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).

The severe acute respiratory syndrome coronavirus (SARS-CoV) possesses a large 29.7-kb positive-stranded RNA genome. The first open reading frame encodes replicase polyproteins 1a and 1ab, which are cleaved to generate 16 "nonstructural" proteins, nsp1 to nsp16, involved in viral replication and/or RNA processing. Among these, nsp10 plays a critical role in minus-strand RNA synthesis in a related coronavirus, murine hepatitis virus. Here, we report the crystal structure of SARS-CoV nsp10 at a resolution of 1.8 Å as determined by single-wavelength anomalous dispersion using phases derived from hexatantalum dodecabromide. nsp10 is a single domain protein consisting of a pair of antiparallel N-terminal helices stacked against an irregular ␤-sheet, a coil-rich C terminus, and two Zn fingers. nsp10 represents a novel fold and is the first structural representative of this family of Zn finger proteins found so far exclusively in coronaviruses. The first Zn finger coordinates a Zn 2؉ ion in a unique conformation. The second Zn finger, with four cysteines, is a distant member of the "gag-knuckle fold group" of Zn 2؉ -binding domains and appears to maintain the structural integrity of the C-terminal tail. A distinct clustering of basic residues on the protein surface suggests a nucleic acid-binding function. Gel shift assays indicate that in isolation, nsp10 binds single-and double-stranded RNA and DNA with high-micromolar affinity and without obvious sequence specificity. It is possible that nsp10 functions within a larger RNA-binding protein complex. However, its exact role within the replicase complex is still not clear.The severe acute respiratory syndrome coronavirus (SARSCoV) is a positive-stranded RNA virus with a large 29.7-kb genome that encodes 14 open reading frames (ORFs) (26,30). The first of these ORFs covers two-thirds of the genome and codes for the replicase polyproteins 1a and 1ab (pp1a and pp1ab) (30). pp1ab is formed by frame shifting of the ribosome into the Ϫ1 frame during translation, just prior to the pp1a stop codon (2, 30). The two polyproteins are cleaved into 16 "nonstructural" proteins, nsp1 to nsp16, by two viral cysteine proteases, a 3CL-like protease and a papain-like protease (10,21,22). These proteolytically generated proteins and/or their cleavage intermediates are involved in viral replication and/or generation of the nested subgenomic mRNAs required for expression of the downstream ORFs within the host cell (32). The remaining ORFs can be categorized into the structural proteins (those that are part of the virion) and the "accessory" proteins. Immunofluorescence microscopy studies in coronaviruses such as murine hepatitis virus (MHV) reveal that some, if not all, of the nsp proteins, including p15 (counterpart to SARS-CoV nsp10), assemble into distinct cytoplasmic, membrane-associated replicase complexes that actively perform viral RNA synthesis (21, 24).We have recently undertaken an initiative to generate a structure-function-interaction map of the entire proteome of SARS-CoV and it...

This paper describes the structure determination of nsp3a, the N-terminal domain of the severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 3. nsp3a exhibits a ubiquitin-like globular fold of residues 1 to 112 and a flexibly extended glutamic acid-rich domain of residues 113 to 183. In addition to the four ␤-strands and two ␣-helices that are common to ubiquitin-like folds, the globular domain of nsp3a contains two short helices representing a feature that has not previously been observed in these proteins. Nuclear magnetic resonance chemical shift perturbations showed that these unique structural elements are involved in interactions with single-stranded RNA. Structural similarities with proteins involved in various cell-signaling pathways indicate possible roles of nsp3a in viral infection and persistence.Severe acute respiratory syndrome (SARS) is a viral infectious disease that has attracted worldwide attention since an outbreak in 2003 (26). It has been postulated that the SARS coronavirus (SARS-CoV) was introduced to the human population from animal CoVs (26). CoVs comprise a large group of enveloped, positive-sense, single-stranded RNA viruses that have been classified in the Nidovirales order. There are three groups of CoVs, based on serological cross-reactivity and phylogenetic relatedness. The SARS-CoV is distantly related to the group 2 viruses and has been classified in group 2b (38).The SARS-CoV represents one of the largest currently known RNA genomes. It is composed of at least 14 functional open reading frames that encode three classes of proteins, i.e., structural proteins (the S, M, E, N, 3a, 7a, and 7b proteins), nonstructural proteins (nsp1 to nsp16), and the accessory proteins (3b, 6, 8, 9b, and 14) (38). With regard to the nonstructural proteins, the translation of the SARS-CoV genome produces two large replicase polyproteins (pp1a and pp1ab), which are processed by two proteases to yield 16 mature nonstructural proteins that mediate RNA replication and processing. Since the SARS outbreak in 2003, knowledge of the structure, activity and function of some of these proteins has increased considerably (30,32,35,41,45); however, the biological roles of many of the SARS-CoV proteins remain unknown. In this paper we describe the nuclear magnetic resonance (NMR) structure determination and a preliminary functional characterization of nsp3a, the N-terminal domain of the largest of the nonstructural proteins, nsp3.SARS-CoV nsp3 is a 213-kDa polypeptide involved in RNA replication and has been proposed to consist of seven domains, nsp3a to nsp3g, which have been identified based on phylogenetic conservation and predicted amino acid secondary structure (38). The biological role of nsp3 is only partially understood, and so far structures have been determined of only the two domains nsp3b, which has been described as an ADP ribose-1Љ-phosphatase (34), and nsp3d, which is a papain-like protease (PLpro) involved in the proteolytic processing of pp1a and pp1ab. nsp3d contains thr...

Mature nonstructural protein-15 (nsp15) from the severe acute respiratory syndrome coronavirus (SARSCoV) contains a novel uridylate-specific Mn 2؉ -dependent endoribonuclease (NendoU). Structure studies of the full-length form of the obligate hexameric enzyme from two CoVs, SARS-CoV and murine hepatitis virus, and its monomeric homologue, XendoU from Xenopus laevis, combined with mutagenesis studies have implicated several residues in enzymatic activity and the N-terminal domain as the major determinant of hexamerization. However, the tight link between hexamerization and enzyme activity in NendoUs has remained an enigma. Here, we report the structure of a trimmed, monomeric form of SARS-CoV nsp15 (residues 28 to 335) determined to a resolution of 2.9 Å. The catalytic loop (residues 234 to 249) with its two reactive histidines (His 234 and His 249) is dramatically flipped by ϳ120°into the active site cleft. Furthermore, the catalytic nucleophile Lys 289 points in a diametrically opposite direction, a consequence of an outward displacement of the supporting loop (residues 276 to 295). In the full-length hexameric forms, these two loops are packed against each other and are stabilized by intimate intersubunit interactions. Our results support the hypothesis that absence of an adjacent monomer due to deletion of the hexamerization domain is the most likely cause for disruption of the active site, offering a structural basis for why only the hexameric form of this enzyme is active.