Kuhn Lab at The Scripps Research Institute.

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.