Molecular dissection of the multifunctional poliovirus RNA-binding protein 3AB.
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.
Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook 11794-5222, USA.
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Xiang
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Cuconati
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Paul
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Cao
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Wimmer
D000086042:3C Viral Proteases; D001483:Base Sequence; D003546:Cysteine Endopeptidases / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D016601:RNA-Binding Proteins / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D014158:Transcription, Genetic; D014758:Viral Core Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014764:Viral Proteins; D014779:Virus Replication / Q000502:physiology
D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D014758:Viral Core Proteins; D014764:Viral Proteins; C090443:polymerase 3Dpol, poliovirus; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases