targeting proteins such as anti-alpha 4b7 immunoglobulin A. Chitosan-HYA nanoPECs were non-cytotoxic on human peripheral blood mononuclear cells (PBMCs), within the investigated nanoparticle concentrations. A dose-dependent reduction of the HIV-1 infection of PBMCs co-cultured with the nanocarriers was observed. Even more interestingly, a synergistic effect was evidenced with the nanocarriers by comparing the IC50 (50% inhibitory concentration) value of the aqueous TF solution (4.35 mu mol L-1) with that of TF loaded nanoPECs (1.71 mu mol L-1) and anti-a4b7 IgA functionalized TF/nanoPECs (1.01 mu mol L-1). This effect could be attributed to the presence of zinc(II) in the formulation of the colloids. All these data establish that the zinc(II) stabilized chitosan-HYA nanoPECs can be potentially efficient and safe colloidal delivery system candidates for enhancing antiviral activities in the treatment of HIV infection and AIDS

Putative functional domains of human cytomegalovirus pUL56 involved in dimerization and benzimidazole D-ribonucleoside activity.

International audienceBenzimidazole D-ribonucleosides inhibit DNA packaging during human cytomegalovirus (HCMV) replication. Although they have been shown to target pUL56 and pUL89 (the large and small subunits of the HCMV terminase, respectively) their mechanism of action is not yet fully understood. We aimed here to better understand HCMV DNA maturation and the mechanism of action of benzimidazole derivatives

A novel mutation in the UL54 gene of human cytomegalovirus isolates that confers resistance to foscarnet

International audienceFoscarnet is currently licensed for the treatment of human cytomegalovirus (HCMV) infection. Mutations proven to confer resistance to foscarnet have mostly been mapped to regions II, III and VI of the HCMV UL54-encoded DNA polymerase. We previously showed that sequential foscarnet-resistant HCMV isolates recovered from a patient with lymphoma had change N495K in region delta-C of the DNA polymerase. To evaluate the impact of change N495K on HCMV sensitivity to foscarnet, a recombinant HCMV strain carrying the mutation was produced by homologous recombination. The recombinant virus showed a 3.4-fold increase in foscarnet resistance, and remained sensitive to ganciclovir and cidofovir. In addition, the recombinant strain showed a reduction of infectious virus yield compared with its parent strain. Change N495K should be added to the list of mutations conferring resistance to foscarnet and be taken into account in the genotypic diagnosis of antiviral resistance

Conserved domains and structure prediction of human cytomegalovirus UL27 protein.

International audienceThe human cytomegalovirus (HCMV) nuclear UL27 protein (pUL27) could be involved at the stage of nuclear egress. Maribavir is a new anti-HCMV drug that targets nuclear egress through direct inhibition of the HCMV serine-threonine kinase, UL97 protein (pUL97). Because maribavir-resistance-related mutations are observed in both proteins, pUL27 is thought to interfere with pUL97 activity; however, its mechanism of action remains unclear

Putative functional domains of human cytomegalovirus pUL56 involved in dimerization and benzimidazole D-ribonucleoside activity

International audienceBACKGROUND: Benzimidazole D-ribonucleosides inhibit DNA packaging during human cytomegalovirus (HCMV) replication. Although they have been shown to target pUL56 and pUL89 (the large and small subunits of the HCMV terminase, respectively) their mechanism of action is not yet fully understood. We aimed here to better understand HCMV DNA maturation and the mechanism of action of benzimidazole derivatives. METHODS: The HCMV pUL56 protein was studied by sequence analysis of the HCMV UL56 gene and herpesvirus counterparts combined with primary structure analysis of the corresponding amino acid sequences. RESULTS: The UL56 sequence analysis of 45 HCMV strains and counterparts among herpesviruses allowed the identification of 12 conserved regions. Moreover, comparison with the product of gene 49 (gp49) of bacteriophage T4 suggested that the pUL56 zinc finger is localized close to the dimerization site of pUL56, providing a spatial organization of the catalytic site that allows recognition and cleavage of DNA. CONCLUSIONS: This study provides a basis to investigate the mechanism of concatemeric DNA cleavage and a biochemical basis for DNA packaging inhibition by benzimidazole derivatives

New functional domains of human cytomegalovirus pUL89 predicted by sequence analysis and three-dimensional modelling of the catalytic site DEXDc.

International audienceBenzimidazole D-ribonucleosides inhibit DNA packaging during human cytomegalovirus (HCMV) replication. Although they have been shown to target pUL56 and pUL89, the large and small subunits of the HCMV terminase respectively, their mechanism of action is not yet fully understood

New functional domains of human cytomegalovirus pUL89 predicted by sequence analysis and three-dimensional modelling of the catalytic site DEXDc

International audienceINTRODUCTION: Benzimidazole D-ribonucleosides inhibit DNA packaging during human cytomegalovirus (HCMV) replication. Although they have been shown to target pUL56 and pUL89, the large and small subunits of the HCMV terminase respectively, their mechanism of action is not yet fully understood. METHODS AND RESULTS: To better understand HCMV DNA maturation and the mechanism of action of benzimidazole derivatives, we studied the HCMV pUL89 protein by a genetic approach combined with primary structure analysis. The pUL89 sequence analysis of 25 HCMV strains and counterparts among herpesviruses allowed identification of 12 conserved regions. We also built a three-dimensional model of the pUL89 ATPasic catalytic site, including ATPase motor motifs 1, II and III, that may facilitate the development of future antiviral drugs active against HCMV. Finally, we identified several putative functional domains in pUL89, such as pUL89 zinc finger (pUL89-ZF), DNA cutting sites and portal binding sites, that are probably involved in CMV DNA cleavage and packaging