Using proteinase trapping to detect revertants of inactive rhinoviral 2A proteinase mutants.
The 2A proteinase of human rhinovirus 2 cleaves itself off the growing polyprotein at its own N terminus during translation; this property was used to develop an in vivo screening system with the lacZ gene fragment of M13mp18. The fusion of an active 2A proteinase to the C-terminus of the alpha-fragment did not affect alpha-complementation, as the proteinase cleaved itself off the alpha-fragment. However, an inactive 2A proteinase remained fused to the alpha-fragment hindering alpha-complementation. Random mutations were then introduced into the 2A gene site by PCR amplification. Mutants defective in alpha-complementation (thus containing an inactive 2A proteinase) were obtained at an efficiency of 5%, mutants showing reduced 2A activity at an efficiency of 1%. Mutants showing reduced or no 2A activity were then subjected to PCR mutagenesis. Three mutants reactivating an inactive 2A proteinase were examined and the compensatory changes determined.
Luderer
M
M
Institute of Biochemistry, University of Vienna, Austria.
Liebig
H D
HD
Sommergruber
W
W
Blaas
D
D
Kuechler
E
E
Skern
T
T
D001435:Bacteriophages / Q000201:enzymology; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000235:genetics*; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005784:Gene Amplification; D005816:Genetic Complementation Test; D016296:Mutagenesis; D009154:Mutation; D016133:Polymerase Chain Reaction; D012229:Rhinovirus / Q000201:enzymology / Q000235:genetics*; D014760:Viral Fusion Proteins / Q000378:metabolism; D014764:Viral Proteins
D004269:DNA, Bacterial; D014760:Viral Fusion Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus