Structural Features of a Picornavirus Polymerase Involved in the Polyadenylation of Viral RNA

Picornaviruses have 3 = polyadenylated RNA genomes, but the mechanisms by which these genomes are polyadenylated during viral replication remain obscure. Based on prior studies, we proposed a model wherein the poliovirus RNA-dependent RNA polymerase (3Dpol) uses a reiterative transcriptionmechanism while replicating the poly(A) and poly(U) portions of viral RNA templates. To further test this model, we examined whether mutations in 3Dpol influenced the polyadenylation of virion RNA.We identified nine alanine substitutionmutations in 3Dpol that resulted in shorter or longer 3 = poly(A) tails in virion RNA. These mutations could disrupt structural features of 3Dpol required for the recruitment of a cellular poly(A) polymerase; how-ever, the structural orientation of these residues suggests a direct role of 3Dpol in the polyadenylation of RNA genomes. Reaction mixtures containing purified 3Dpol and a template RNAwith a defined poly(U) sequence provided data consistent with a tem-plate-dependent reiterative transcriptionmechanism for polyadenylation. The phylogenetically conserved structural features of 3Dpol involved in the polyadenylation of virion RNA include a thumb domain alpha helix that is positioned in the minor groove of the double-stranded RNA product and lysine and arginine residues that interact with the phosphates of both the RNA tem-plate and product strands. Picornaviruses, like many other positive-strand RNA viruses,have RNA genomes with variable-length 3 = poly(A) tails (1). The poly(A) tails of picornaviruses are important for viability (2), with the length of the poly(A) tails influencing the magnitudes o