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Updated: Aug 17, 2026
Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells
Published on: June 3, 2012
Identification of the influenza virus transcriptase by affinity-labeling with pyridoxal 5'-phosphate
Abstract:
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.
Insights
Pyridoxal 5'-phosphate (PLP) identifies the fowl plaque virus (FPV) transcriptase. This enzyme
Area of Science:
- Virology
- Molecular Biology
- Enzymology
Background:
- Fowl plaque virus (FPV) possesses a transcriptase essential for its replication.
- Identifying the specific viral protein responsible for transcription is crucial for understanding viral mechanisms.
Purpose of the Study:
- To identify the viral transcriptase of fowl plaque virus (FPV).
- To elucidate the role of specific viral proteins in transcription initiation and elongation.
Main Methods:
- In vitro transcription assays using fowl plaque virus (FPV).
- Kinetic analysis of pyridoxal 5 omino-phosphate (PLP) inhibition.
- Chemical labeling with [3H]borohydride and protein identification.
Main Results:
- Pyridoxal 5 omino-phosphate (PLP) competitively inhibited nucleoside triphosphate addition, affecting both initiation and elongation.
- The core protein PB1 was preferentially labeled by [3H]borohydride, indicating it binds nucleotides.
- GTP and CTP protected PB1 from labeling, confirming its role in nucleotide binding.
Conclusions:
- The FPV core protein PB1 is identified as the transcriptase.
- PB1 is responsible for both initiation and elongation of viral RNA synthesis.
- PB1 likely associates with the 3 omino ends of template RNAs within the virion.
