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Multiple mutations involved in the phenotype of a temperature-sensitive small-plaque mutant of poliovirus
Abstract:
A temperature-sensitive small-plaque mutant of poliovirus type 1, ts247, has been analyzed previously. Several mutations were detected in the P3 region of the genome by analysis of proteins and by T1 oligonucleotide mapping of viral RNA. We have now studied spontaneous reversion of ts247 to the wild-type phenotype. This was found to be a two-step event, reversion to a ts+ phenotype (revertant R247-51) being distinct from acquisition of normal plaque size (revertant R247-12). The mutation responsible for the ts phenotype of ts247, implicated also in virus aggregation and heat lability, could not be detected by biochemical studies. Analysis of homotypic recombinants obtained by crossing ts247 with a guanidine-resistant derivative of a temperature-sensitive replicase mutant mapped this mutation to the P1 region or to the 5' end of the P2 region of the genome. The small-plaque phenotype of ts247 and R247-51 was correlated with an abnormality in polypeptide 3C (protease); direct sequencing of viral RNA revealed a U to C change at nucleotide 5658, which altered an isoleucine to threonine in the protease of ts247 and R247-51 but not of R247-12. Two other mutations were present in the region of the genome coding for polypeptide 3D of ts247 and of both classes of revertants. They thus seemed to play no role in the phenotype of ts247. One mutation, an A to G change at nucleotide 7135, was silent at the protein level, whereas the other, an A to G change at nucleotide 6264, determined a major amino acid change from glutamate to glycine in the viral replicase.
Insights
Spontaneous reversion of poliovirus ts247 occurs in two steps, affecting temperature sensitivity and plaque size independently. A mutation in the 3C protease gene causes the temperature-sensitive phenotype, while other mutations in the replicase gene do not impact ts247
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Poliovirus type 1 temperature-sensitive mutant ts247 exhibits a small-plaque phenotype.
- Previous studies identified mutations in the P3 region of the ts247 genome.
- Spontaneous reversion of ts247 to wild-type was investigated to understand its genetic basis.
Purpose of the Study:
- To elucidate the genetic mechanisms underlying the temperature-sensitive (ts) and small-plaque phenotypes of poliovirus ts247.
- To identify the specific mutations responsible for ts247's altered characteristics.
- To differentiate the genetic events leading to temperature-resilience and normal plaque morphology.
Main Methods:
- Analysis of spontaneous revertants of ts247.
- Homotypic recombination between ts247 and a guanidine-resistant mutant.
- Direct RNA sequencing to identify nucleotide changes.
- Correlation of genetic mutations with protein alterations and phenotypic characteristics.
Main Results:
- Reversion of ts247 to wild-type is a two-step process, yielding distinct temperature-resilient (R247-51) and normal-plaque (R247-12) revertants.
- A U-to-C nucleotide change at position 5658 in the 3C protease gene is responsible for the ts phenotype and is present in ts247 and R247-51.
- Two mutations in the 3D replicase gene region were found in ts247 and both revertants, suggesting they are not responsible for the ts phenotype.
Conclusions:
- The temperature-sensitive phenotype of poliovirus ts247 is primarily determined by a mutation in the 3C protease gene.
- The small-plaque phenotype is linked to the same 3C protease mutation.
- Mutations in the viral replicase gene do not contribute to the observed temperature-sensitive or small-plaque phenotypes of ts247.