Properties of purified recombinant poliovirus protein 3aB as substrate for viral proteinases and as co-factor for RNA polymerase 3Dpol.
The poliovirus-specific polypeptide 3AB (B = VPg) was expressed in Escherichia coli and purified to near homogeneity. Corresponding to its known association with membranes in poliovirus-infected HeLa cells, 3AB expressed in E. coli was also membrane-associated, and it could be solubilized only in detergent-containing buffers. In soluble form, 3AB was resistant to digestion with the virus-specific proteinases 3Cpro and 3CDpro. However, it was cleaved by these enzymes to 3A and VPg when bound to the bacterial membranes, an observation suggesting that 3AB may deliver the genome-linked protein VPg to the membrane-associated poliovirus replication complex. The specific activity of 3CDpro in processing 3AB was significantly higher than that of 3Cpro. Soluble 3AB was found to stimulate nearly 100-fold poly (A)-dependent, primer-dependent poly(U) synthesis, catalyzed by purified poliovirus RNA polymerase 3Dpol. We propose that 3AB has a dual function in poliovirus genome replication: as a precursor for VPg, and as a co-factor for 3Dpol.
Lama
J
J
Department of Microbiology, School of Medicine, State University of New York at Stony Brook 11794-5222.
Paul
A V
AV
Harris
K S
KS
Wimmer
E
E
D000086042:3C Viral Proteases; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism; D017955:Poliovirus / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014758:Viral Core Proteins / Q000378:metabolism*; D014764:Viral Proteins
D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases