IDENTIFICATION OF 2 SEPARATE DOMAINS IN THE INFLUENZA-VIRUS PB1 PROTEIN INVOLVED IN THE INTERACTION WITH THE PB2 AND PA SUBUNITS - A MODEL FOR THE VIRAL-RNA POLYMERASE STRUCTURE

The domains of the PB1 subunit of the influenza virus polymerase invol ved in the interaction with the PB2 and PA subunits have been defined by mutational analysis of PB1 protein, The experimental approach inclu ded in vivo competition of the PB1 activity, two-hybrid interaction as says and in vitro binding to PB1-specific matrices, Mutants of the PB1 gene including N-terminal, C-terminal and internal deletions and sing le amino acid insertions were constructed. They were unable to support polymerase activity in a reconstituted transcription-replication syst em and were tested for their competition activity when expressed in ex cess over wild-type PB1 protein. The pattern of competition obtained s uggested that the N-terminal 78 amino acids and the sequences between positions 506 and 659 in the PB1 protein are involved in the interacti on with the other components of the polymerase. We identified the N-te rminal region of PB1 protein as responsible for the interaction with t he PA subunit by two-hybrid assays in mammalian cells, N- and C-termin al fragments of the PB1 protein were expressed as His-tagged proteins and purified on Ni2+-NTA resin, Such PB1-specific matrices were used i n binding assays in vitro with metabolically labelled PB2 and PA prote ins and mutants thereof, The results obtained indicated that the N-ter minal and the C-terminal regions of PB1 are responsible for binding to PA and PB2 subunits, respectively, With this information and previous ly published results we propose a preliminary model for the architectu re of the influenza virus RNA polymerase.