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In vitro expression and enzyme activity analysis of mutant human cytomegalovirus DNA polymerases
ELSEVIER
Journal of Clinical Virology Journal of Clinical Virology 12 (1999) 109-115
Molecular Targets for Antiviral Development -
Posters
G1-01 In vitro expression and enzyme activity analysis of mutant human cytomegalovirus DNA polymerases SOPHIE ALAIN H6pital Lariboisi~re, Service de Bact~riologie-Virologie, Paris, France Aminoacid substitutions F-412-V/C, R-413-G (region IV) and A-987-G (region V) of human cytomegalovirus (HCMV) DNA polymerase (UL54) confer resistance to both ganciclovir and cidofovir. These data suggest the involvement of these aminoacids in triphosphate binding. The aim of the study was to analyse the effects of mutations in highly conserved positions around these aminoacids on substrate binding and enzyme activity. The UL54 coding sequence of Towne strain associated or not with its specific polyadenylation site was cloned into PGEM Zf 11 + downstream from a T7 promoter in the presence or absence of a 40bp AT-rich leader sequence from the alfalfa mosaic virus (AMV) and expressed in an in vitro coupled transcription/translation reticulocyte lysate system (TNT, Promega). DNA polymerase activity assays were performed using 32P dCTP and the protocol described by Cihlar et al. (Protein Expression and Purification, 1997) with some modifications. Constructs including the AMV leader sequence and the HCMV polyadenylation sites led to the higher polymerase activity and were used for in vitro mutagenesis. Mutations were introduced by using GeneEditor in vitro Site-directed mutagenesis System (Promega). Five mutants were obtained: 3 new mutants T-405-1, T-405-S, V-983-A and 2 controls F-412-C and A-987-G. First polymerase assays detected low polymerase activity for the mutant A-987-G and no activity for mutant V-983-A. Experiments are in progress to measure the activity of our mutant DNA polymerases and of control mutant and wild enzymes. Marker transfer experiments are in progress to analyse phenotypic susceptibility of our mutants to antiviral drugs.
G1-02 Inibition of human cytomegalovirus replication by a phosphodiester antisense oligonucleotide carried by SGal-beta-cyclodextrin SOUAD A B D O U UMR UHP-CNRS 7565 - GEVSM - Facult~ de Pharmacie, Nancy, France The use of antisense oligodeoxynucleotide as tools for modulating gene expression represents a novel strategy for designing drugs to treat diseases. Although progress has been rapid, the vulnerability of oligonucleotides to nucleases' attak, combined with their intracellular distribution and uptake properties limit their therapeutic potential. Cyclodextrins, consisting of cyclic oligosaccharidyl molecules, are well known for their property to form inclusion complexes with guest molecules and thus to ensure their transport and improve their pharmacological properties. We have investigated the ability of S-Gal-beta-cyclodextrin to enhance the antiviral activity of an human anti-cytomegalovirus phosphodiester oligonucleotide (anti-u136;PO), directed against the splice donor region of immediate early genes (u136-u137). The antiviral effect of (anti-u136;PO) free or complexed, was evaluated in cell culture against human cytomegalovirus strain reference AD169 and clinical isolates, and compared with that of ganciclovir (GCV). The (anti-u136;PO) showed higher antiviral activity than GCV. Indeed, according to the viral strain, its 50 % inhibitory concentration (IC50) is 2 to 18 fold lower than that of the GCV. Moreover the IC50 of [anti-u136;PO:S-Gal-beta-CD] complex exhibits a 3 to 5 fold enhancement comparatively to that of free oligonucleotide. This effect is neither due to a cytotoxicity of the molecules, nor to an antiviral effect of the cyclodextrin. In situ studies by flow cytometry and fluorescence microscopy showed that, after complexation, the oligonucleotide uptake is improved and could be due to an improvement of the oligonucleotide endocytosis. The intracellular distribution of free or complexed oligonucleotide is vesicular, cytosolic and perinuclear.
Journal of Clinical Virology Journal of Clinical Virology 12 (1999) 109-115
Molecular Targets for Antiviral Development -
Posters
G1-01 In vitro expression and enzyme activity analysis of mutant human cytomegalovirus DNA polymerases SOPHIE ALAIN H6pital Lariboisi~re, Service de Bact~riologie-Virologie, Paris, France Aminoacid substitutions F-412-V/C, R-413-G (region IV) and A-987-G (region V) of human cytomegalovirus (HCMV) DNA polymerase (UL54) confer resistance to both ganciclovir and cidofovir. These data suggest the involvement of these aminoacids in triphosphate binding. The aim of the study was to analyse the effects of mutations in highly conserved positions around these aminoacids on substrate binding and enzyme activity. The UL54 coding sequence of Towne strain associated or not with its specific polyadenylation site was cloned into PGEM Zf 11 + downstream from a T7 promoter in the presence or absence of a 40bp AT-rich leader sequence from the alfalfa mosaic virus (AMV) and expressed in an in vitro coupled transcription/translation reticulocyte lysate system (TNT, Promega). DNA polymerase activity assays were performed using 32P dCTP and the protocol described by Cihlar et al. (Protein Expression and Purification, 1997) with some modifications. Constructs including the AMV leader sequence and the HCMV polyadenylation sites led to the higher polymerase activity and were used for in vitro mutagenesis. Mutations were introduced by using GeneEditor in vitro Site-directed mutagenesis System (Promega). Five mutants were obtained: 3 new mutants T-405-1, T-405-S, V-983-A and 2 controls F-412-C and A-987-G. First polymerase assays detected low polymerase activity for the mutant A-987-G and no activity for mutant V-983-A. Experiments are in progress to measure the activity of our mutant DNA polymerases and of control mutant and wild enzymes. Marker transfer experiments are in progress to analyse phenotypic susceptibility of our mutants to antiviral drugs.
G1-02 Inibition of human cytomegalovirus replication by a phosphodiester antisense oligonucleotide carried by SGal-beta-cyclodextrin SOUAD A B D O U UMR UHP-CNRS 7565 - GEVSM - Facult~ de Pharmacie, Nancy, France The use of antisense oligodeoxynucleotide as tools for modulating gene expression represents a novel strategy for designing drugs to treat diseases. Although progress has been rapid, the vulnerability of oligonucleotides to nucleases' attak, combined with their intracellular distribution and uptake properties limit their therapeutic potential. Cyclodextrins, consisting of cyclic oligosaccharidyl molecules, are well known for their property to form inclusion complexes with guest molecules and thus to ensure their transport and improve their pharmacological properties. We have investigated the ability of S-Gal-beta-cyclodextrin to enhance the antiviral activity of an human anti-cytomegalovirus phosphodiester oligonucleotide (anti-u136;PO), directed against the splice donor region of immediate early genes (u136-u137). The antiviral effect of (anti-u136;PO) free or complexed, was evaluated in cell culture against human cytomegalovirus strain reference AD169 and clinical isolates, and compared with that of ganciclovir (GCV). The (anti-u136;PO) showed higher antiviral activity than GCV. Indeed, according to the viral strain, its 50 % inhibitory concentration (IC50) is 2 to 18 fold lower than that of the GCV. Moreover the IC50 of [anti-u136;PO:S-Gal-beta-CD] complex exhibits a 3 to 5 fold enhancement comparatively to that of free oligonucleotide. This effect is neither due to a cytotoxicity of the molecules, nor to an antiviral effect of the cyclodextrin. In situ studies by flow cytometry and fluorescence microscopy showed that, after complexation, the oligonucleotide uptake is improved and could be due to an improvement of the oligonucleotide endocytosis. The intracellular distribution of free or complexed oligonucleotide is vesicular, cytosolic and perinuclear.