AN ASPARTIC-ACID AT AMINO-ACID-108 IS REQUIRED TO RESCUE INFECTIOUS VIRUS AFTER TRANSFECTION OF A POLIOVIRUS CDNA CONTAINING A CGDD BUT NOTSGDD AMINO-ACID MOTIF IN 3D(POL)

The poliovirus RNA-dependent RNA polymerase (3D(pol)) contains a regio n of homology centered around the amino acid motif YGDD (amino acids 3 26 to 329), which has been postulated to be,involved in the catalytic activity of the enzyme, Previous studies from this laboratory have use d oligonucleotide site directed mutagenesis to substitute the tyrosine amino acid at this motif with other amino acids (S. A. Jablonski and C, D, Morrow, J, Virol, 67:373-381, 1993), The viruses recovered with 3D(pol) genes with a methionine mutation also contained a second mutat ion at amino acid 108 resulting in a glutamic acid-to-aspartic acid ch ange (3D-E-108 to 3D-D-108) in the poliovirus RNA polymerase, On the b asis of these results, we suggested that the amino acid at position 10 8 might interact with the YGDD region of the poliovirus polymerase, To further investigate this possibility, we have constructed a series of constructs in which the poliovirus RNA polymerases contained a mutati on at amino acid 108 (3D-E-108 to 3D-D-108) as well as a mutation in w hich the tyrosine amino acid (3D-Y-326) was substituted with cysteine (3D-C-326) or serine (3D-S-326), The mutant 3D(pol) polymerases were e xpressed in Escherichia coli, and in vitro enzyme activity was analyze d, Enzymes containing the 3D-D-108 mutation with the wild-type amino a cid (3D-Y-326) demonstrated in vitro enzyme activity similar to that o f the wild-type enzyme containing 3D-E-108, In contrast, enzymes with the 3D-C-326 or 3D-S-326 mutation had less in vitro activity than the wild type, The inclusion of the second mutation at amino acid 3D-D-108 did not significantly affect the in vitro activity of the polymerases containing 3D-C-326 or 3D-S-326 mutation, Transfections of poliovirus cDNAs containing the substitution at amino acid 326 with or without t he second mutation at amino acid 108 were performed, Consistent with p revious findings, we found that transfection of poliovirus cDNAs conta ining the 3D-C-326 or 3D-S-326 mutation in 3D(pol) did not result in t he production of virus. Surprisingly, transfection of the poliovirus c DNAs containing the 3D-D-108/C-326 double mutation, but not the 3D-D-1 08/S-326 mutation, resulted in the production of virus, The virus obta ined from transfection of poliovirus cDNAs containing 3D-D-108/C-326 m utation replicated with kinetics similar to that of the wild-type viru s. RNA sequence analysis of the region of the 3D(pol) containing the 3 D-C-326 mutation revealed that the codon fbr cysteine (UGC) reverted t o the codon for tyrosine (UAC). The results of these studies establish that under the appropriate conditions, poliovirus has the capacity to revert mutations within the YGDD amino acid motif of the poliovirus 3 D(pol) gene and further strengthen the idea that interaction between a mino acid 108 and the YGDD region of 3D(pol) is required for viral rep lication.