ONE OF 2 NTP BINDING-SITES IN POLIOVIRUS RNA-POLYMERASE REQUIRED FOR RNA REPLICATION

The poliovirus RNA-dependent RNA polymerase (3D(pol)) has been shown t o contain two NTP binding: sites by chemical cross-linking of oxidized nucleotide to the intact protein. Only one site (Lys-61) was shown to be essential for RNA chain elongation activity by purified enzyme; ho wever, a full-length viral RNA, coding for an altered lysine residue ( K276L) in the second site, generated virus with a minute plaque phenot ype that rapidly reverted to a wild-type phenotype with Arg-276 replac ing Leu-276 in 3D. Viruses with lysine to leucine substitutions in oth er positions of the second binding site of their polymerase proteins g rew with wild-type phenotype, To test the significance of the second b inding site, poliovirus 3D(pol) was generated with lysine (wild type), leucine, or arginine at residue 276 and tested for NTP cross-linking using P-32-oxidized GTP. Analysis of cyanogen bromide peptides of each 3D preparation showed that the second NTP binding site had severely r educed NTP binding in mu 276(Leu) but not in the revertant mu 276(Arg) , despite the reported requirement for lysine in the cross-linking rea ction. To eliminate the possibility that P-32-oxidized GTP cross-linke d to Arg at residue 276, a model system was designed with unmodified a mino acid or acetylated (alpha-amino) amino acid and P-32-oxidized GTP . Cross-linking to lysine, but not leucine or arginine, was observed t hus eliminating the possibility that NTP could be cross-linked to resi due 276 in 3D, We conclude that NTP binding at the second site in poli ovirus 3D is at lysine residues at positions other than 276 (278 or 28 3), and nucleotide binding at these sites has no bearing on elongation activity or replication of the virus, Nucleotide binding only at the site including Lys-61 is essential for RNA replication.