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.
Xiang
W
W
Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook 11794-5222, USA.
Cuconati
A
A
Paul
A V
AV
Cao
X
X
Wimmer
E
E
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