title: Adaptive mutations in the nuclear export protein of human-derived H5N1 strains facilitate a polymerase activity-enhancing conformation.
abstract: The nuclear export protein (NEP) (NS2) of the highly pathogenic human-derived H5N1 strain A/Thailand/1(KAN-1)/2004 with the adaptive mutation M16I greatly enhances the polymerase activity in human cells in a concentration-dependent manner. While low NEP levels enhance the polymerase activity, high levels are inhibitory. To gain insights into the underlying mechanism, we analyzed the effect of NEP deletion mutants on polymerase activity after reconstitution in human cells. This revealed that the polymerase-enhancing function of NEP resides in the C-terminal moiety and that removal of the last three amino acids completely abrogates this activity. Moreover, compared to full-length NEP, the C-terminal moiety alone exhibited significantly higher activity and seemed to be deregulated, since even the highest concentration did not result in an inhibition of polymerase activity. To determine transient interactions between the N- and C-terminal domains in cis, we fused both ends of NEP to a split click beetle luciferase and performed fragment complementation assays. With decreasing temperature, increased luciferase activity was observed, suggesting that intramolecular binding between the C- and N-terminal domains is preferentially stabilized at low temperatures. This stabilizing effect was significantly reduced with the adaptive mutation M16I or a combination of adaptive mutations (M16I, Y41C, and E75G), which further increased polymerase activity also at 34°C. We therefore propose a model in which the N-terminal moiety of NEP exerts an inhibitory function by back-folding to the C-terminal domain. In this model, adaptive mutations in NEP decrease binding between the C- and N-terminal domains, thereby allowing the protein to "open up" and become active already at a low temperature.
authors.affiliation: Institute for Virology, University Medical Center Freiburg, Freiburg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Reuther
authors.forename: Sebastian
authors.initials: S
authors.lastname: Giese
authors.forename: Veronika
authors.initials: V
authors.lastname: Götz
authors.forename: Normann
authors.initials: N
authors.lastname: Kilb
authors.forename: Benjamin
authors.initials: B
authors.lastname: Mänz
authors.forename: Linda
authors.initials: L
authors.lastname: Brunotte
authors.forename: Martin
authors.initials: M
authors.lastname: Schwemmle
mesh_terms: D002467:Cell Nucleus / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D021381:Protein Transport; D013696:Temperature; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics*; D014779:Virus Replication
chemical_list: D017361:Viral Nonstructural Proteins