COMPLETE PROTEIN LINKAGE MAP OF POLIOVIRUS P3 PROTEINS - INTERACTION OF POLYMERASE 3D(POL) WITH VPG AND WITH GENETIC-VARIANTS OF 3AB

Poliovirus has evolved to maximize its genomic information by producin g multifunctional viral proteins. The P3 nonstructural proteins harbor various activities when paired with different binding partners. These viral polypeptides regulate host cell macromolecular synthesis and fu nction as proteinases, as RNA binding proteins, or as RNA-dependent RN A polymerase, A cleavage product of the P3 region is the genome-linked protein VPg that is essential in the initiation of RNA synthesis. We have used an inducible yeast two-hybrid system to analyze directly pro tein-protein interactions among P3 proteins. Sixteen signals of homo- or heterodimer interactions have been observed and have been divided i nto three groups. Of interest is the newly discovered affinity of VPg to 3D(pol) that suggests direct interaction between these molecules in genome replication. A battery of 3AB variants (eight clustered-charge -to-alanine changes and five single-amino-acid mutations) has been use d to map the binding determinants of 3AB-3AB interaction which were fo und to differ from the amino acids critical for the 3AB-3D(pol) intera ction. The viral proteinase 3C(pro) was not found to interact with oth er 3C(pro) molecules or with any other P3 polypeptide in yeast cells, a result confirmed by glutaraldehyde crosslinking. The weak apparent i nteraction between 3AB and 3CD(pro) scored in the yeast two-hybrid sys tem was in contrast to a strong signal by far-Western blotting. The re sults elucidate, in part, previous results of biochemical and genetic analyses. The role of the interactions in RNA replication is addressed .