title: Analysis of picornavirus 2A(pro) proteins: separation of proteinase from translation and replication functions.
abstract: The poliovirus (PV) genome was manipulated by replacing its 2A-encoding sequence with the corresponding sequence of coxsackie B4 virus (CBV4) or human rhinovirus type 2 (HRV2). In vitro translation of the resulting chimeric PV genomes revealed a normal cis-cleavage activity for both heterologous 2A(pro) proteinases in the chimeric PV polyproteins. However, only the genome containing the 2A-encoding sequence of CBV4 (PV/CBV4-2A) yielded viable virus in transfected cells, producing a mixture of large and small plaques on HeLa cell monolayers. The large-plaque variants were found to contain single-amino-acid mutations at a specific site near the C terminus of the CBV4 2A(pro) protein. When the same single-amino-acid mutations were directly introduced into the parental PV/CBV4-2A genome, chimeric viruses with a large-plaque phenotype and a wild-type PV-like growth pattern were obtained upon transfection, an observation demonstrating that these point mutations alone had a drastic effect on the growth of the PV/CBV4 chimeric virus. On the other hand, the chimeric genome containing the 2A-encoding sequence of HRV2 (PV/HRV2-2A) produced a null phenotype in transfected HeLa cells, although low-level replication of this chimeric genome was evident. We conclude that only 2A(pro) of the more closely related enterovirus CBV4 is able to functionally substitute for that of PV in vivo, and a subtle genetic modification of the CBV4 2A(pro) protein results in a drastic improvement in the growth of the chimeric PV/CBV4-2A virus. In addition, this chimeric cDNA approach enabled us to dissect multiple biological functions encoded by the 2A(pro) proteins.
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D029822:Enterovirus B, Human / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D016679:Genome, Viral; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010849:Picornaviridae / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D017354:Point Mutation; D017955:Poliovirus / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012229:Rhinovirus / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins; D014779:Virus Replication
chemical_list: D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus