Molecular dissection of the multifunctional poliovirus RNA-binding protein 3AB. |
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| Authors: | W Xiang A Cuconati A V Paul X Cao and E Wimmer |
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| Affiliation: | Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook 11794-5222, USA. |
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| Abstract: | Genome replication of poliovirus, as yet unsolved, involves numerous viral polypeptides that arise from proteolysis of the viral polyprotein. One of these proteins is 3AB, an RNA-binding protein with multiple functions, that serves also as the precursor for the genome-linked protein VPg (= 3B). Eight clustered charged amino acid-to-alanine mutants in the 3AB coding region of poliovirus were constructed and analyzed, together with three additional single-amino acid exchange mutants in VPg, for viral phenotypes. All mutants expressed severe inhibition in RNA synthesis, but none were temperature sensitive (ts). The 3AB polypeptides of mutants with a lethal phenotype were overexpressed in Escherichia coli, purified to near homogeneity, and studied with respect to four functions: (1) ribonucleoprotein complex formation with 3CDpro and the 5'-terminal cloverleaf of the poliovirus genome; (2) binding to the genomic and negative-sense RNA; (3) stimulation of 3CDpro cleavage; and (4) stimulation of RNA polymerase activity of 3Dpol. The results have allowed mapping of domains important for RNA binding and the formation of certain protein-protein complexes, and correlation of these processes with essential steps in viral genome replication. |
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