title: Ankyrin Repeat Proteins of Orf Virus Influence the Cellular Hypoxia Response Pathway.
abstract: Hypoxia-inducible factor (HIF) is a transcriptional activator with a central role in regulating cellular responses to hypoxia. It is also emerging as a major target for viral manipulation of the cellular environment. Under normoxic conditions, HIF is tightly suppressed by the activity of oxygen-dependent prolyl and asparaginyl hydroxylases. The asparaginyl hydroxylase active against HIF, factor inhibiting HIF (FIH), has also been shown to hydroxylate some ankyrin repeat (ANK) proteins. Using bioinformatic analysis, we identified the five ANK proteins of the parapoxvirus orf virus (ORFV) as potential substrates of FIH. Consistent with this prediction, coimmunoprecipitation of FIH was detected with each of the ORFV ANK proteins, and for one representative ORFV ANK protein, the interaction was shown to be dependent on the ANK domain. Immunofluorescence studies revealed colocalization of FIH and the viral ANK proteins. In addition, mass spectrometry confirmed that three of the five ORFV ANK proteins are efficiently hydroxylated by FIH in vitro While FIH levels were unaffected by ORFV infection, transient expression of each of the ORFV ANK proteins resulted in derepression of HIF-1α activity in reporter gene assays. Furthermore, ORFV-infected cells showed upregulated HIF target gene expression. Our data suggest that sequestration of FIH by ORFV ANK proteins leads to derepression of HIF activity. These findings reveal a previously unknown mechanism of viral activation of HIF that may extend to other members of the poxvirus family.
  
  
  OBJECTIVE
  The protein-protein binding motif formed from multiple repeats of the ankyrin motif is common among chordopoxviruses. However, information on the roles of these poxviral ankyrin repeat (ANK) proteins remains limited. Our data indicate that the parapoxvirus orf virus (ORFV) is able to upregulate hypoxia-inducible factor (HIF) target gene expression. This response is mediated by the viral ANK proteins, which sequester the HIF regulator FIH (factor inhibiting HIF). This is the first demonstration of any viral protein interacting directly with FIH. Our data reveal a new mechanism by which viruses reprogram HIF, a master regulator of cellular metabolism, and also show a new role for the ANK family of poxvirus proteins.
authors: Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand.
authors: Da-Yuan
authors: DY
authors: Chen
authors: School of Biological Sciences, University of Adelaide, Adelaide, South Australia, Australia.
authors: Jacqueline-Alba
authors: JA
authors: Fabrizio
authors: School of Biological Sciences, University of Adelaide, Adelaide, South Australia, Australia.
authors: Sarah E
authors: SE
authors: Wilkins
authors: Protein Discovery Centre, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia.
authors: Keyur A
authors: KA
authors: Dave
authors: Protein Discovery Centre, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia.
authors: Jeffrey J
authors: JJ
authors: Gorman
authors: Department of Renal Medicine, School of Medicine, Flinders University, Flinders Medical Centre, Bedford Park, South Australia, Australia.
authors: Jonathan M
authors: JM
authors: Gleadle
authors: Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand.
authors: Stephen B
authors: SB
authors: Fleming
authors: School of Biological Sciences, University of Adelaide, Adelaide, South Australia, Australia daniel.peet@adelaide.edu.au andy.mercer@otago.ac.nz.
authors: Daniel J
authors: DJ
authors: Peet
authors: Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand daniel.peet@adelaide.edu.au andy.mercer@otago.ac.nz.
authors: Andrew A
authors: AA
authors: Mercer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017089:Ankyrin Repeat; D015687:Cell Hypoxia; D019295:Computational Biology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D006900:Hydroxylation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000378:metabolism; D007985:Leydig Cells; D008297:Male; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D009923:Orf virus / Q000235:genetics* / Q000378:metabolism; D061251:Primary Cell Culture; D011485:Protein Binding; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D012756:Sheep; D015398:Signal Transduction
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011994:Recombinant Proteins; D012097:Repressor Proteins; D006899:Mixed Function Oxygenases
keywords: ankyrin repeat; factor inhibiting HIF; hypoxia-inducible factor; orf virus; parapoxvirus