Journal article
Phage display of the Equine arteritis virus nsp1 ZF domain and examination of its metal interactions
Technical University of Denmark1
Sektion for Eksotiske Virussygdomme, Division of Virology, National Veterinary Institute, Technical University of Denmark2
Division of Virology, National Veterinary Institute, Technical University of Denmark3
National Veterinary Institute, Technical University of Denmark4
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.
Language: | Chinese |
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Year: | 2004 |
Pages: | 159-169 |
ISSN: | 18790984 and 01660934 |
Types: | Journal article |
DOI: | 10.1016/j.jviromet.2004.04.002 |
IMAC Nsp1 arterivirus nickel nidovirus replicase zinc finger