Identification of the influenza virus transcriptase by affinity-labeling with pyridoxal 5'-phosphate.
Pyridoxal 5'-phosphate (PLP), a reversible inhibitor of in vitro transcription by fowl plaque virus, has been used to identify the transcriptase. Kinetic analyses showed that PLP competitively inhibits the addition of each nucleoside triphosphate in ApG-primed reactions, suggesting that both initiation and elongation are affected. The irreversible inhibition by PLP following reduction with borohydride was prevented by preincubation with the first substrate: GTP in unprimed reactions or CTP in the presence of ApG. On reaction of FPV proteins with PLP and [3H]borohydride the core protein PB1 was preferentially labeled and the labeling was selectively blocked by GTP or ApG + CTP. These data suggest that PB1 has the nucleotide-binding site of the transcriptase, is responsible for both initiation and elongation, and is apparently associated with the 3' ends of template RNAs in virions.
D000345:Affinity Labels; D000818:Animals; D002642:Chick Embryo; D009980:Influenza A virus / Q000201:enzymology*; D007700:Kinetics; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D018894:Reverse Transcriptase Inhibitors; D014158:Transcription, Genetic; D014758:Viral Core Proteins; D014764:Viral Proteins / Q000378:metabolism
D000345:Affinity Labels; D018894:Reverse Transcriptase Inhibitors; D014758:Viral Core Proteins; D014764:Viral Proteins; D011732:Pyridoxal Phosphate