The active sites of the influenza cap-dependent endonuclease are on different polymerase subunits.
The cap-dependent endonuclease of the influenza viral RNA polymerase, which produces the capped RNA primers that initiate viral mRNA synthesis, is comprised of two active sites, one for cap binding and one for endonuclease cleavage. We identify the amino acid sequences that constitute these two active sites and demonstrate that they are located on different polymerase subunits. Binding of the 5' terminal sequence of virion RNA (vRNA) to the polymerase activates a tryptophan-rich, cap-binding sequence on the PB2 subunit. At least one of the tryptophans functions in cap binding, indicating that this active site is probably similar to that of other known cap-binding proteins. Endonuclease cleavage, which is activated by the subsequent binding of the 3' terminal sequence of vRNA, resides in a PB1 sequence that contains three essential acidic amino acids, similar to the active sites of other enzymes that cut polynucleotides to produce 3'-OH ends. These results, coupled with those of our previous study, provide a molecular map of the five known essential active sites of the influenza viral polymerase.
Li
M L
ML
Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas at Austin, 78712, USA.
Rao
P
P
Krug
R M
RM
D000600:Amino Acids, Dicarboxylic; D020134:Catalytic Domain; D004720:Endonucleases / Q000378:metabolism*; D008954:Models, Biological; D009975:Orthomyxoviridae / Q000201:enzymology*; D012315:RNA Caps / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D014364:Tryptophan; D014764:Viral Proteins / Q000378:metabolism*
D000600:Amino Acids, Dicarboxylic; C057027:PB2 protein, Influenzavirus A; D012315:RNA Caps; D012367:RNA, Viral; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1; D014364:Tryptophan; D012324:RNA-Dependent RNA Polymerase; D004720:Endonucleases