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Herpes simplex virus DNA polymerase as the site of phosphonoacetate sensitivity: temperature-sensitive mutants
Abstract:
Temperature-sensitive (ts) mutants in a number of complementation groups of herpes simplex virus type 1 (HSV-1) are deficient in DNA polymerase induction at the restrictive temperature. Twenty-two mutants in 15 complementation groups were tested for sensitivity to phosphonoacetate (PAA), a compound that inhibits HSV replication in vivo and the DNA polymerase in vitro. One mutant, tsD9, was resistant to PAA (Pr), whereas all others were sensitive. Revertants of tsD9 to the ts+ phenotype simultaneously lost PAA resistance. Additional Pr mutants were isolated from ts mutants belonging to several complementation groups of HSV-1. Double mutants (ts Pr phenotype) were used in three-factor recombination analyses to locate the PAA locus on the genetic map at a position indistinguishable from the ts lesion in tsD9. In all cases, resistance or sensitivity to PAA in vivo was correlated with resistance or sensitivity of DNA polymerase in vitro. These data are compatible with the temperature-sensitive lesion of tsD9 and the determinant of PAA sensitivity both residing in the structural gene for DNA polymerase.
Insights
Herpes simplex virus type 1 (HSV-1) DNA polymerase mutations affect phosphonoacetate (PAA) sensitivity. This study links temperature-sensitive lesions to PAA resistance, suggesting both are in the viral DNA polymerase gene.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Herpes simplex virus type 1 (HSV-1) temperature-sensitive (ts) mutants often show impaired DNA polymerase induction.
- Phosphonoacetate (PAA) is a known inhibitor of HSV replication and DNA polymerase activity.
Purpose of the Study:
- To investigate the relationship between temperature-sensitive mutations in HSV-1 and sensitivity to phosphonoacetate (PAA).
- To determine if PAA sensitivity is linked to specific genetic loci, potentially including the viral DNA polymerase gene.
Main Methods:
- Testing 22 HSV-1 ts mutants across 15 complementation groups for PAA sensitivity.
- Isolating and characterizing additional PAA-resistant (Pr) mutants.
- Utilizing three-factor recombination analysis with double mutants (ts Pr phenotype) to map the PAA locus.
- Correlating in vivo PAA sensitivity with in vitro DNA polymerase activity.
Main Results:
- One mutant, tsD9, exhibited PAA resistance (Pr), while others were sensitive.
- Revertants of tsD9 to ts+ also lost PAA resistance.
- Three-factor recombination mapped the PAA locus to the same position as the ts lesion in tsD9.
- In vivo PAA sensitivity directly correlated with in vitro DNA polymerase sensitivity.
Conclusions:
- The temperature-sensitive lesion in tsD9 and the determinant for PAA sensitivity are likely located within the structural gene for HSV-1 DNA polymerase.
- PAA resistance can serve as a marker for mutations within the HSV-1 DNA polymerase gene.