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 cl)NA. 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 å Arg substitution at position 60 resulted in cold sensitivity for growth at 33°. Replacement of Lys 60 with lie, either singly or in combination with substitutions at position 61, resulted in viruses that produced three- to fivefold more 31) 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 Gin-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. © 1990.