The Structure of the RNA-Dependent RNA Polymerase of a Permutotetravirus Suggests a Link between Primer-Dependent and Primer-Independent Polymerases

Thosea asigna virus (TaV), an insect virus belonging to the Permutatetraviridae family, has a positive-sense single-stranded RNA (ssRNA) genome with two overlapping open reading frames, encoding for the replicase and capsid proteins. The particular TaV replicase includes a structurally unique RNA-dependent RNA polymerase (RdRP) with a sequence permutation in the palm sub-domain, where the active site is anchored. This non-canonical arrangement of the RdRP palm is also found in double-stranded RNA viruses of the Birnaviridae family. Both virus families also share a conserved VPg sequence motif at the polymerase N-terminus which in birnaviruses appears to be used to covalently link a fraction of the replicase molecules to the 5’-end of the genomic segments. Birnavirus VPgs are presumed to be used as primers for replication initiation. Here we have solved the crystal structure of the TaV RdRP, the first non-canonical RdRP of a ssRNA virus, in its apo- form and bound to different substrates. The enzyme arranges as a stable dimer maintained by mutual interactions between the active site cleft of one molecule and the flexible N-terminal tail of the symmetrically related RdRP. The latter, partially mimicking the RNA template backbone, is involved in regulating the polymerization activity. As expected from previous sequence-based bioinformatics predictions, the overall architecture of the TaV enzyme shows important resemblances with birnavirus polymerases. In addition, structural comparisons and biochemical analyses reveal unexpected similarities between the TaV RdRP and those of Flaviviruses. In particular, a long loop protruding from the thumb domain towards the central enzyme cavity appears to act as a platform for de novo initiation of RNA replication. Our findings strongly suggest an unexpected evolutionary relationship between the RdRPs encoded by these distant ssRNA virus groups.Work in Barcelona was supported by grant BIO2011-24333 from the Spanish Ministry of Economy and Competitiveness and by the SILVER Large Scale Collaborative Project, grant agreement number 260644, of the European Union 7th Framework. Work in Madrid was supported by grant AGL2011-24758 from the Spanish Ministry of Economy and Competitiveness. X-ray data were collected at the ESRF (Grenoble, France) within a Block Allocation Group (BAG Barcelona) and at the SLS (Villigen, Switzerland). Financial support was provided by the ESRF and SLS. DSF was supported by a pre-doctoral fellowship from fundació “La Caixa”Peer Reviewe

The T=1 capsid protein of Penicillium chrysogenum virus is formed by a repeated helix-rich core indicative of gene duplication

et al.Penicillium chrysogenum virus (PcV), a member of the Chrysoviridae family, is a double-stranded RNA (dsRNA) fungal virus with a multipartite genome, with each RNA molecule encapsidated in a separate particle. Chrysoviruses lack an extracellular route and are transmitted during sporogenesis and cell fusion. The PcV capsid, based on a T=1 lattice containing 60 subunits of the 982-amino-acid capsid protein, remains structurally undisturbed throughout the viral cycle, participates in genome metabolism, and isolates the virus genome from host defense mechanisms. Using three-dimensional cryoelectron microscopy, we determined the structure of the PcV virion at 8.0 Å resolution. The capsid protein has a high content of rod-like densities characteristic of α-helices, forming a repeated α-helical core indicative of gene duplication. Whereas the PcV capsid protein has two motifs with the same fold, most dsRNA virus capsid subunits consist of dimers of a single protein with similar folds. The spatial arrangement of the α-helical core resembles that found in the capsid protein of the L-A virus, a fungal totivirus with an undivided genome, suggesting a conserved basic fold. The encapsidated genome is organized in concentric shells; whereas the inner dsRNA shells are well defined, the outermost layer is dense due to numerous interactions with the inner capsid surface, specifically, six interacting areas per monomer. The outermost genome layer is arranged in an icosahedral cage, sufficiently well ordered to allow for modeling of an A-form dsRNA. The genome ordering might constitute a framework for dsRNA transcription at the capsid interior and/or have a structural role for capsid stability. Copyright © 2010, American Society for Microbiology. All Rights Reserved.This work was supported by grants from the Spanish Ministry of Science and Innovation (BFU 2008-02328/BMC and S-0505-Mat-0238 to J.L.C. and BIO2008-02361 to J.R.C.) and the NIH Intramural Research Program with support from the Center for Information Technology.Peer Reviewe

Human metastatic cholangiocarcinoma patient-derived xenografts and tumoroids for preclinical drug evaluation

Cholangiocarcinoma (CCA) is usually diagnosed at advanced stages, with limited therapeutic options. Preclinical models focused on unresectable metastatic CCA are necessary to develop rational treatments. Pathogenic mutations in IDH1/2, ARID1A/B, BAP1, and BRCA1/2 have been identified in 30\\%–50\\% of patients with CCA. Several types of tumor cells harboring these mutations exhibit homologous recombination deficiency (HRD) phenotype with enhanced sensitivity to PARP inhibitors (PARPi). However, PARPi treatment has not yet been tested for effectiveness in patient-derived models of advanced CCA.We have established a collection of patient-derived xenografts from patients with unresectable metastatic CCA (CCA\_PDX). The CCA\_PDXs were characterized at both histopathologic and genomic levels. We optimized a protocol to generate CCA tumoroids from CCA\_PDXs. We tested the effects of PARPis in both CCA tumoroids and CCA\_PDXs. Finally, we used the RAD51 assay to evaluate the HRD status of CCA tissues.This collection of CCA\_PDXs recapitulates the histopathologic and molecular features of their original tumors. PARPi treatments inhibited the growth of CCA tumoroids and CCA\_PDXs with pathogenic mutations of BRCA2, but not those with mutations of IDH1, ARID1A, or BAP1. In line with these findings, only CCA\_PDX and CCA patient biopsy samples with mutations of BRCA2 showed RAD51 scores compatible with HRD.Our results suggest that patients with advanced CCA with pathogenic mutations of BRCA2, but not those with mutations of IDH1, ARID1A, or BAP1, are likely to benefit from PARPi therapy. This collection of CCA\_PDXs provides new opportunities for evaluating drug response and prioritizing clinical trials.The authors would like to thank the patients and their families for their support. This work was supported by grants from the Fundaci o Marat o TV3 awarded to T. Macarulla, M. Mel e, and S. Peir o; BeiGene research grant awarded toT. Macarulla and S. Peir o; AECC (INVES20036TIAN), Ram on y Cajal investigator program (RYC2020-029098-I), Proyecto de IþDþi (PID2019-108008RJ-I00), and FERO Foundation grant awarded to T.V. Tian; Proyecto de Investigaci on en Salud from the Instituto de Salud Carlos III (ISCIII) (PI20/00898) awarded to T. Macarulla; FIS/FEDER from the Instituto de Salud Carlos III (ISCIII) (PI12/01250; CP08/00223; PI16/00253 and CB16/12/00449) awarded to S. Peir o; and Ram on y Cajal investigator program (RYC-2017-22249) awarded to M. Mel e. Q. Serra-Camprubí is a recipient of the Ph.D. fellowship from La Caixa Foundation (LCF/PR/PR12/51070001). A. LlopGuevara was supported by the AECC (INVES20095LLOP) and V. Serra by the ISCIII (CPII19/00033). E.J. Arenas was funded by the AECC (POSTD211413AREN).J. Arribas is funded by the Instituto de Salud Carlos III (AC15/00062, CB16/12/00449, and PI22/00001). This publication is based upon the work of COST Action CA18122, European Cholangiocarcinoma Network, supported by the COST (European Cooperation in Science and Technology, www.cost.eu), a funding agency for research and innovation networks. The authors would like to thank Dr. V.A. Raker for manuscript editing and Drs. N. Herranz and J. Mateo for scientific discussions. The authors acknowledge the infrastructure and support of the FERO Foundation, La Caixa Foundation, and the Cellex Foundation.Peer Reviewed"Article signat per 31 autors/es: Queralt Serra-Camprubí; Helena Verdaguer; Winona Oliveros; Núria Lupión-Garcia; Núria Lupión-Garcia;Alba Llop-Guevara; Cristina Molina; Maria Vila-Casadesús; Anthony Turpin; Cindy Neuzillet; Joan Frigola; Jessica Querol; Mariana Yáñez-Bartolomé; Florian Castet; Carles Fabregat-Franco; Carmen Escudero-Iriarte; Marta Escorihuela; Enrique J. Arenas; Cristina Bernadó-Morales; Noemí Haro; Francis J. Giles; Óscar J. Pozo; Josep M. Miquel ; Paolo G. Nuciforo; Ana Vivancos; Marta Melé; Violeta Serra ; Joaquín Arribas; Josep Tabernero; Sandra Peiró; Teresa Macarulla; Tian V. Tian"Postprint (published version

The structure of a protein primer-polymerase complex in the initiation of genome replication

Picornavirus RNA replication is initiated by the covalent attachment of a UMP molecule to the hydroxyl group of a tyrosine in the terminal protein VPg. This reaction is carried out by the viral RNA-dependent RNA polymerase (3D). Here, we report the X-ray structure of two complexes between foot-and-mouth disease virus 3D, VPg1, the substrate UTP and divalent cations, in the absence and in the presence of an oligoadenylate of 10 residues. In both complexes, VPg fits the RNA binding cleft of the polymerase and projects the key residue Tyr3 into the active site of 3D. This is achieved by multiple interactions with residues of motif F and helix α8 of the fingers domain and helix α13 of the thumb domain of the polymerase. The complex obtained in the presence of the oligoadenylate showed the product of the VPg uridylylation (VPg-UMP). Two metal ions and the catalytic aspartic acids of the polymerase active site, together with the basic residues of motif F, have been identified as participating in the priming reaction. © 2006 European Molecular Biology Organization | All Rights Reserved.Work in Barcelona was supported by Grants BIO2002-00517 and BFU2005-02376/BMC. Work in Madrid by Grants BMC 2001.1823.C02-01, BFU2005-00863/BMC and Fundación R Areces. CF and AA were supported by I3P fellowships from Ministerio de Educación y Ciencia. RA was supported by an FPI fellowship from Comunidad de Madrid. The financial support was provided by the ESRFPeer Reviewe

Additional binding sites for anionic phospholipids and calcium ions in the crystal structures of complexes of the C2 domain of protein kinase Cα

The C2 domain of protein kinase Cα (PKCα) corresponds to the regulatory sequence motif, found in a large variety of membrane trafficking and signal transduction proteins, that mediates the recruitment of proteins by phospholipid membranes. In the PKCα isoenzyme, the Ca2+-dependent binding to membranes is highly specific to 1,2-sn-phosphatidyl-L-serine. Intrinsic Ca2+ binding tends to be of low affinity and non-cooperative, while phospholipid membranes enhance the overall affinity of Ca2+ and convert it into cooperative binding. The crystal structure of a ternary complex of the PKCα-C2 domain showed the binding of two calcium ions and of one 1,2-dicaproyl-sn-phosphatidyl-L-serine (DCPS) molecule that was coordinated directly to one of the calcium ions. The structures of the C2 domain of PKCα crystallised in the presence of Ca2+ with either 1,2-diacetyl-sn-phosphatidyl-L-serine (DAPS) or 1,2-dicaproyl-sn-phosphatidic acid (DCPA) have now been determined and refined at 1.9 Å and at 2.0 Å, respectively. DAPS, a phospholipid with short hydrocarbon chains, was expected to facilitate the accommodation of the phospholipid ligand inside the Ca2+-binding pocket. DCPA, with a phosphatidic acid (PA) head group, was used to investigate the preference for phospholipids with phosphatidyl-L-serine (PS) head groups. The two structures determined show the presence of an additional binding site for anionic phospholipids in the vicinity of the conserved lysine-rich cluster. Site-directed mutagenesis, on the lysine residues from this cluster that interact directly with the phospholipid, revealed a substantial decrease in C2 domain binding to vesicles when concentrations of either PS or PA were increased in the absence of Ca2+. In the complex of the C2 domain with DAPS a third Ca2+, which binds an extra phosphate group, was identified in the calcium-binding regions (CBRs). The interplay between calcium ions and phosphate groups or phospholipid molecules in the C2 domain of PKCα is supported by the specificity and spatial organisation of the binding sites in the domain and by the variable occupancies of ligands found in the different crystal structures. Implications for PKCα activity of these structural results, in particular at the level of the binding affinity of the C2 domain to membranes, are discussed. © 2002 Elsevier Science Ltd. All rights reserved.This research was supported by grants PB98-0389 to the Universidad de Murcia, and BIO099-0865 to the IBMB and by 1FD97-1558 from DGESIC (Spain) to a collaborative project between the Universidad de Murcia and the IBMB. Data were collected at the EMBL protein crystallography beamlines at ESRF (Grenoble) within a Block Allocation Group (BAG Barcelona), as at ESRF BM14. This work was supported financially by the ESRF and by grant HPRI-CT-1999-00022 of the European Union.Peer Reviewe

Ubiquitination mediates Kv1.3 endocytosis as a mechanism for protein Kinase C-dependent modulation

The voltage-dependent potassium channel Kv1.3 plays essential physiological functions in the immune system. Kv1.3, regulating the membrane potential, facilitates downstream Ca2+ -dependent pathways and becomes concentrated in specific membrane microdomains that serve as signaling platforms. Increased and/or delocalized expression of the channel is observed at the onset of several autoimmune diseases. In this work, we show that adenosine (ADO), which is a potent endogenous modulator, stimulates PKC, thereby causing immunosuppression. PKC activation triggers down-regulation of Kv1.3 by inducing a clathrin-mediated endocytic event that targets the channel to lysosomal-degradative compartments. Therefore, the abundance of Kv1.3 at the cell surface decreases, which is clearly compatible with an effective anti-inflammatory response. This mechanism requires ubiquitination of Kv1.3, catalyzed by the E3 ubiquitin-ligase Nedd4-2. Postsynaptic density protein 95 (PSD-95), a member of the MAGUK family, recruits Kv1.3 into lipid-raft microdomains and protects the channel against ubiquitination and endocytosis. Therefore, the Kv1.3/PSD-95 association fine-tunes the anti-inflammatory response in leukocytes. Because Kv1.3 is a promising multi-therapeutic target against human pathologies, our results have physiological relevance. In addition, this work elucidates the ADO-dependent PKC-mediated molecular mechanism that triggers immunomodulation by targeting Kv1.3 in leukocytes

Structure of the complex of an Fab fragment of a neutralizing antibody with foot-and-mouth disease virus: Positioning of a highly mobile antigenic loop

Data from cryo-electron microscopy and X-ray crystallography have been combined to study the interactions of foot-and-mouth disease virus serotype C (FMDV-C) with a strongly neutralizing monoclonal antibody (mAb) SD6. The mAb SD6 binds to the long flexible GH-loop of viral protein 1 (VP1) which also binds to an integrin receptor. The structure of the virus-Fab complex was determined to 30 Å resolution using cryo-electron microscopy and image analysis. The known structure of FMDV-C, and of the SD6 Fab co-crystallized with a synthetic peptide corresponding to the GH-loop of VP1, were fitted to the cryo-electron microscope density map. The SD6 Fab is seen to project almost radially from the viral surface in an orientation which is only compatible with monovalent binding of the mAb. Even taking into account the mAb hinge and elbow flexibility, it is not possible to model bivalent binding without severely distorting the Fabs. The bound GH-loop is essentially in what has previously been termed the 'up' position in the best fit Fab orientation. The SD6 Fab interacts almost exclusively with the GH-loop of VP1, making very few other contacts with the viral capsid. The position and orientation of the SD6 Fab bound to FMDV-C is in accord with previous immunogenic data.Peer Reviewe

El ritual funerari a la necròpolis del Coll del Moro (Gandesa, Terra Alta)

La necròpolis del Coll del Moro es troba a uns 5 km de Gandesa, situada en uns turons planers que formen una carena al llarg de la qual transcorre la carretera nacional 420, de Tarragona a Alcolea del Pinar. No es tracta d'una única àrea d'enterrament, sinó aixovars d'enterrament, que es reparteix en tres sectors (Calars, Camp Teuler i les Maries), situats en zones boscoses que trobem entre les extenses vinyes típiques de la comarca que ens ocupa