| Title:
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Phage display of the Equine arteritis virus nsp1 ZF domain and examination of its metal interactions |
| Type:
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Journal articleJournal article |
| Participant(s):
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Forfatter:
Oleksiewicz, Martin B.
Technical University of Denmark, National Veterinary Institute
Forfatter:
Snijder, E.J.
Technical University of Denmark
Technical University of Denmark
Email:
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| Abstract:
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A putative zinc finger (ZF) domain in the Equine arteritis virus (EAV) nsp 1 protein was described recently to be required for viral transcription. The nsp 1 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 Ni2+ 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-nsp 1 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 Zn2+, Ni2+ or Cu2+, but not Mg2+, 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 nsp 1 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. |
| Published:
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in journal: Journal of Virological Methods (ISSN: 0166-0934) (DOI: http://dx.doi.org/10.1016/j.jviromet.2004.04.002), vol: 119, issue: 2, pages: 159-169, 2004 |
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