title: Proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein 3.
abstract: 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.
authors: Molecular and Integrative Neurosciences Department, Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA.
authors: Benjamin W
authors: BW
authors: Neuman
authors: Jeremiah S
authors: JS
authors: Joseph
authors: Kumar S
authors: KS
authors: Saikatendu
authors: Pedro
authors: P
authors: Serrano
authors: Amarnath
authors: A
authors: Chatterjee
authors: Margaret A
authors: MA
authors: Johnson
authors: Lujian
authors: L
authors: Liao
authors: Joseph P
authors: JP
authors: Klaus
authors: John R
authors: JR
authors: Yates
authors: Kurt
authors: K
authors: Wüthrich
authors: Raymond C
authors: RC
authors: Stevens
authors: Michael J
authors: MJ
authors: Buchmeier
authors: Peter
authors: P
authors: Kuhn
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D003035:Cobalt / Q000378:metabolism; D009696:Nucleic Acids / Q000378:metabolism; D010802:Phylogeny; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D040901:Proteomics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014771:Virion / Q000235:genetics / Q000378:metabolism
chemical_list: D009696:Nucleic Acids; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C087633:nonstructural protein, coronavirus; D003035:Cobalt; D011494:Protein Kinases