Abstract
The ankyrin (ANK) repeat was named after the cytoskeletal protein ankyrin and is found in a wide range of eukaryotic and bacterial proteins where it mediates protein-protein interactions. Viruses, especially the Poxviridae and Polydnaviridae families, also encode ANK repeat containing proteins. ANK repeat proteins form the largest family of Chordopoxvirus proteins but knowledge of their functions remains limited. Nearly 80% of these viral proteins carry an F-box-like motif near the C-terminus. Recent research demonstrated that the F-box-like motif of at least some poxviral ANK proteins is able to bind cellular SKP1 protein, thus the ANK proteins might associate with host cellular ubiquitin ligase complexes.
Hypoxia Inducible Factor-1a (HIF-1a) is a key component of the transcriptional factor complex, HIF, which regulates cellular and systemic responses to hypoxia via stimulating expression of numerous genes. It is regulated in part by Factor Inhibiting HIF (FIH-1), an asparaginyl hydroxylase whose binding site in HIF-1a is similar to the ANK repeat consensus sequence. Consistent with this, some mammalian ANK repeat containing proteins have been shown to interact with FIH-1 and undergo asparaginyl hydroxylation. The role of asparaginyl hydroxylation of cellular ANK repeat proteins remains elusive but these observations raised the possibility that viral ANK repeat proteins might interact with FIH-1 in infected cells.
Here it is demonstrated that the five orf virus (ORFV) ANK repeat proteins ov008, ov123, ov126, ov128, and ov129 interact with endogenous FIH-1. Particularly, over-expression of ov008 affects the levels of FIH-1. During ORFV infection, binding between ov008 and FIH-1 is weak but can be enhanced by treatment with the hydroxylase inhibitor dimethyloxaloylglycine (DMOG). These results suggest that the interaction of ov008 and FIH-1 leads to the specific hydroxylation of ov008 by endogenous FIH-1 during ORFV infection. Analysis of truncated forms of ov008 revealed that the ANK repeat domain is the site of interaction between ov008 and FIH-1. The 1st and 4th ANK repeats are critical for FIH-1 recognition and binding. In addition, mutation of the asparaginyl residue of predicted binding sequence in 1st repeat of ov008 ANK repeat domain resulted in decreased FIH-1 co-precipitation. Together the results suggest the 1st ANK repeat of ov008 determines the interaction of ov008 with FIH-1 while the 4th repeat may have a role in maintain the overall structure of the ov008 ANK repeat domain.
No differences were observed in the replication of wild type ORFV or a recombinant ORFV with ov008 gene deleted. Neither the expression of ov008 nor ORFV infection leads to detectable increases in HIF-1a levels under normoxic or hypoxic conditions.
This is the first demonstration of any viral protein interacts with FIH-1. The results reveal a possible link between poxviral ANK repeat proteins and the HIF pathway. Further investigations will be required to determine if expression of ov008 has any effect on HIF activity or other biological reactions that associate with FIH-1.