Structural basis of the influenza A virus RNA polymerase PB2 RNA-binding domain containing the pathogenicity-determinant lysine 627 residue

Abstract

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 RNA primers/ 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 "φ"-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 φ loop and found that lysine 627 is located in the φ 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 RNA binding of viral RNA polymerases. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.