Genetic analysis of poliovirus protein 3A: characterization of a non-cytopathic mutant virus defective in killing Vero cells.
A mutational and genetic analysis of the poliovirus protein 3A has led to the identification of a single amino acid mutant virus with a restrictive phenotype to form plaques in Vero cells. This mutant (I46T 3A) can be grown and amplified in HeLa cells, where virus replication takes place at wild-type levels. However, Vero cells infected with this virus cannot complete the growth cycle. I46T 3A virus has a defect in the ability to kill Vero cells, as estimated by FACS analysis of propidium iodide uptake by dead cells. Since these defects are observed under conditions where no abnormalities in the rate of synthesis and processing of the mutant polyprotein occur, the inability to induce the cytopathic effect in infected Vero cells denotes the existence of a defect in the activity of 3A, but not the level of expression of the viral genome. As a consequence of this impaired capability to generate the cytopathic effect, I46T 3A mutant viruses cannot be titrated by plaque assay in Vero cells. Only revertant viruses with the wild-type sequence arise and form lysis plaques in Vero cells. Our results suggest a role for the 3A protein (or a precursor thereof) in the virus-induced cytopathic effect. The mutant virus characterized in this work may be a useful tool to understand how poliovirus kills infected cells and carries out the final step of its life-cycle, the release of virus progeny.
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Canto Blanco, Spain. juan@liai.org
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D019943:Amino Acid Substitution; D000818:Animals; D016923:Cell Death; D002457:Cell Extracts; D002522:Chlorocebus aethiops; D003588:Cytopathogenic Effect, Viral; D003673:Defective Viruses / Q000235:genetics* / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D007532:Isoleucine / Q000235:genetics / Q000502:physiology; D010641:Phenotype; D017354:Point Mutation; D017955:Poliovirus / Q000235:genetics* / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism; D013912:Threonine / Q000235:genetics / Q000502:physiology; D014709:Vero Cells; D014758:Viral Core Proteins / Q000235:genetics* / Q000502:physiology; D014764:Viral Proteins / Q000096:biosynthesis / Q000378:metabolism
C109349:3A protein, Poliovirus; D002457:Cell Extracts; D011506:Proteins; D014758:Viral Core Proteins; D014764:Viral Proteins; D007532:Isoleucine; D013912:Threonine