Distinct regions of influenza virus PB1 polymerase subunit recognize vRNA and cRNA templates

The influenza virus RNA polymerase is a heterotrimer comprising the PB1, PB 2 and PA subunits. PB1 is the core of the complex and accounts for the poly merase activity. We have studied the interaction of PB1 with model cRNA tem plate by in vitro binding and Northwestern analyses. The binding to model c RNA was specific and showed an apparent K-d of similar to 7 x 10(-8) M. In contrast to the interaction with vRNA, PB1 was able to bind equally the 5' and 3' arm of the cRNA panhandle. The N-terminal 139 amino acids of PB1 and sequences between positions 267 and 493 proved positive for binding to cRN A, whereas the interaction with vRNA template previously was mapped to the N- and C-terminal regions. Competition experiments using the 5' and 3' arms of either the vRNA or cRNA panhandle indicated that the N-terminal binding site is shared by both templates, The data indicate that the PB1 RNA-bindi ng sites are constituted by: (i) residues located at the N-terminus (probab ly common for vRNA and cRNA binding) and, either (ii) residues from the cen tral part of PB1 (for cRNA) or (iii) residues from the C-terminal region of PB1 (for vRNA), and suggest that PB1 undergoes a conformational change upo n binding to cRNA versus vRNA templates.