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Sequence and transcriptional analysis of the guinea-pig cytomegalovirus DNA polymerase gene
1Division of Infectious Diseases, Children's Hospital Research Foundation, Cincinnati, Ohio 45229, USA. MarkS60365@aol.com
Abstract:
Although the guinea-pig cytomegalovirus (GPCMV) displays a similar pathogenesis to human cytomegalovirus (HCMV), there have unfortunately been few molecular analyses of the GPCMV genome to date. The guinea-pig has proved useful for the testing of drugs active against CMV infection, and insights derived from characterization of the specific virally encoded molecular targets of antiviral therapies would allow this model system to be more fully developed. Because the DNA polymerase serves as an important target for nucleoside antiviral agents active against herpesviruses, experiments were undertaken to identify, clone and sequence the GPCMV DNA polymerase gene (pol). A 3285 bp ORF capable of encoding a 1094 amino acid protein was identified spanning portions of the HindIII Q and P fragments of the genome. This ORF contained extensive homology to other herpesvirus DNA pol genes. Northern blot analyses identified two 3' coterminal pol-specific mRNAs of 3.9 and 1.9 kb at early times post-infection. Primer extension and nuclease protection analyses mapped the 5' end of the 3.9 kb transcript to a site 275 bases upstream of the pol initiation codon. Comparison of the GPCMV pol-encoded sequence to those of other herpesvirus polymerases identified non-conservative amino acid substitutions in a domain involved in substrate recognition.
Insights
Researchers identified and sequenced the guinea-pig cytomegalovirus (GPCMV) DNA polymerase gene (pol). This characterization provides crucial molecular targets for developing antiviral therapies against CMV infections in this important animal model.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Human cytomegalovirus (HCMV) and guinea-pig cytomegalovirus (GPCMV) share similar pathogenesis.
- GPCMV is a valuable model for testing antiviral drugs, but its genome requires further molecular characterization.
- Viral DNA polymerase is a key target for nucleoside antiviral agents against herpesviruses.
Purpose of the Study:
- To identify, clone, and sequence the GPCMV DNA polymerase (pol) gene.
- To characterize the molecular targets for antiviral therapies in the GPCMV model.
Main Methods:
- Identification and sequencing of a 3285 bp open reading frame (ORF) encoding the GPCMV DNA polymerase.
- Northern blot analysis to detect pol-specific messenger RNA (mRNA) transcripts.
- Primer extension and nuclease protection assays to map transcript start sites.
Main Results:
- A 3285 bp ORF encoding a 1094 amino acid protein, homologous to other herpesvirus DNA polymerases, was identified.
- Two GPCMV pol-specific mRNAs (3.9 kb and 1.9 kb) were detected at early infection stages.
- Non-conservative amino acid substitutions were found in the GPCMV polymerase's substrate recognition domain compared to other herpesviruses.
Conclusions:
- The GPCMV DNA polymerase gene has been successfully identified and sequenced.
- This characterization provides essential molecular information for developing targeted antiviral strategies in the GPCMV model.
- Identified sequence variations may influence drug efficacy and warrant further investigation.