Characterization of influenza virus PB1 protein binding to viral RNA: Two separate regions of the protein contribute to the interaction domain

The interaction of the PB1 subunit of the influenza virus polymerase with t he viral RNA (vRNA) template has been studied in vitro. The experimental ap proach included the in vitro binding of labeled model vRNA to PBI protein i mmobilized as an immunoprecipitate, as well as Northwestern analyses. The b inding to model vRNA was specific, and an apparent K-d of about 2 x 10(-8) M was determined. Although interaction with the isolated 3' arm of the panh andle was detectable, interaction with the 5' arm was prominent and the bin ding was optimal with a panhandle analog structure (5'+3' probe). When pres ented with a panhandle analog mixed probe, PBL was able to retain the 3' ar m as efficiently as the 5' arm. The sequences of the PBI protein involved i n vRNA binding were identified by in vitro interaction tests with PB1 delet ion mutants. Two separate regions of the PB1 protein sequence proved positi ve for binding: the N-terminal 83 amino acids and the C-proximal sequences located downstream of position 493, All mutants able to interact with model vRNA were capable of binding the 5' arm more efficiently than the 3' arm o f the panhandle. Taken together, these results suggest that two separate re gions of the PB1 protein constitute a vRNA binding site that interacts pref erentially with the 5' arm of the panhandle structure.