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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.
Publication Date: 2004-05-26 PubMed ID: 15158598DOI: 10.1016/j.jviromet.2004.04.002Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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This study explores the functionality of a potential zinc finger domain in the Equine arteritis virus (EAV) nsp1 protein, which has been recently discovered to be essential for the virus’s transcription processes. The researchers used Phage display and a newly developed binding assay. A better understanding of this process could provide insights into virus transcription mechanisms.
Methodology and Experiment Design
- The researchers utilised the M13KE gIII phage to express the zinc finger (ZF) domain of the nsp1 protein on its surface. The ZF domain was fused to the N terminus of the phage pIII protein.
- A binding assay was created for the study. This assay was based on the usage of immobilized Ni(2+) ions, also known as Ni-NTA. The purpose of the binding assay was to evaluate the functionality of the ZF domain.
Findings and Observation
- Phages that presented the ZF domain showed significantly better binding capabilities to the Ni-NTA than phages which displayed negative-control peptides, even though they also had metal-coordinating residues.
- The binding capacity of ZF-displaying phages could be inhibited through the usage of an anti-nsp1 serum or by mutating residues deemed critical for zinc coordination.
- The researchers discovered that low levels of Tween 20 (0.1%) could abolish the binding, but when Zn(2+), Ni(2+), or Cu(2+), but not Mg(2+), were included in the binding buffer, it rescued the binding. This result suggested that the secondary structure formation was involved in the binding of the ZF to the Ni-NTA.
Conclusion and Significance
- The results obtained provided the first empirical proof that the potential nsp1 ZF domain can coordinate divalent metal ions, and that this property is related to the domain’s secondary structure.
- The Ni-NTA binding assay which was developed during this study could potentially be used in future research for the study of other ZF domains.
These findings potentially open new pathways to better understand the mechanisms of viral transcription, which could be instrumental in developing new strategies to interfere with these mechanisms and treat viral infections.
Cite This Article
APA
Oleksiewicz MB, Snijder EJ, Normann P.
(2004).
Phage display of the Equine arteritis virus nsp1 ZF domain and examination of its metal interactions.
J Virol Methods, 119(2), 159-169.
https://doi.org/10.1016/j.jviromet.2004.04.002 Publication
Researcher Affiliations
- Danish Veterinary Institute, Lindholm, 4771 Kalvehave, Denmark. mboz@novonordisk.com
MeSH Terms
- Animals
- Equartevirus / chemistry
- Equartevirus / metabolism
- Nickel / metabolism
- Peptide Library
- Protein Structure, Tertiary / physiology
- Viral Nonstructural Proteins / chemistry
- Viral Nonstructural Proteins / genetics
- Viral Nonstructural Proteins / metabolism
- Zinc Fingers / physiology
Citations
This article has been cited 2 times.- Xue F, Sun Y, Yan L, Zhao C, Chen J, Bartlam M, Li X, Lou Z, Rao Z. The crystal structure of porcine reproductive and respiratory syndrome virus nonstructural protein Nsp1beta reveals a novel metal-dependent nuclease. J Virol 2010 Jul;84(13):6461-71.
- Sun Y, Xue F, Guo Y, Ma M, Hao N, Zhang XC, Lou Z, Li X, Rao Z. Crystal structure of porcine reproductive and respiratory syndrome virus leader protease Nsp1alpha. J Virol 2009 Nov;83(21):10931-40.
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