Replication of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) requires proteolytic processing of the replicase polyprotein by two viral cysteine proteases, a chymotrypsin-like protease (3CLpro) and a papain-like protease (PLpro). These proteases are important targets for development of antiviral drugs that would inhibit viral replication and reduce mortality associated with outbreaks of SARS-CoV. In this work, we describe the 1.85-Å crystal structure of the catalytic core of SARS-CoV PLpro and show that the overall architecture adopts a fold closely resembling that of known deubiquitinating enzymes. Key features, however, distinguish PLpro from characterized deubiquitinating enzymes, including an intact zinc-binding motif, an unobstructed catalytically competent active site, and the presence of an intriguing, ubiquitinlike N-terminal domain. To gain insight into the active-site recognition of the C-terminal tail of ubiquitin and the related LXGG motif, we propose a model of PLpro in complex with ubiquitinaldehyde that reveals well defined sites within the catalytic cleft that help to account for strict substrate-recognition motifs.membrane-associated protease ͉ ubiquitin-like domain

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...

The crystal structure of a conserved domain of nonstructural protein 3 (nsP3) from severe acute respiratory syndrome coronavirus (SARS-CoV) has been solved by single-wavelength anomalous dispersion to 1.4 A resolution. The structure of this "X" domain, seen in many single-stranded RNA viruses, reveals a three-layered alpha/beta/alpha core with a macro-H2A-like fold. The putative active site is a solvent-exposed cleft that is conserved in its three structural homologs, yeast Ymx7, Archeoglobus fulgidus AF1521, and Er58 from E. coli. Its sequence is similar to yeast YBR022W (also known as Poa1P), a known phosphatase that acts on ADP-ribose-1''-phosphate (Appr-1''-p). The SARS nsP3 domain readily removes the 1'' phosphate group from Appr-1''-p in in vitro assays, confirming its phosphatase activity. Sequence and structure comparison of all known macro-H2A domains combined with available functional data suggests that proteins of this superfamily form an emerging group of nucleotide phosphatases that dephosphorylate Appr-1''-p.

Protein kinases are highly tractable targets for drug discovery. However, the biological function and therapeutic potential of the majority of the 500+ human protein kinases remains unknown. We have developed physical and virtual collections of small molecule inhibitors, which we call chemogenomic sets, that are designed to inhibit the catalytic function of almost half the human protein kinases. In this manuscript we share our progress towards generation of a comprehensive kinase chemogenomic set (KCGS), release kinome profiling data of a large inhibitor set (Published Kinase Inhibitor Set 2 (PKIS2)), and outline a process through which the community can openly collaborate to create a KCGS that probes the full complement of human protein kinases.

Infection by severe acute respiratory syndrome coronavirus (SARS-CoV) is initiated by the recognition of ACE-2 receptor on the surface of respiratory epithelial cells by the "spike" glycoprotein present on the viral surface (27,29,34). Subsequent progression of infection involves a series of complex, tightly regulated processes that begin by the entry of genomic RNA into the cytosol and culminate with the budding of infectious progeny (14, 15). These mature, fully formed virions are functionally as well as morphologically indistinguishable from their parents and have a quasi-fluid-like, pleomorphic, bilipid envelope whose surface is studded with three main structural transmembrane proteins: the matrix (M), the small envelope (E), and the trimeric spike (S) glycoproteins (16,40,54). The envelopes of these particles encase the ϳ29-kb genomic RNA that is thought to be organized as a helical filamentous ribonucleoprotein (RNP) complex. Several copies of the N protein self-associate and form a template for binding RNA during nucleocapsid formation (13,16,18,35,61). As noted in studies done using murine hepatitis virus (MHV), the initial steps of virus assembly, including the formation of the RNP complex and its eventual packaging into the virion lumen, occurs in a temporally regulated manner, mainly at the endoplasmic reticulum-Golgi intermediate compartments just prior to budding (1,8,22,55). Successful targeting of the RNP into the virion lumen is thought to be facilitated by its anchoring onto the membrane-embedded M protein by specific interaction between their respective C-terminal tails (10,23,32,39,56). Despite extensive studies on several model coronaviruses spanning 25 years, our structural understanding of these assembly events remains sketchy (5,7,8,15,24,34).SARS-CoV N protein is translated from the smallest of the eight subgenomic RNAs (the bicistronic sg-mRNA 9) (15,26,54) that spans the genomic 3Ј-most open reading frame, ORF9a (Fig. 1a). Coronaviral N proteins are typically ca. 45 to 50 kDa, very basic (with typical pIs of ϳ10), prone to aggregate into large homopolymers (16), phosphorylated at multiple sites (3, 50, 58), and extremely labile to proteolytic degradation (39,57,61). These characteristics have hindered in vitro structural studies on full-length N. The N-terminal domains of coronaviral N proteins (N-NTDs) typically share about 30 to 40% sequence identity (Fig. 1c). As in most nidoviruses, the fulllength SARS-CoV N protein (430 residues) has three main protein domains: an N-terminal RNA-binding domain (i.e., the N-NTD), a poorly structured central serine-rich region that is thought to house the primary sites of phosphorylation (33,58), and a C-terminal domain (N-CTD [52]) that is mainly involved in oligomerization and self-association (4; Fig. 1b). In addition, a few coronaviruses have about 20 residues upstream of the NTD that are rich in serine, glycine, and arginine (SRG motif; Fig. 1b). N protein is also known to undergo sumoylation (28).