Determination of the poliovirus RNA polymerase error frequency at eight sites in the viral genome
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1Department of Immunology and Medical Microbiology, College of Medicine, University of Florida, Gainesville 32610-0266.
Abstract:
The poliovirus RNA polymerase error frequency was measured in vivo at eight sites in the poliovirus genome. The frequency at which specific G residues in poliovirion RNA changed to another base during one round of viral RNA replication was determined. Poliovirion RNA uniformly labeled with 32Pi was hybridized to a synthetic DNA oligonucleotide that was complementary to a sequence in the viral genome that contained a single internal G residue. The nonhybridized viral RNA was digested with RNase T1, and the protected RNA oligonucleotide was purified by gel electrophoresis. The base substitution frequency at the internal G residue was measured by finding the fraction of this RNA oligonucleotide that was resistant to RNase T1 digestion. A mean value of 2.0 x 10(-3) +/- 1.2 x 10(-3) was obtained at two sites. A modification of the above procedure involved the use of 5'-end-labeled RNA oligonucleotides. The mean value of the error frequency determined at eight sites in the viral genome by using this technique was 4.1 x 10(-3) +/- 0.6 x 10(-3). Sequencing two of the RNase T1-resistant RNA oligonucleotides confirmed that the internal G was changed to a C, A, or U residue in most of these oligonucleotides. Thus, our results indicated that the polymerase had a high error frequency in vivo and that there was no significant variation in the values determined at the specific sites examined in this study.
Insights
Poliovirus RNA polymerase exhibits a high error frequency in vivo, with mutations occurring at specific genomic sites during replication. This study quantifies these errors, revealing significant base substitutions.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Poliovirus RNA polymerase is crucial for viral replication.
- Understanding polymerase error rates is vital for studying viral evolution and pathogenesis.
- Previous studies have suggested polymerase infidelity, but in vivo error frequencies were not well-characterized.
Purpose of the Study:
- To quantify the in vivo error frequency of poliovirus RNA polymerase.
- To determine the frequency of specific base substitutions, particularly G to other bases, during viral RNA replication.
- To assess variations in error frequency across different sites in the poliovirus genome.
Main Methods:
- Uniformly 32P-labeled poliovirus RNA was hybridized with complementary DNA oligonucleotides containing a single internal guanine (G) residue.
- RNase T1 digestion was used to identify and quantify non-hybridized RNA fragments, indicating base substitutions.
- Modified procedures using 5'-end-labeled RNA oligonucleotides were employed for broader genomic site analysis.
- Sequencing of resistant RNA oligonucleotides confirmed the nature of base substitutions.
Main Results:
- A mean error frequency of 2.0 x 10^-3 was observed at two specific genomic sites.
- Analysis at eight sites revealed a mean error frequency of 4.1 x 10^-3.
- Sequencing confirmed that the internal G residue was frequently substituted with cytosine (C), adenine (A), or uracil (U).
Conclusions:
- Poliovirus RNA polymerase demonstrates a high error frequency in vivo.
- The observed error rates were consistent across the examined genomic sites, suggesting a uniform polymerase infidelity.
- These findings contribute to understanding poliovirus mutation dynamics and potential evolutionary pathways.