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Isolation and analysis of an aciclovir-resistant murine cytomegalovirus mutant
T Minematsu1, S Mori, Y Eizuru
1Department of Microbiology, Miyazaki Medical College, Kihara, Kiyotake, Mizayaka 889-1692, Japan.
Abstract:
An aciclovir (ACV)-resistant murine cytomegalovirus (MCMV) was isolated from the Smith strain and the mutant was analysed. Attempts were also made to identify directly the mutated gene. The 50% inhibitory concentration (IC(50)) of ACV for the mutant strain was approximately 30 times higher than that for 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 mutation of cytosine (C) to guanine (G) occurred at the 2476th nucleotide position in the DNA polymerase gene region, resulting in an amino acid substitution of proline (Pro) with alanine (Ala) at codon 826. 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 III digestion of the selected PCR product and electrophoresis.
Insights
Researchers identified a specific mutation in murine cytomegalovirus (MCMV) conferring resistance to aciclovir (ACV). This single base change in the DNA polymerase gene provides a target for detecting ACV-resistant MCMV strains.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Murine cytomegalovirus (MCMV) is a model organism for studying human cytomegalovirus (HCMV).
- Aciclovir (ACV) resistance in herpesviruses can arise from mutations in viral DNA polymerase.
- Understanding resistance mechanisms is crucial for effective antiviral therapy.
Purpose of the Study:
- To isolate and characterize an aciclovir (ACV)-resistant murine cytomegalovirus (MCMV) strain.
- To identify the genetic basis of ACV resistance in MCMV.
- To develop a method for detecting the identified mutation.
Main Methods:
- Isolation and phenotypic characterization of ACV-resistant MCMV.
- Determination of drug sensitivity (IC50) to ACV, ganciclovir (GCV), cidofovir (CDV), and foscarnet (PFA).
- Molecular analysis including PCR, DNA sequencing, marker transfer, and restriction enzyme digestion (Hae III).
Main Results:
- An ACV-resistant MCMV mutant was isolated with an approximately 30-fold higher IC50 for ACV compared to the wild-type.
- The mutant showed similar sensitivity to GCV but slight resistance to CDV and PFA.
- A single nucleotide mutation (C to G at position 2476) in the DNA polymerase gene was identified, causing a Pro to Ala substitution at codon 826.
- This specific mutation was confirmed to confer ACV resistance and was detectable by Hae III digestion of PCR products.
Conclusions:
- A specific mutation in the MCMV DNA polymerase gene is responsible for aciclovir resistance.
- This finding provides a molecular basis for understanding and detecting ACV resistance in MCMV.
- The identified mutation offers a potential diagnostic marker for ACV-resistant MCMV infections.