The role of the novel influenza A protein PB1-F2 in viral pathogenesis in the avian species

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Influenza A virus is an orthomyxovirus and has a genome composed of 8 negative sense RNA segments. Until recently it was believed that influenza A virus encoded 10 proteins; however, in 2001 an eleventh ORF was identified in the laboratory strain A Puerto Rico 8 34 (H1N1) (PR8). This 87 amino acid protein, termed PB1-F2, is encoded on segment 2 of the virus, which also encodes the PB1 protein that functions as part of the polymerase complex. PB1-F2 was subsequently shown to localise to mitochondria and to cause apoptosis in immune cells. Thus it may function to aid immune evasion. PB1-F2 was found to be conserved in 64 of 75 influenza A virus isolates on the Genbank database; in the majority of cases the ORF encodes a 90 aa protein. A closer examination of the segment 2 sequences reveals that the ORF is present in a truncated form in many isolates, often as a 57 aa ORF. To date, all the information on PB1-F2 is based on human influenza and the role of this protein in viral pathogenesis in other animal species has not been determined. In this study we will investigate the role of PB1-F2 in replication and pathogenicity of avian influenza viruses. Using an avian influenza virus reverse genetics system established in our laboratory, we will generate a panel of viruses expressing different forms of PB1-F2 in order to determine the role of this protein in viral pathogenesis. We are also interested in whether PB1-F2 plays a role in the generation of pandemic viruses. Along with the surface antigens, segment 2 is transferred from avian viruses into a human virus backbone when a pandemic virus is created, indicating that either PB1 of PB1-F2 are important in this process. We are going to generate a panel of influenza viruses with segment 2 derived from viruses isolated at either side of a pandemic junction to elucidate whether the introduction of an avian segment 2 increases the fitness of the pandemic virus. We will attempt to separate the functions of PB1 and PB1-F2 in this process by changing only the PB1-F2 and testing whether these viruses induce apoptosis in human immune cells, and replicate in human airway cells to the same extent as their wild type equivalents. In addition, we will investigate the biochemical and functional properties of PB1-F2 by looking at how the protein is post-translationally modified, whether it interacts with other virus proteins and if it gets incorporated into virus particles.