Fine mapping of the subunit binding sites of influenza virus RNA polymerase

Background: Influenza virus RNA polymerase consists of three subunits, PB1, PB2 and PA, and catalyzes both transcription and replication of the RNA genome. PB1 is a catalytic subunit of RNA polymerization and a core of subunit assembly. Previously, we mapped the PB1-PB2 binding sites (1-259 of PB2 and 501-757 of PB1), and the PB1-PA binding sites (1- 140 of PB1 and 201-716 of PA). In order to determine the fine map of the subunit binding sites, we continued the same line of experiments. Methods: Serial deletion mutants of each subunit were expressed with N-terminal HA-tag, and co-immunoprecipitated with the non-tagged wild-type subunits by anti-HA or subunit-specific antibodies. Results: PBIN599 and PB1C10 were co-immunoprecipitated with PB2wt, but PB1N625 and PB1N700 were weakly co-immunoprecipitated with PB2wt. PB1C25 or PB1C40 were not co-immunoprecipitated with PB2wt. PB2N50 and PB2N75 were co-immunoprecipitated with PB1wt. However, PB2N104 or PB2N150 were not co-immunoprecipitated with PB1wt. PB1C535 and PB1C617 were co-immunoprecipitated with PAwt, but PB1N25 was not. PAN300, PAN400, PAN500 and PA501-692 were co-immunoprecipitated with PB1wt, but PAC49 was not. Conclusions: PB1-PB2 binding sites are mapped in PB1(600-747) and PB2(76-104), and PB1PA binding sites are in PB1(1-25) and PA(668-692). (C) 2001 Elsevier Science B.V All rights reserved.