title: The ubiquitination of the influenza A virus PB1-F2 protein is crucial for its biological function.
abstract: The aim of the present study was to identify what influences the short half-life of the influenza A virus PB1-F2 protein and whether a prolonged half-life affects the properties of this molecule. We hypothesized that the short half-life of PB1-F2 could conceal the phenotype of the protein. Because proteasome degradation might be involved in PB1-F2 degradation, we focused on ubiquitination, a common label for proteasome targeting. A cluster of lysine residues was demonstrated as an ubiquitination acceptor site in evolutionary and functionally distinct proteins. The PB1-F2 sequence alignment revealed a cluster of lysines on the carboxy terminal end of PB1-F2 in almost all of the GenBank sequences available to date. Using a proximity ligation assay, we identified ubiquitination as a novel posttranslational modification of PB1-F2. Changing the lysines at positions 73, 78, and 85 to arginines suppressed the ubiquitination of A/Puerto Rico/8/1934 (H1N1)-derived PB1-F2. The mutation of the C-terminal lysine residue cluster positively affected the overall expression levels of avian A/Honk Kong/156/1997 (H5N1)- and mammalian A/Puerto Rico/8/1934 (H1N1)-derived PB1-F2. Moreover, increased PB1-F2 copy numbers strengthened the functions of this virus in the infected cells. The results of a minigenome luciferase reporter assay revealed an enhancement of viral RNA-dependent RNA polymerase activity in the presence of stabilized PB1-F2, regardless of viral origin. IFNβ antagonism was enhanced in 293T cells transfected with a plasmid expressing stabilized K→R mutant variants of PB1-F2. Compared with PB1-F2 wt, the loss of ubiquitination enhanced the antibody response after DNA vaccination. In summary, we revealed that PB1-F2 is an ubiquitinated IAV protein, and this posttranslational modification plays a central role in the regulation of the biological functions of this protein.
authors: Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05, Bratislava, Slovak Republic.
authors: Ivan
authors: I
authors: Košík
authors: Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05, Bratislava, Slovak Republic.
authors: Margaréta
authors: M
authors: Práznovská
authors: Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05, Bratislava, Slovak Republic.
authors: Martina
authors: M
authors: Košíková
authors: Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05, Bratislava, Slovak Republic.
authors: Zuzana
authors: Z
authors: Bobišová
authors: Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05, Bratislava, Slovak Republic.
authors: Jaroslav
authors: J
authors: Hollý
authors: Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05, Bratislava, Slovak Republic.
authors: Eva
authors: E
authors: Varečková
authors: Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05, Bratislava, Slovak Republic.
authors: František
authors: F
authors: Kostolanský
authors: Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05, Bratislava, Slovak Republic.
authors: Gustáv
authors: G
authors: Russ
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation / Q000502:physiology; D002460:Cell Line; D005260:Female; D006801:Humans; D009980:Influenza A virus / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D054875:Ubiquitination; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*
chemical_list: C442816:PB1-F2 protein, Influenza A virus; D014764:Viral Proteins