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Proteomics Analysis Unravels the Functional Repertoire of Coronavirus Nonstructural Protein 3 ^,

Benjamin W. Neuman,^1,5, Jeremiah S. Joseph,^2, Kumar S. Saikatendu,² Pedro Serrano,³ Amarnath Chatterjee,³ Margaret A. Johnson,³ Lujian Liao,² Joseph P. Klaus,¹ John R. Yates III,² Kurt Wüthrich,^3,4 Raymond C. Stevens,³ Michael J. Buchmeier,^1* and Peter Kuhn^2,3*

Molecular and Integrative Neurosciences Department,¹ Department of Cell Biology,² Department of Molecular Biology,³ Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037,⁴ School of Biological Sciences, University of Reading, Whiteknights, RG6 6AJ Reading, United Kingdom⁵

Received 11 December 2007/ Accepted 16 March 2008

Severe acute respiratory syndrome (SARS) coronavirus infection and growth are dependent on initiating signaling and enzyme actions upon viral entry into the host cell. Proteins packaged during virus assembly may subsequently form the first line of attack and host manipulation upon infection. A complete characterization of virion components is therefore important to understanding the dynamics of early stages of infection. Mass spectrometry and kinase profiling techniques identified nearly 200 incorporated host and viral proteins. We used published interaction data to identify hubs of connectivity with potential significance for virion formation. Surprisingly, the hub with the most potential connections was not the viral M protein but the nonstructural protein 3 (nsp3), which is one of the novel virion components identified by mass spectrometry. Based on new experimental data and a bioinformatics analysis across the Coronaviridae, we propose a higher-resolution functional domain architecture for nsp3 that determines the interaction capacity of this protein. Using recombinant protein domains expressed in Escherichia coli, we identified two additional RNA-binding domains of nsp3. One of these domains is located within the previously described SARS-unique domain, and there is a nucleic acid chaperone-like domain located immediately downstream of the papain-like proteinase domain. We also identified a novel cysteine-coordinated metal ion-binding domain. Analyses of interdomain interactions and provisional functional annotation of the remaining, so-far-uncharacterized domains are presented. Overall, the ensemble of data surveyed here paint a more complete picture of nsp3 as a conserved component of the viral protein processing machinery, which is intimately associated with viral RNA in its role as a virion component.

Published ahead of print on 26 March 2008.

Supplemental material for this article may be found at http://jvi.asm.org/.

These authors contributed equally to this paper.