MOLECULAR DISSECTION OF THE MULTIFUNCTIONAL POLIOVIRUS RNA-BINDING PROTEIN 3AB

Genome replication of poliovirus, as yet unsolved, involves numerous v iral polypeptides that arise from proteolysis of the viral polyprotein . One of these proteins is 3AB, an RNA-binding protein with multiple f unctions, that serves also as the precursor for the genome-linked prot ein VPg (=3B). Eight clustered charged amino acid-to-alanine mutants i n the 3A8 coding region of poliovirus were constructed and analyzed, t ogether with three additional single-amino acid exchange mutants in VP g, for viral phenotypes. All mutants expressed severe inhibition in RN A synthesis, but none were temperature sensitive (ts). The 3AB polypep tides of mutants with a lethal phenotype were overexpressed in Escheri chia coli, purified to near homogeneity, and studied with respect to f our functions: (1) ribonucleoprotein complex formation with 3CD(pro) a nd the 5'-terminal cloverleaf of the poliovirus genome; (2) binding to the genomic and negative-sense RNA; (3) stimulation of 3CD(pro) cleav age; and (4) stimulation of RNA polymerase activity of 3D(pol). The re sults have allowed mapping of domains important for RNA binding and th e formation of certain protein-protein complexes, and correlation of t hese processes with essential steps in viral genome replication.