Pyridoxal phosphate as a probe of reovirus transcriptase.

The ribonucleoprotein core of reovirus is a multienzyme complex that transcribes messenger ribonucleic acid (mRNA) from double-stranded RNA templates. So far, the core has resisted attempts to disassemble it and identify the polypeptide species responsible for RNA polymerase activity. As an alternative approach, we tested pyridoxal 5-phosphate (PLP) as a potential affinity labeling reagent for reovirus transcriptase in vitro; PLP has been used as an affinity reagent for cellular and viral nucleic acid polymerases. We found that PLP inhibited reovirus transcriptase reversibly (apparent Ki = 0.2 mM), but the inhibition was noncompetitive with respect to each of the four ribonucleoside triphosphates. This interaction required both the aldehyde and phosphate moieties in PLP, since pyridoxamine and pyridoxal were relatively inactive. To identify the polypeptides involved, we labeled the PLP--core complex by reductive alkylation with [3H]borohydride. At PLP concentrations close to the apparent Ki, labeling was selective for the two largest virion polypeptides, lambda 1 and lambda 2. At saturation, there were only 10 high-affinity PLP binding sites per core in each of the lambda polypeptide species. These findings implicate either or both lambda polypeptide species in viral transcription and they indicate that a special population, representing no more than 10% of the total lambda molecules in each core, participates in RNA synthesis.