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.

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).

Pladienolide, herboxidiene and spliceostatin have been identified as splicing modulators that target SF3B1 in the SF3b subcomplex. Here we report that PHF5A, another component of this subcomplex, is also targeted by these compounds. Mutations in PHF5A-Y36, SF3B1-K1071, SF3B1-R1074 and SF3B1-V1078 confer resistance to these modulators, suggesting a common interaction site. RNA-seq analysis reveals that PHF5A-Y36C has minimal effect on basal splicing but inhibits the global action of splicing modulators. Moreover, PHF5A-Y36C alters splicing modulator-induced intron-retention/exon-skipping profile, which correlates with the differential GC content between adjacent introns and exons. We determine the crystal structure of human PHF5A demonstrating that Y36 is located on a highly conserved surface. Analysis of the cryo-EM spliceosome Bact complex shows that the resistance mutations cluster in a pocket surrounding the branch point adenosine, suggesting a competitive mode of action. Collectively, we propose that PHF5A–SF3B1 forms a central node for binding to these splicing modulators.

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...

Muscle-invasive bladder cancer (MIBC) is an aggressive disease with limited therapeutic options. Although immunotherapies are approved for MIBC, the majority of patients fail to respond, suggesting existence of complementary immune evasion mechanisms. Here, we report that the PPARγ/RXRα pathway constitutes a tumor-intrinsic mechanism underlying immune evasion in MIBC. Recurrent mutations in RXRα at serine 427 (S427F/Y), through conformational activation of the PPARγ/RXRα heterodimer, and focal amplification/overexpression of PPARγ converge to modulate PPARγ/RXRα-dependent transcription programs. Immune cell-infiltration is controlled by activated PPARγ/RXRα that inhibits expression/secretion of inflammatory cytokines. Clinical data sets and an in vivo tumor model indicate that PPARγHigh/RXRαS427F/Y impairs CD8+ T-cell infiltration and confers partial resistance to immunotherapies. Knockdown of PPARγ or RXRα and pharmacological inhibition of PPARγ significantly increase cytokine expression suggesting therapeutic approaches to reviving immunosurveillance and sensitivity to immunotherapies. Our study reveals a class of tumor cell-intrinsic “immuno-oncogenes” that modulate the immune microenvironment of cancer.

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.

Activation of the fibroblast growth factor receptor FGFR4 by FGF19 drives hepatocellular carcinoma (HCC), a disease with few, if any, effective treatment options. While a number of pan-FGFR inhibitors are being clinically evaluated, their application to FGF19-driven HCC may be limited by dose-limiting toxicities mediated by FGFR1-3 receptors. To evade the potential limitations of pan-FGFR inhibitors, we generated H3B-6527, a highly selective covalent FGFR4 inhibitor, through structure-guided drug design. Studies in a panel of 40 HCC cell lines and 30 HCC PDX models showed that FGF19 expression is a predictive biomarker for H3B-6527 response. Moreover, coadministration of the CDK4/6 inhibitor palbociclib in combination with H3B-6527 could effectively trigger tumor regression in a xenograft model of HCC. Overall, our results offer preclinical proof of concept for H3B-6527 as a candidate therapeutic agent for HCC cases that exhibit increased expression of FGF19. .