Distinct regions of influenza virus PB1 polymerase subunit recognize vRNA and cRNA templates.
The influenza virus RNA polymerase is a heterotrimer comprising the PB1, PB2 and PA subunits. PB1 is the core of the complex and accounts for the polymerase activity. We have studied the interaction of PB1 with model cRNA template by in vitro binding and Northwestern analyses. The binding to model cRNA was specific and showed an apparent Kd of approximately 7x10(-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 cRNA, 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-binding sites are constituted by: (i) residues located at the N-terminus (probably common for vRNA and cRNA binding) and, either (ii) residues from the central 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 upon binding to cRNA versus vRNA templates.
Centro Nacional de Biotecnología (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain.
S
S
González
J
J
Ortín
D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D009690:Nucleic Acid Conformation; D009975:Orthomyxoviridae / Q000201:enzymology*; D011233:Precipitin Tests; D011487:Protein Conformation; D015347:RNA Probes / Q000235:genetics / Q000378:metabolism; D018075:RNA, Complementary / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D013698:Templates, Genetic; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D015347:RNA Probes; D018075:RNA, Complementary; D012367:RNA, Viral; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1