title: Disruption of Src homology 3-binding motif within non-structural protein 1 of influenza B virus unexpectedly enhances viral replication in human cells.
abstract: The influenza virus non-structural protein 1 (NS1) is a multifunctional virulence factor that plays a crucial role during infection by blocking the innate antiviral immune response of infected cells. In contrast to the well-studied NS1 protein of influenza A virus, knowledge about structure and functions of the influenza B virus homologue B/NS1, which shares less than 25 % sequence identity, is still limited. Here, we report on a reverse genetic analysis to study the role of a highly conserved class II Src homology 3 domain-binding motif matching the consensus PxxPx(K/R) that we identified at positions 122-127 of the B/NS1 protein. Surprisingly, glycine substitutions in the Src homology 3 domain-binding motif increased virus replication up to three orders of magnitude in human lung cells. Enhanced mutant virus propagation was accompanied by increased gene expression and apoptosis induction linking this motif to the control of programmed cell death. A MS-based interactome study revealed that the glycine substitutions facilitate binding of B/NS1 to heat shock protein 90-beta (HSP90β). Moreover, recruitment of the viral polymerase basic protein 2 to the B/NS1-HSP90β complex was observed. Pharmacological inhibition of HSP90 reduced mutant virus propagation suggesting that the mutation-induced involvement of HSP90β enhanced viral replication. This study not only functionally characterizes a conserved motif within the B/NS1 protein, but also illustrates a rare example in which mutation of a highly conserved sequence within a viral protein does not result in high fitness costs, but rather increases viral replication via recruitment of a host factor.
authors.affiliation: Unit 17, Influenza and Other Respiratory Viruses, Robert Koch Institute, Seestr. 10, Berlin 13353, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Sadewasser
authors.affiliation: Unit 17, Influenza and Other Respiratory Viruses, Robert Koch Institute, Seestr. 10, Berlin 13353, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Saenger
authors.affiliation: Unit 17, Influenza and Other Respiratory Viruses, Robert Koch Institute, Seestr. 10, Berlin 13353, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Paki
authors.affiliation: ZBS 6 - Proteomics and Spectroscopy, Robert Koch Institute, Seestr. 10, Berlin 13353, Germany.
authors.forename: Torsten
authors.initials: T
authors.lastname: Schwecke
authors.affiliation: Unit 17, Influenza and Other Respiratory Viruses, Robert Koch Institute, Seestr. 10, Berlin 13353, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Wolff
mesh_terms: D019943:Amino Acid Substitution; D002460:Cell Line; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009981:Influenza B virus / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D007251:Influenza, Human / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D011485:Protein Binding; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D018909:src Homology Domains
chemical_list: D018841:HSP90 Heat-Shock Proteins; C085820:INS1 protein, influenza virus; D017361:Viral Nonstructural Proteins