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.The severe acute respiratory syndrome coronavirus (SARSCoV) is an enveloped virus with a 29.7-kb positive-strand RNA genome (35). Replication of this genome and transcription are mediated by a large membrane-anchored RNA processing complex. Components of this complex are derived from the 16 nonstructural proteins (nsp1 to nsp16) that are processed from the open reading frame 1a (ORF1a) and ORF1b. The polyprotein 1a (pp1a) is translated from ORF1a, while the polyprotein 1ab (pp1ab) is formed by a Ϫ1 ribosomal frameshift upstream of the ORF1a stop codon, causing read-through into ORF1b. SARS-CoV encodes two proteinases, a "main proteinase" (nsp5) and a papain-like proteinase (PL2 pro domain of nsp3). These two proteins proteolytically cleave pp1a and pp1ab into the 16 mature nsp's (61). Specifically, SARS-CoV PL2 pro cleaves pp1a at the three sites 177 LNGG 2 AVT 183 , 815 LKGG 2 API 821 , and 2737 LKGG 2 KIV 2743 to release nsp1, nsp2, and nsp3, respectively.In current coronavirus terminology, the term "nonstructural protein" usually refers to peptides processed from pp1a and pp1ab, while "structural protein" refers to the N, M, S, and E proteins, which interact to coordinate the structure of the virion lipi...

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