A "Slide-back" Mechanism for the Initiation of Protein-primed RNA Synthesis by the RNA Polymerase of Poliovirus.

Poliovirus RNA replication is initiated when a molecule of UMP is covalently linked to the hydroxyl group of a tyrosine in the terminal protein VPg. This reaction can be reproduced in vitro with an assay that utilizes two purified viral proteins, RNA polymerase 3D[sup pol] and viral protein 3CD[sup pro], synthetic VPg, UTP, and Mg[sup 2+]. The template for the reaction is either poliovirus RNA or transcripts of a small RNA hairpin, termed cre(2C), located in the coding sequence of protein 2C[sup ATPase]. The products of the reaction are VPgpU and VPgpUpU, the primers used by 3D[sup pol] for RNA synthesis. With mutant template RNAs in this assay we determined the precise initiation site. Our results indicate that 1) 3D[sup pol] does not possess strict specificity toward the nucleotide it links to VPg, 2) A-5 of the conserved ¹GXXXAAAXXXXXXA[sup 14] sequence in the loop is the template nucleotide for the linkage of both the first and second UMPs to VPg, 3) VPgpUpU is synthesized by a "slideback" mechanism, and 4) A-6 provides specificity to the reaction during the slide-back step and also modulates the uridylylation reaction. In additional experiments we determined the effect of mutations in the [sup 5]AAA[sup 7] sequence of cre(2C) on viral growth, RNA replication, and on the activity of the 2C[sup ATPase] protein. Furthermore, we observed that the spacing between G-1 and A-5 and the size of the loop affect the yield but not the nature of the VPglinked products. [ABSTRACT FROM AUTHOR]