Structural Basis of the Influenza A Virus RNA Polymerase

Because the influenza A virus has an RNA genome, its RNAdependent RNA polymerase, comprising the PA, PB1, and PB2 subunits, is essential for viral transcription and replication. The binding of RNAprimers/promoters to the polymerases is an initiation step in viral transcription. In our current study, we reveal the 2.7 Å tertiary structure of the C-terminal RNA-binding domain of PB2 by x-ray crystallography. This domain incorporates lysine 627 of PB2, and this residue is associated with the high pathogenicity and host range restriction of influenza A virus. We found from our current analyses that this lysine is located in a unique “ ”-shaped structure consisting of a helix and an encircled loop within the PB2 domain. By electrostatic analysis, we identified a highly basic groove along with this loop and found that lysine 627 is located in the loop. A PB2 domain mutant in which glutamic acid is substituted at position 627 shows significantly lower RNA binding activity. This is the first report to show a relationship between RNA binding activity and the pathogenicity-determinant lysine 627. Using the Matras program for protein three-dimensional structural comparisons, we further found that the helix bundles in the PB2 domain are similar to that of activator 1, the 40-kDa subunit of DNA replication clamp loader (replication factor C), which is also an RNA-binding protein. This suggests a functional and structural relationship between the RNA-binding mechanisms underlying both influenza A viral transcription and cellular DNA replication. Our present results thus provide important new information for developing novel drugs that target the primer/promoter RNAbinding of viral RNA polymerases. In 1918, a pandemic expansion of influenza A virus resulted in ten million deaths worldwide (1), and strategies to prevent any future expansions of this virus are therefore an important endeavor (2, 3). Although inhibitors of neuraminidase and the M2 ion channel are widely used as anti-influenza A virus drugs (4, 5), some adverse effects of these agents and also the emergence of drug-resistant viruses have been reported (6, 7). Because the transcription and replication of influenza A virus requires the activity of its RNA-dependent RNA polymerase, this enzyme represents a very promising target for anti-viral drug development. InfluenzaA virus digests hostmRNAvia the PB1 subunit of its RNA polymerase (8). A 5 -RNA fragment of 10–15 nucleotides is used as a primer for transcription initiation (8) through primer and promoter RNA binding activities within the C-terminal region of its RNA polymerase PB2 domain (9–11). The PA subunit has protease activity and binds to PB (2). The determination of the tertiary structure of a protein is a valuable tool in the development of a targeting drug, and low resolution structures have previously been determined for the PA, PB1, and PB2 subunits of influenza A virus RNA polymerase by electron microscopy (12). In addition, the structureofPB2nuclear localization signal domain (amino acid (aa)3 residues 687–759), the PB2 cap (7-methyl guanosine triphosphate)-binding domain (aa 318– 483), and the C-terminal domain of PA with the N-terminal domain of PB1 have also been reported (13–17). The C-terminal domainofPB2 includes theLys627 residue,whichplaysa role in the high pathogenicity and host range restriction of the influenza virus.The structureofPB2hasnotbeenpreviously elucidated, and the structure-function relationships between this domain and Lys627 represent a significant advance in our understanding of the pathogenicity of influenza A.