, Picornaviruses have a peptide termed VPg covalently linked to the 5 (cid:1) -end of the genome. Attachment of VPg to the genome occurs in at least two steps. First, Tyr-3 of VPg, or some precur-sorthereof,isusedasaprimerbytheviralRNA-dependentRNA polymerase, 3Dpol, to produce VPg-pUpU. Second, VPg-pUpU is used as a primer to produce full-length genomic RNA. Production of VPg-pUpU is templated by a single adenylate residue located in the loop of an RNA stem-loop structure termed oriI by using a slide-back mechanism. Recruitment of 3Dpol to and its stability on oriI have been suggested to require an interaction between the back of the thumb subdomain of 3Dpol and an undefined region of the 3C domain of viral protein 3CD. We have performed surface acidic-to-alanine-scanning mutagene-sisof3Ctoidentifythesurfaceof3Cwithwhich3Dpolinteracts. This analysis identified numerous viable poliovirus mutants with reduced growth kinetics that correlated to reduced kinetics of RNA synthesis that was attributable to a change in VPg-pUpU production. Importantly, these 3C derivatives were all capable of binding to oriI as well as wild-type 3C. Synthetic lethality was observed for these mutants polio-virusmutantcontaining3Dpol-R455A,aresidueonthebackofthe VPg uridylylation. guidemoleculardockingofthestructuresforapoliovirus3Cdimer