title: Phage display of the Equine arteritis virus nsp1 ZF domain and examination of its metal interactions.
abstract: A putative zinc finger (ZF) domain in the Equine arteritis virus (EAV) nsp1 protein was described recently to be required for viral transcription. The nsp1 ZF (50 aa) was expressed on the surface of M13KE gIII phage, fused to the N terminus of the phage pIII protein. To evaluate the functionality of the ZF domain, a binding assay was developed, based on the use of immobilized Ni(2+) ions (Ni-NTA). Phages displaying ZF bound significantly better to Ni-NTA than did phages displaying negative-control peptides, which also contained metal-coordinating residues. Also, binding of ZF-displaying phages could be inhibited by an anti-nsp1 serum, or by mutation of residues predicted to be important for zinc coordination. Finally, binding was abolished by low concentrations (0.1%) Tween 20, and rescued by including Zn(2+), Ni(2+) or Cu(2+), but not Mg(2+), in the binding buffer, suggesting that formation of secondary structure was involved in binding of the ZF to Ni-NTA. These findings provide the first experimental evidence that the putative nsp1 ZF domain can coordinate divalent metal ions, and that this property is associated with the secondary structure of the domain. The Ni-NTA binding assay developed in the present study may have general applications in the study of other ZF domains.
authors.affiliation: Danish Veterinary Institute, Lindholm, 4771 Kalvehave, Denmark. mboz@novonordisk.com
authors.forename: Martin B
authors.initials: MB
authors.lastname: Oleksiewicz
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Snijder
authors.forename: P
authors.initials: P
authors.lastname: Normann
mesh_terms: D000818:Animals; D007240:Equartevirus / Q000737:chemistry / Q000378:metabolism*; D009532:Nickel / Q000378:metabolism*; D019151:Peptide Library; D017434:Protein Structure, Tertiary / Q000502:physiology; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers / Q000502:physiology*
chemical_list: D019151:Peptide Library; D017361:Viral Nonstructural Proteins; D009532:Nickel