title: The influenza A virus PB1-F2 protein targets the inner mitochondrial membrane via a predicted basic amphipathic helix that disrupts mitochondrial function.
abstract: The 11th influenza A virus gene product is an 87-amino-acid protein provisionally named PB1-F2 (because it is encoded by an open reading frame overlapping the PB1 open reading frame). A significant fraction of PB1-F2 localizes to the inner mitochondrial membrane in influenza A virus-infected cells. PB1-F2 appears to enhance virus-induced cell death in a cell type-dependent manner. For the present communication we have identified and characterized a region near the COOH terminus of PB1-F2 that is necessary and sufficient for its inner mitochondrial membrane localization, as determined by transient expression of chimeric proteins consisting of elements of PB1-F2 genetically fused to enhanced green fluorescent protein (EGFP) in HeLa cells. Targeting of EGFP to mitochondria by this sequence resulted in the loss of the inner mitochondrial membrane potential, leading to cell death. The mitochondrial targeting sequence (MTS) is predicted to form a positively charged amphipathic alpha-helix and, as such, is similar to the MTS of the p13(II) protein of human T-cell leukemia virus type 1. We formally demonstrate the functional interchangeability of the two sequences for mitochondrial localization of PB1-F2. Mutation analysis of the putative amphipathic helix in the PB1-F2 reveals that replacement of five basic amino acids with Ala abolishes mitochondrial targeting, whereas mutation of two highly conserved Leu to Ala does not. These findings demonstrate that PB1-F2 possesses an MTS similar to other viral proteins and that this MTS, when fused to EGFP, is capable of independently compromising mitochondrial function and cellular viability.
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: James S
authors.initials: JS
authors.lastname: Gibbs
authors.forename: Daniela
authors.initials: D
authors.lastname: Malide
authors.forename: Felicita
authors.initials: F
authors.lastname: Hornung
authors.forename: Jack R
authors.initials: JR
authors.lastname: Bennink
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Yewdell
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D009980:Influenza A virus / Q000502:physiology*; D007425:Intracellular Membranes / Q000502:physiology / Q000821:virology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D017931:DNA Primers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins