RNA-DEPENDENT ACTIVATION OF PRIMER RNA PRODUCTION BY INFLUENZA-VIRUS POLYMERASE - DIFFERENT REGIONS OF THE SAME PROTEIN SUBUNIT CONSTITUTE THE 2 REQUIRED RNA-BINDING SITES

The capped RNA primers required for the initiation of influenza virus mRNA synthesis are produced by the viral polymerase itself, which cons ists of three proteins PB1, PB2 and PA. Production of primers is activ ated only when the 5'- and 3'-terminal sequences of virion RNA (vRNA) bind sequentially to the polymerase, indicating that vRNA molecules fu nction not only as templates for mRNA synthesis but also as essential cofactors which activate catalytic functions. Using thio U-substituted RNA and UV crosslinking, we demonstrate that the 5' and 3' sequences of vRNA bind to different amino acid sequences In the same protein sub unit, the PB1 protein, Mutagenesis experiments proved that these two a mino acid sequences constitute the functional RNA-binding sites. The 5 ' sequence of vRNA binds to an amino acid sequence centered around two arginine residues at positions 571 and 572, causing an allosteric alt eration which activates two new functions of the polymerase complex. I n addition to the PB2 protein subunit acquiring the ability to bind 5' -capped ends of RNAs, the PB1 protein itself acquires the ability to b ind the 3' sequence of vRNA, via a ribonucleoprotein 1 (RNP1)-like mot if, amino acids 249-256, which contains two phenylalanine residues req uired for binding. Binding to this site induces a second allosteric al teration which results in the activation of the endonuclease that prod uces the capped RNA primers needed for mRNA synthesis, Hence, the PB1 protein plays a central role in the catalytic activity of the viral po lymerase, not only in the catalysis of RNA-chain elongation but also i n the activation of the enzyme activities that produce capped RNA prim ers.