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Cytomegalovirus stimulates the expression of cellular enzymes involved in nucleotides biosynthesis
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Abstracts
Molecular Targets for Antiviral D e v e l o p m e n t - Posters G1-03 Cytomegalovirus stimulates the expression of cellular enzymes involved in nucleotides biosynthesis DAVID LEMBO Dept. of Public Health and Microbiology, Medical School, Turin, Italy To ensure a sufficient supply of dNTPs for the viral DNA polymerase in non dividing cells, many members of the Herpesviridae family encode nucleotides biosynthetic enzymes such as thymidine kinase (TK), dihydrofolate reductase (DHFR), thymidilate synthetase (TS) and an active form of ribonucleotide reductase (RR). Unlike others herpesviruses, cytomegalovirus does not encode these enzymes and thus must largely rely upon host cell metabolism to supply precursors and enzyme activities for viral DNA replication. It appears that productive replication of CMV in terminally differentiated cells depends on its ability to stimulate cellular DNA synthetic machinery. According to this view, here we report that infection of quiescent fibroblast with human (HCMV)or murine (MCMV) cytomegalovirus stimulates the expression of cellular DHFR, TS, and the small subunit of RR. Suppression of the virus-induced DHFR and TS activity by the specific inhibitors methotrexate and D1694 prevents the replication of both HCMV and MCMV, indicating that these enzymes are cellular targets of virus infection. The TS inhibitor D 1694 displayed a strong anticytomegaloviral activity (ED50 0.0046uM; ED90 0.0081 uM) at drug concentrations well below those producing cytotoxic effect on quiescent cells. Moreover, addition of exogenous thymidine (10 uM)inhibits the antiviral activity of D1694 indicating that TS is the sole cellular target. Finally, D1694 was also able to suppress DNA synthesis of two clinical isolates of HCMV resistant to ganciclovir, due to mutation in the UL 97 gene, to the same extent observed with HCMV AD 169.
G1-04 Genetic Analysis of an Aciclovir-Resistant Murine Cytomegalovirus Mutant TOSHIO MINEMATSU Miyazaki Medical College, Kiyotake, Japan We isolated an acidovir (ACV)-resistant murine cytomegalovirus (MCMV) from the Smith strain, and analysed the mutant. Attempts were also made to identify directly the mutated gene. The 50% inhibitory concentration (IC50) of ACV for the mutant strain was about thirty times more than that of the wild-type strain. The mutant strain was equally sensitive to ganciclovir (GCV) but slightly resistant to cidofovir (CDV) and foscarnet (PFA) when compared with the wild-type. Molecular analysis of the mutant strain revealed that a single base substitution of cytosine' (C) with guanine (G) occurred at 2476 nucleotide position in the DNA polymerase gene region, resulting in an amino acid substitution of proline (Pro) with alanine (Ala) at codon 826 in the DNA polymerase gene product. The marker transfer experiment confirmed that this mutation conferred ACV resistance to MCMV. This mutation at codon 826 was easily identified by means of Hae II1 digestion of the selected PCR product.
Abstracts
Molecular Targets for Antiviral D e v e l o p m e n t - Posters G1-03 Cytomegalovirus stimulates the expression of cellular enzymes involved in nucleotides biosynthesis DAVID LEMBO Dept. of Public Health and Microbiology, Medical School, Turin, Italy To ensure a sufficient supply of dNTPs for the viral DNA polymerase in non dividing cells, many members of the Herpesviridae family encode nucleotides biosynthetic enzymes such as thymidine kinase (TK), dihydrofolate reductase (DHFR), thymidilate synthetase (TS) and an active form of ribonucleotide reductase (RR). Unlike others herpesviruses, cytomegalovirus does not encode these enzymes and thus must largely rely upon host cell metabolism to supply precursors and enzyme activities for viral DNA replication. It appears that productive replication of CMV in terminally differentiated cells depends on its ability to stimulate cellular DNA synthetic machinery. According to this view, here we report that infection of quiescent fibroblast with human (HCMV)or murine (MCMV) cytomegalovirus stimulates the expression of cellular DHFR, TS, and the small subunit of RR. Suppression of the virus-induced DHFR and TS activity by the specific inhibitors methotrexate and D1694 prevents the replication of both HCMV and MCMV, indicating that these enzymes are cellular targets of virus infection. The TS inhibitor D 1694 displayed a strong anticytomegaloviral activity (ED50 0.0046uM; ED90 0.0081 uM) at drug concentrations well below those producing cytotoxic effect on quiescent cells. Moreover, addition of exogenous thymidine (10 uM)inhibits the antiviral activity of D1694 indicating that TS is the sole cellular target. Finally, D1694 was also able to suppress DNA synthesis of two clinical isolates of HCMV resistant to ganciclovir, due to mutation in the UL 97 gene, to the same extent observed with HCMV AD 169.
G1-04 Genetic Analysis of an Aciclovir-Resistant Murine Cytomegalovirus Mutant TOSHIO MINEMATSU Miyazaki Medical College, Kiyotake, Japan We isolated an acidovir (ACV)-resistant murine cytomegalovirus (MCMV) from the Smith strain, and analysed the mutant. Attempts were also made to identify directly the mutated gene. The 50% inhibitory concentration (IC50) of ACV for the mutant strain was about thirty times more than that of the wild-type strain. The mutant strain was equally sensitive to ganciclovir (GCV) but slightly resistant to cidofovir (CDV) and foscarnet (PFA) when compared with the wild-type. Molecular analysis of the mutant strain revealed that a single base substitution of cytosine' (C) with guanine (G) occurred at 2476 nucleotide position in the DNA polymerase gene region, resulting in an amino acid substitution of proline (Pro) with alanine (Ala) at codon 826 in the DNA polymerase gene product. The marker transfer experiment confirmed that this mutation conferred ACV resistance to MCMV. This mutation at codon 826 was easily identified by means of Hae II1 digestion of the selected PCR product.