A genetic locus in mutant poliovirus genomes involved in overproduction of RNA polymerase and 3C proteinase.
A mutagenic oligonucleotide cassette was used to introduce single and tandem amino acid substitutions into the proteinase 3C coding region of an infectious poliovirus type 1 cDNA. The sites targeted for mutagenesis, residues 60, 61, and 66, are located within a putative helical loop structure which may be involved in substrate recognition by the enzyme. Fourteen viable 3C proteinase mutants were isolated. A Lys----Arg substitution at position 60 resulted in cold sensitivity for growth at 33 degrees. Replacement of Lys 60 with Ile, either singly or in combination with substitutions at position 61, resulted in viruses that produced three- to fivefold more 3D RNA polymerase than wild-type poliovirus. 3C-mediated processing of the remaining sites within the polyprotein was not noticeably affected. The overproduction of 3D is a consequence of more efficient processing of the carboxy-terminal Gln-Gly amino acid pair of 3C. Together with a previous report in which substitution of Val 54 with an Ala residue results in a poliovirus that produces decreased levels of 3D, these observations provide evidence that the putative loop region (residues 51-66) may be a functional domain involved in recognition of the carboxy-terminal Gln-Gly cleavage site of 3C.
Dewalt
P G
PG
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717.
Blair
W S
WS
Semler
B L
BL
D000086042:3C Viral Proteases; D002874:Chromosome Mapping; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000235:genetics*; D004247:DNA / Q000032:analysis; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics*; D005814:Genes, Viral; D006367:HeLa Cells; D009154:Mutation; D017955:Poliovirus / Q000201:enzymology / Q000235:genetics*; D012367:RNA, Viral / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D014764:Viral Proteins
D012367:RNA, Viral; D014764:Viral Proteins; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases