The poliovirus polypeptide 3AB, the precursor of the genome-bound VPg protein, stimulates in vitro the synthesis of poly(U) directed by the viral polymerase 3Dpol (Lama, J., Paul, A., Harris, K., and Wimmer, E. (1994) J. Biol. Chem. 269, 66-70), suggesting that 3AB could be modulating the activity of the viral polymerase in poliovirus-infected cells. To address the exact function of 3AB in the viral replication cycle, a biochemical and molecular genetic analysis of 3AB has been carried out. 3AB protein bound RNA probes in two different assays, and amino acid positions implicated in the RNA binding activity of 3AB were determined. Mutant proteins with reduced RNA binding activity were unable to stimulate 3Dpol polymerase activity. Purified protein 3A showed no RNA binding or 3Dpol stimulatory activity, but 3A and VPg mutations conferred a synergistic effect on the 3AB functions. Polioviruses encoding for these mutant 3ABs were constructed. These mutant viruses translated their RNA genomes in vitro and processed their polyproteins as wild type virus did. Cells infected with 3AB mutant viruses showed over 90% inhibition in the accumulation of plus and minus viral RNA strands and more than 100-fold reduction of virus yield at 4 h postinfection. Our results suggest that 3AB protein functions in vivo as a co-factor of the viral polymerase and that the activity of 3AB may be regulated by proteolytic processing.