Uridylylation of the potyvirus VPg by viral replicase NIb correlates with the nucleotide binding capacity of VPg.
Poty- and picornaviruses share similar genome organizations and polyprotein processing strategies. By analogy to picornaviruses it has been proposed that the genome-linked protein VPg may serve as a primer for genome replication of potyviruses. The multifunctional VPg of potato virus A (PVA; genus Potyvirus) was found to be uridylylated by NIb, the RNA polymerase of PVA. The nucleotidylation activity of NIb is more efficient in the presence of Mn(2+) than Mg(2+) and does not require an RNA template. Our results suggest that the nucleotidylation reaction exhibits weak preference for UTP over the other NTPs. An NTP-binding experiment with oxidized [alpha-(32)P]UTP revealed that PVA VPg contains an NTP-binding site. Deletion of a 7-amino acid-long putative NTP-binding site from VPg reduced nucleotide-binding capacity and debilitated uridylylation reaction. These results provide evidence that VPg may play a similar role in RNA synthesis of potyviruses as it does in the case of picornaviruses.
Puustinen
Pietri
P
Department of Applied Biology, P. O. Box 27, University of Helsinki, Helsinki 00014, Finland.
Mäkinen
Kristiina
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D010849:Picornaviridae / Q000378:metabolism; D017800:Potyvirus / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D016622:Silver Staining; D014357:Trypsin / Q000494:pharmacology; D014529:Uridine / Q000737:chemistry*; D014758:Viral Core Proteins / Q000737:chemistry* / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
D000596:Amino Acids; D003432:Cross-Linking Reagents; D009711:Nucleotides; D010455:Peptides; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D014764:Viral Proteins; D008345:Manganese; D012313:RNA; C061396:nuclear inclusion protein b, mosaic viruses; D012321:DNA-Directed RNA Polymerases; D014357:Trypsin; D008274:Magnesium; D014529:Uridine