Analysis of the functional significance of amino acid residues in the putative NTP-binding pattern of the poliovirus 2C protein.
The amino acid sequence of the poliovirus 2C protein contains two highly conserved stretches, GSPGTGKS136 and MDD177, which correspond to the consensus 'A' and 'B' motifs (GXXXXGKS/T and DD/E, respectively) found in nucleoside triphosphate-binding proteins. To assess the functional importance of these amino acid sequences, we changed conserved and non-conserved amino acids. The replacement of the non-conserved Thr133 residue with Ser or Ala did not markedly change the virus phenotype. Similarly, replacement of the non-conserved Pro131 residue by Ala did not abolish virus viability, but changes of this residue to Thr or Asn were not tolerated. No viable mutant could be isolated after transfection of cultured cells with transcripts mutated at the conserved Lys135, Ser136 or Asp177 residues. However, true revertants were selected from Arg135 and Ser135 mutants, from Glu177 and Gly177 mutants, and from Ala136 mutants. Thr136 mutants not only gave rise to true revertants, but also to two independent isolates of a suppressor mutant, Asn140----Tyr. All the lethal mutations resulted in severe inhibition of viral RNA synthesis in vivo, although no translational deficiency was detected in a cell-free system. This is the first direct evidence for the functional significance of the nucleoside triphosphate-binding pattern in the poliovirus 2C protein.
Teterina
N L
NL
Unité de Virologie Moléculaire (CNRS UA 545), Institut Pasteur, Paris, France.
Kean
K M
KM
Gorbalenya
A E
AE
Agol
V I
VI
Girard
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D017955:Poliovirus / Q000235:genetics* / Q000502:physiology; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D017361:Viral Nonstructural Proteins; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication
D002352:Carrier Proteins; D009838:Oligodeoxyribonucleotides; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C076023:2C protein, viral