Effects of mutations in poliovirus 3Dpol on RNA polymerase activity and on polyprotein cleavage.
A series of short insertion mutations was introduced into the poliovirus gene for 3Dpol at a number of different locations. When substituted for wild-type sequences in a full-length, infectious cDNA and tested for infectivity, all 3D mutants were nonviable. The mutant cDNAs were introduced into a bacterial plasmid designed to direct the expression of poliovirus 3CD, a viral protein composed of contiguous protease and RNA polymerase sequences. Bacteria transformed with these plasmids all expressed similar amounts of 3CD, and all mutant proteins cleaved themselves to generate wild-type 3Cpro and mutant 3Dpol polypeptides with approximately the same efficiency as wild-type 3CD. The released mutant 3Dpol proteins were all defective in RNA-dependent RNA polymerase activity in vitro. Uncleaved 3CD is a protease required for processing the viral capsid protein precursor, P1. In an in vitro assay of P1 cleavage activity, some of the mutant 3CD proteins expressed in Escherichia coli showed normal activity, while others were clearly inactive. Thus, alterations in the sequence and/or folding of different regions of the 3D protein have differential effects on its various activities.
Burns
C C
CC
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.
Lawson
M A
MA
Semler
B L
BL
Ehrenfeld
E
E
D000595:Amino Acid Sequence; D000818:Animals; D002213:Capsid / Q000235:genetics; D004251:DNA Transposable Elements; D004279:DNA, Viral / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D017955:Poliovirus / Q000201:enzymology / Q000235:genetics*; D014176:Protein Biosynthesis; D011817:Rabbits; D014158:Transcription, Genetic
D004251:DNA Transposable Elements; D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases