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-1␣ 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.IMPORTANCE 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.KEYWORDS factor inhibiting HIF, hypoxia-inducible factor, ankyrin repeat, orf virus, parapoxvirus C ells must be able to respond rapidly to low oxygen levels in order to survive, and triggering of the heterodimeric transcription factor hypoxia-inducible factor (HIF) is key to the cellular adaptation to hypoxia. Under normoxic conditions, the HIF alpha subunits (HIF-1␣ and HIF-2␣) are tightly suppressed by posttranslational modifications, which are relaxed with decreasing oxygen availability. These posttranslational modifi-

Asparagine hydroxylation is likely to be a reversible post-translational modification

“…To determine if we could observe asparagine dehydroxylation in a cell-free reaction, we generated hydroxylated protein by incubating the recombinant C-terminal domain of HIF2α (CAD) with recombinant FIH. To prevent re-hydroxylation by FIH, hydroxylated CAD was re-purified and subsequently incubated with cellular lysates derived from FIH-knockout HeLa cells (Chen et al, 2017). After 3 hours the CAD was affinity purified, digested and analysed using mass spectrometry.…”

“…10 μM CAD was hydroxylated by incubation with 10 μM FIH, 112.5 mM NaCl, 65 mM Tris-HCl, 4 mM sodium ascorbate, 500 μM DTT, 30 μM FeSO4 and 40 μM 2-oxoglutarate for 2.5 h at 37°C, and repurified using a HisTrap HP column (GE Healthcare). FIH -/-HeLa cells (Chen et al, 2017) were lysed in ice-cold lysis buffer (1% Triton-x100, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl), supplemented with HALT EDTA-free protease inhibitors). Protein concentration was ~0.5mg/ml.…”

Ankyrins in human health and disease – an update of recent experimental findings

“…This is a new mechanism used by viruses to de-repress HIF and develop infection. However, the possible influence of the ORFV ANK-HIF interaction on viral replication has yet to be explored [2]. The numerous genes encoding ANK-containing proteins present in the genome of various pathogenic microbial agents have been acquired from eukaryotic organisms due to horizontal gene transfer [3].…”

Orf virus: A promising new therapeutic agent

“…The ANK repeat proteins encoded by ORFV mediate the degradation of host antiviral factor via the ubiquitin/proteasome system, facilitating ORFV replication and cross‐species infections . Recently, ANK repeat proteins were demonstrated to influence the cellular hypoxia response pathway . During ORFV infection, the viral ANK repeat proteins enhance hypoxia‐inducible factor (HIF) target gene expression and sequester the factor inhibiting HIF (FIH).…”

“…52 Recently, ANK repeat proteins were demonstrated to influence the cellular hypoxia response pathway. 53 During ORFV infection, the viral ANK repeat proteins enhance hypoxia-inducible factor (HIF) target gene expression and sequester the factor inhibiting HIF (FIH).…”