GENETIC DISSECTION OF INTERACTION BETWEEN POLIOVIRUS 3D POLYMERASE AND VIRAL PROTEIN 3AB

Poliovirus RNA dependent RNA polymerase 3D and viral protein 3AB are b oth thought to be required for the initiation of RNA synthesis. These two proteins physically associate with each other and with viral RNA r eplication complexes found on virus-induced membranes in infected cell s. An understanding of the interface between 3D and 3AB would provide a first step in visualizing the architecture of the multiprotein compl ex that is assembled during poliovirus infection to replicate and pack age the viral RNA genome. The identification of mutations in 3D that d iminish 3D-3AB interactions without affecting other functions of 3D po lymerase is needed to study the function of the 3D-3AB interaction in infected cells. We describe the use of the yeast two-hybrid system to isolate and characterize mutations in 3D polymerase that cause it to i nteract less efficiently with 3AB than wild-type polymerase. One mutat ion, a substitution of leucine for valine at position 391 (V391L), res ulted in a 3AB-specific interaction defect in the two-hybrid system, c ausing a reduction in the interaction of 3D polymerase with 3AB but no t with another viral protein br a host protein tested. In vitro, purif ied 3D-V391L polymerase bound to membrane-associated 3AB with reduced affinity. Poliovirus that contained the 3D-V391L mutation was temperat ure sensitive, displaying a Pronounced conditional defect in RNA synth esis. We conclude that interaction between 3AB and 3D or 3D-containing polypeptides plays a role in RNA synthesis during poliovirus infectio n.