PHOTOAFFINITY-LABELING OF INFLUENZA-VIRUS RNA-POLYMERASE PB1 SUBUNIT WITH 8-AZIDO GTP

8-Azido GTP (8-N-3 GTP) was demonstrated to be polymerized into RNA by influenza virus-associated RNA polymerase at about one tenth the rate of GTP incorporation, The K-m value for the azido analogue of GTP in primer-dependent RNA synthesis was 94 mu M whereas K-m for the natural substrate, GTP, was 6.7 mu M. Upon exposure of a mixture of 8-N-3 [alpha-P-32]GTP and influenza virus ribonucleoprotein (RNP) complexes to ultraviolet light, the PB1 subunit of viral RNA polymerase was selectively radio-labeled, The photo-labeling of PB1 was competed strongly by GTP and to lesser extents by other nucleoside 5'-triphosphates, These results altogether support the prediction that the substrate-binding site (S site) of influenza RNA polymerase is located on the PB1 protein, In the presence of ApG primer, the 8-N-3 GTP binding was reduced to about 40% level, suggesting that the GTP analogue can bind not only to the S site but also to the primer- and product-binding site (P site).