title: 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 RNA-dependent 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 A 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 "phi"-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 phi loop and found that lysine 627 is located in the phi 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.
authors.affiliation: Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, and Institute for Health Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan. kuzuhara@ph.bunri-u.ac.jp
authors.forename: Takashi
authors.initials: T
authors.lastname: Kuzuhara
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kise
authors.forename: Hiroko
authors.initials: H
authors.lastname: Yoshida
authors.forename: Takahiro
authors.initials: T
authors.lastname: Horita
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Murazaki
authors.forename: Akie
authors.initials: A
authors.lastname: Nishimura
authors.forename: Noriko
authors.initials: N
authors.lastname: Echigo
authors.forename: Hiroko
authors.initials: H
authors.lastname: Utsunomiya
authors.forename: Hideaki
authors.initials: H
authors.lastname: Tsuge
mesh_terms: D018360:Crystallography, X-Ray; D009980:Influenza A virus / Q000201:enzymology*; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D051818:Replication Protein C / Q000737:chemistry / Q000378:metabolism; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: C057027:PB2 protein, Influenzavirus A; D012367:RNA, Viral; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase; D051818:Replication Protein C