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We report that ribavirin exerts an inhibitory and mutagenic activity on SARS-CoV-2-infecting Vero cells, with a therapeutic index higher than 10. Deep sequencing analysis of the mutant spectrum of SARS-CoV-2 replicating in the absence or presence of ribavirin indicated an increase in the number of mutations, but not in deletions, and modification of diversity indices, expected from a mutagenic activity. Notably, the major mutation types enhanced by replication in the presence of ribavirin were A→G and U→C transitions, a pattern which is opposite to the dominance of G→A and C→U transitions previously described for most RNA viruses. Implications of the inhibitory activity of ribavirin, and the atypical mutational bias produced on SARS-CoV-2, for the search for synergistic anti-COVID-19 lethal mutagen combinations are discussed.

Sterols are unevenly distributed within cellular membranes. How their biosynthetic and transport machineries are organized to generate heterogeneity is largely unknown. We previously showed that the yeast sterol transporter Osh2 is recruited to endoplasmic reticulum (ER)–endocytic contacts to facilitate actin polymerization. We now find that a subset of sterol biosynthetic enzymes also localizes at these contacts and interacts with Osh2 and the endocytic machinery. Following the sterol dynamics, we show that Osh2 extracts sterols from these subdomains, which we name ERSESs (ER sterol exit sites). Further, we demonstrate that coupling of the sterol synthesis and transport machineries is required for endocytosis in mother cells, but not in daughters, where plasma membrane loading with accessible sterols and endocytosis are linked to secretion.

Despite the high virological response rates achieved with current directly-acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. Identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultra-deep sequencing (UDS) methods for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV from 220 patients who failed therapy, which are not bona fide resistance-associated substitutions (RAS). They were present frequently in basal and post-treatment virus of patients who failed therapy to different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Coherently, their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. Also, they have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value of treatment response are discussed.

Despite the high virological response rates achieved with current directly acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. The identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultradeep sequencing (UDS) methods, for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV, which are not bona fide resistance-associated substitutions (RAS), from 220 patients who failed therapy. They were present frequently in basal and posttreatment virus of patients who failed different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. They also have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value for treatment response, are discussed., The work at CBMSO was supported by grants SAF2014-52400-R from Ministerio de Economía y Competitividad (MINECO), SAF2017-87846-R and BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MICIU), PI18/00210 from Instituto de Salud Carlos III, S2013/ABI-2906 (PLATESA from Comunidad de Madrid/FEDER), and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/00121 and CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). The work in Barcelona was supported by Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF) grant number PI19/00301 and by the Centro para el Desarrollo Tecnológico Industrial (CDTI) from the MICIU, grant number IDI-20151125. Work at CAB was supported by MINECO grant BIO2016-79618R and PID2019-104903RB-I00 (funded by the EU under the FEDER program) and by the Spanish State research agency (AEI) through project number MDM-2017-0737 Unidad de Excelencia “María de Maeztu”-Centro de Astrobiología (CSIC-INTA). Work at IBMB was supported by MICIN grant BIO2017-83906-P (funded by the EU under the FEDER program). C.G.-C. is supported by predoctoral contract PRE2018-083422 from MICIU. B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo), cofinanced by Fondo Social Europeo (FSE).

The Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of proteinprotein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided in trans (i.e., via expression from a separate RNA molecule), while others are required in cis (i.e., expressed from the template RNA molecule). In vitro studies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymatic cis-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that this cis-acting role of 3D is distinct from the catalytic activity, which is predominantly trans acting. Immunofluorescence studies suggest that both cis-and trans-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts in cis with RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further., This work, including the efforts of Eleni-Anna Loundras and Joseph Ward, was funded by Biotechnology and Biological Sciences Research Council (BBSRC) (BB/F01614X/1). This work, including the efforts of Morgan R. Herod, David J. Rowlands, and Nicola J. Stonehouse, was funded by Biotechnology and Biological Sciences Research Council (BBSRC) (BB/K003801/1). This work, including the efforts of Nuria Verdaguer, was funded by Ministerio de Economía y Competitividad (MINECO) (BIO2014-54588-P). This work, including the efforts of Cristina Ferrer-Orta, was funded by Ministerio de Economía y Competitividad (MINECO) (MDM-2014-0435).

Most emerging and re-emerging human and animal viral diseases are associated with RNA viruses. All these pathogens, with the exception of retroviruses, encode a specialized enzyme called RNA-dependent RNA polymerase (RdRP), which catalyze phosphodiester-bond formation between ribonucleotides (NTPs) in an RNA template-dependent manner. These enzymes function either as single polypeptides or in complex with other viral or host components to transcribe and replicate the viral RNA genome. The structures of RdRPs and RdRP catalytic complexes, currently available for several members of (+) ssRNA, (-)ssRNA and dsRNA virus families, have provided high resolution snapshots of the functional steps underlying replication and transcription of viral RNA genomes and their regulatory mechanisms.

Most emerging and re-emerging human and animal viral diseases are associated with RNA viruses. All these pathogens, with the exception of retroviruses, encode a specialized enzyme called RNA-dependent RNA polymerase (RdRP), which catalyze phosphodiester-bond formation between ribonucleotides (NTPs) in an RNA template-dependent manner. These enzymes function either as single polypeptides or in complex with other viral or host components to transcribe and replicate the viral RNA genome. The structures of RdRPs and RdRP catalytic complexes, currently available for several members of (+) ssRNA, (-)ssRNA and dsRNA virus families, have provided high resolution snapshots of the functional steps underlying replication and transcription of viral RNA genomes and their regulatory mechanisms.

Viral RNA-dependent RNA polymerases (RdRps) are major determinants of high mutation rates and generation of mutant spectra that mediate RNA virus adaptability. The RdRp of the picornavirus foot-and-mouth disease virus (FMDV), termed 3D, is a multifunctional protein that includes a nuclear localization signal (NLS) in its N-terminal region. Previous studies documented that some amino acid substitutions within the NLS altered nucleotide recognition and enhanced the incorporation of the mutagenic purine analogue ribavirin in viral RNA, but the mutants tested were not viable and their response to lethal mutagenesis could not be studied. Here we demonstrate that NLS amino acid substitution M16A of FMDV serotype C does not affect infectious virus production but accelerates ribavirin-mediated virus extinction. The mutant 3D displays polymerase activity, RNA binding, and copying processivity that are similar to those of the wild-type enzyme but shows increased ribavirin-triphosphate incorporation. Crystal structures of the mutant 3D in the apo and RNA-bound forms reveal an expansion of the template entry channel due to the replacement of the bulky Met by Ala. This is a major difference with other 3D mutants with altered nucleotide analogue recognition. Remarkably, two distinct loop β9-α11 conformations distinguish 3Ds that exhibit higher or lower ribavirin incorporation than the wild-type enzyme. This difference identifies a specific molecular determinant of ribavirin sensitivity of FMDV. Comparison of several polymerase mutants indicates that different domains of the molecule can modify nucleotide recognition and response to lethal mutagenesis. The connection of this observation with current views on quasispecies adaptability is discussed., Work in Madrid was supported by grants BFU2011-23604, SAF2014-52400-R, and AGL2014-52395-C2-1-R from the Spanish MINECO and S2013/ABI-2906 (PLATESA from Comunidad de Madrid/FEDER). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Work in Barcelona was supported by grants BIO2017-83906-P and Maria de Maeztu Unit of Excellence MDM-2014-0435, from the Spanish MINECO. X-ray data were collected at ALBA Synchrotron (Cerdanyola de Valles, Spain) XALOC beamline with the collaboration of ALBA staff and at ESRF beamline ID29 (Grenoble, France). Financial support was provided by ALBA and ESRF. The kinetic studies at the University of Missouri were partially supported by grant AI076119 (National Institutes of Health, USA) to S.G.S. C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/ 00121) cofinanced by the European Regional Development Fund (ERDF). Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged.

© 2015 by the author; licensee MDPI, Basel, Switzerland. RNA viruses typically encode their own RNA-dependent RNA polymerase (RdRP) to ensure genome replication within the infected cells. RdRP function is critical not only for the virus life cycle but also for its adaptive potential. The combination of low fidelity of replication and the absence of proofreading and excision activities within the RdRPs result in high mutation frequencies that allow these viruses a rapid adaptation to changing environments. In this review, we summarize the current knowledge about structural and functional aspects on RdRP catalytic complexes, focused mainly in the Picornaviridae family. The structural data currently available from these viruses provided high-resolution snapshots for a range of conformational states associated to RNA template-primer binding, rNTP recognition, catalysis and chain translocation. As these enzymes are major targets for the development of antiviral compounds, such structural information is essential for the design of new therapies., Núria Verdaguer acknowledges funding from the Spanish Ministry of Economy and Competitiveness (BIO2011-24333). We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

Membrane fusion is essential in a myriad of eukaryotic cell biological processes, including the synaptic transmission. Rabphilin-3A is a membrane trafficking protein involved in the calcium-dependent regulation of secretory vesicle exocytosis in neurons and neuroendocrine cells, but the underlying mechanism remains poorly understood. Here, we report the crystal structures and biochemical analyses of Rabphilin-3A C2B–SNAP25 and C2B–phosphatidylinositol 4,5-bisphosphate (PIP2) complexes, revealing how Rabphilin-3A C2 domains operate in cooperation with PIP2/Ca2+ and SNAP25 to bind the plasma membrane, adopting a conformation compatible to interact with the complete SNARE complex. Comparisons with the synaptotagmin1–SNARE show that both proteins contact the same SNAP25 surface, but Rabphilin-3A uses a unique structural element. Data obtained here suggest a model to explain the Ca2+-dependent fusion process by membrane bending with a myriad of variations depending on the properties of the C2 domain-bearing protein, shedding light to understand the fine-tuning control of the different vesicle fusion events., Work in Barcelona was supported by Grants BIO2014-54588-P [Ministry of Economy and Competitiveness (MINECO), Spain–European Fund for Economic and Regional Development (FEDER)] and Maria de Maeztu Unit of Excellence MDM-2014-0435. Work in Murcia was supported by Grants BFU2014-52269-P (MINECO, Spain–FEDER) and Fundación Séneca Region de Murcia 19409/PI/14. X-ray data were collected at ALBA-CELLS (beamline XALOC) (Cerdanyola del Valles, Barcelona, Spain) with the collaboration of ALBA staff and at ESRF beamline ID23.2 (Grenoble, France). Financial support was also provided by ALBA and ESRF.

The selective pressures acting on viruses that replicate under enhanced mutation rates are largely unknown. Here, we describe resistance of foot-and-mouth disease virus to the mutagen 5-fluorouracil (FU) through a single polymerase substitution that prevents an excess of A to G and U to C transitions evoked by FU on the wild-type foot-and-mouth disease virus, while maintaining the same level of mutant spectrum complexity. The polymerase substitution inflicts upon the virus a fitness loss during replication in absence of FU but confers a fitness gain in presence of FU. The compensation of mutational bias was documented by in vitro nucleotide incorporation assays, and it was associated with structural modifications at the N-terminal region and motif B of the viral polymerase. Predictions of the effect of mutations that increase the frequency of G and C in the viral genome and encoded polymerase suggest multiple points in the virus life cycle where the mutational bias in favor of G and C may be detrimental. Application of predictive algorithms suggests adverse effects of the FU-directed mutational bias on protein stability. The results reinforce modulation of nucleotide incorporation as a lethal mutagenesis-escape mechanism (that permits eluding virus extinction despite replication in the presence of a mutagenic agent) and suggest that mutational bias can be a target of selection during virus replication, Work in Madrid was supported by grants [BFU2011-23604, SAF2014-52400-R, S2013/ABI2906 (PLATESA from Comunidad de Madrid/FEDER)] and Fundacion R. Areces and grant BFU2012-40020 to U.B. CIBERehd is funded by Instituto de Salud Carlos III. Work in Barcelona was supported by grants [BIO2011-24333, BIO2014-54588-P and MDM-2014-0435 from the Spanish MINECO]. X-ray data were collected at ALBA Synchrotron (Cerdanyola de Valles, Spain) XALOC beamline with the collaboration of ALBA staff. Financial support was provided by ALBA. C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/00121) cofinanced by the European Regional Development Fund (ERDF). The kinetic studies at the University of Missouri were partially supported by grant [AI076119] (National Institute of Health, USA) to S.G.S.

Foot-and-mouth disease virus (FMDV) is an RNA virus belonging to the Picornaviridae family that contains three small viral proteins (VPgs), named VPg1, VPg2 and VPg3, linked to the 5&prime, end of the viral genome. These VPg proteins act as primers for RNA replication, which is initiated by the consecutive binding of two UMP molecules to the hydroxyl group of Tyr3 in VPg. This process, termed uridylylation, is catalyzed by the viral RNA-dependent RNA polymerase named 3Dpol. 5-Fluorouridine triphosphate (FUTP) is a potent competitive inhibitor of VPg uridylylation. Peptide analysis showed FUMP covalently linked to the Tyr3 of VPg. This fluorouridylylation prevents further incorporation of the second UMP residue. The molecular basis of how the incorporated FUMP blocks the incorporation of the second UMP is still unknown. To investigate the mechanism of inhibition of VPg uridylylation by FUMP, we have prepared a simplified 15-mer model of VPg1 containing FUMP and studied its x-ray crystal structure in complex with 3Dpol. Unfortunately, the fluorouridylylated VPg1 was disordered and not visible in the electron density maps, however, the structure of 3Dpol in the presence of VPg1-FUMP showed an 8 &Aring, movement of the &beta, 9-&alpha, 11 loop of the polymerase towards the active site cavity relative to the complex of 3Dpol with VPg1-UMP. The conformational rearrangement of this loop preceding the 3Dpol B motif seems to block the access of the template nucleotide to the catalytic cavity. This result may be useful in the design of new antivirals against not only FMDV but also other picornaviruses, since all members of this family require the uridylylation of their VPg proteins to initiate the viral RNA synthesis.

Increasing amounts of data show that conformational dynamics are essential for protein function. Unveiling the mechanisms by which this flexibility affects the activity of a given enzyme and how it is controlled by other effectors opens the door to the design of a new generation of highly specific drugs. Viral RNA-dependent RNA polymerases (RdRPs) are not an exception. These enzymes, essential for the multiplication of all RNA viruses, catalyze the formation of phosphodiester bonds between ribonucleotides in an RNA-template-dependent fashion. Inhibition of RdRP activity will prevent genome replication and virus multiplication. Thus, RdRPs, like the reverse transcriptase of retroviruses, are validated targets for the development of antiviral therapeutics. X-ray crystallography of RdRPs trapped in multiple steps throughout the catalytic process, together with NMR data and molecular dynamics simulations, have shown that all polymerase regions contributing to conserved motifs required for substrate binding, catalysis and product release are highly flexible and some of them are predicted to display correlated motions. All these dynamic elements can be modulated by external effectors, which appear as useful tools for the development of effective allosteric inhibitors that block or disturb the flexibility of these enzymes, ultimately impeding their function. Among all movements observed, motif B, and the B-loop at its N-terminus in particular, appears as a new potential druggable site. © 2013 Elsevier Ltd. All rights reserved., Primary research described in this review was supported by grants BIO2008-02556 and BIO2011-24333, from the Spanish Ministerio de Ciencia e Innovacion and Ministerio de Economía y Competitividad, respectively, and by the SILVER Cooperation project GA No. 260644 of the European Union, Seventh Framework Program.

© 2015, American Society for Microbiology. The N-terminal region of the foot-and-mouth disease virus (FMDV) 3D polymerase contains the sequence MRKTKLAPT (residues 16 to 24) that acts as a nuclear localization signal. A previous study showed that substitutions K18E and K20E diminished the transport to the nucleus of 3D and 3CD and severely impaired virus infectivity. These residues have also been implicated in template binding, as seen in the crystal structures of different 3D-RNA elongation complexes. Here, we report the biochemical and structural characterization of different mutant polymerases harboring substitutions at residues 18 and 20, in particular, K18E, K18A, K20E, K20A, and the double mutant K18A K20A (KAKA). All mutant enzymes exhibit low RNA binding activity, low processivity, and alterations in nucleotide recognition, including increased incorporation of ribavirin monophosphate (RMP) relative to the incorporation of cognate nucleotides compared with the wild-type enzyme. The structural analysis shows an unprecedented flexibility of the 3D mutant polymerases, including both global rearrangements of the closed-hand architecture and local conformational changes at loop β9-α11 (within the polymerase motif B) and at the template-binding channel. Specifically, in 3D bound to RNA, both K18E and K20E induced the opening of new pockets in the template channel where the downstream templating nucleotide at position +2 binds. The comparisons of free and RNA-bound enzymes suggest that the structural rearrangements may occur in a concerted mode to regulate RNA replication, processivity, and fidelity. Thus, the Nterminal region of FMDV 3D that acts as a nuclear localization signal (NLS) and in template binding is also involved in nucleotide recognition and can affect the incorporation of nucleotide analogues. IMPORTANCE : The study documents multifunctionality of a nuclear localization signal (NLS) located at the N-terminal region of the foot-andmouth disease viral polymerase (3D). Amino acid substitutions at this polymerase region can impair the transport of 3D to the nucleus, reduce 3D binding to RNA, and alter the relative incorporation of standard nucleoside monophosphate versus ribavirin monophosphate. Structural data reveal that the conformational changes in this region, forming part of the template channel entry, would be involved in nucleotide discrimination. The results have implications for the understanding of viral polymerase function and for lethal mutagenesis mechanisms., Research in Barcelona was supported by grant BIO2011-24333 from the Spanish MINECO and by SILVER Cooperation project GA number 260644 of the European Union, Seventh Framework Program. Work in Madrid was supported by grants BFU2011-23604 and BIO2011-24351 from MINECO, PLATESA-S2013/ABI-2906 from CAM, and Fundación Ramón Areces. Financial support was provided by the ESRF and Alba. C.F.-O. is a recipient of a JAE postdoctoral contract supported by the Fondo Social Europeo

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 ESRF

Proteins containing C2 domains are the sensors for Ca2+ and PI (4,5)P2 in a myriad of secretory pathways. Here, the use of a freemounting system has enabled us to capture an intermediate state of Ca 2+ binding to the C2A domain of rabphilin 3A that suggests a different mechanism of ion interaction. We have also determined the structure of this domain in complex with PI(4,5)P2 and IP3 at resolutions of 1.75 and 1.9 A, respectively, unveiling that the polybasic cluster formed by strands β3-β4 is involved in the interaction with the phosphoinositides. A comparative study demonstrates that the C2A domain is highly specific for PI(4,5)P2/PI (3,4,5)P3, whereas the C2B domain cannot discriminate among any of the diphosphorylated forms. Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1 indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes the interaction with PI (4,5)P2. Together, these results provide a structural explanation for the ability of different C2 domains to pull plasma and vesicle membranes close together in a Ca2+-dependent manner and reveal how this family of proteins can use subtle structural changes to modulate their sensitivity and specificity to various cellular signals., Work in Barcelona was supported by Grant BIO2011-24333 [Ministerio de Economía y Competitividad (MINECO), Spain-Fondo Europeo de Desarrollo Regional (FEDER)] and work in Murcia by Grants BFU2011-22828 (MINECO, Spain-FEDER) and 08700/PI/08 (Fundación Seneca, Region de Murcia).C.F.-O. is recipient of a Junta para la Amplificación de Estudios postdoctoral contract from Consejo Superior de Investigaciones Cientificas-Fondo Social Europeo. J.G. is recipient of a Juan de la Cierva postdoctoral contract from MINECO

RNA-dependent RNA polymerases (RdRPs) are the central players in both transcription and viral genome replication. Using NMR spectroscopy, Yang and colleagues (in this issue of Structure) show that the conformational changes in the structural motif D of poliovirus RdRP correlate with the nature of the bound nucleotide (correct versus incorrect), with a conserved lysine within this motif playing a key role. © 2012 Elsevier Ltd.

Genome replication in picornavirus is catalyzed by a virally encoded RNA dependent RNA polymerase, termed 3D. These viruses also use a small protein primer, named VPg to initiate RNA replication. Polymerase 3D also catalyzes the covalent linkage of UMP to a N-terminal tyrosine on VPg. Seven different crystal structures of foot-and-mouth disease virus (FMDV) 3D catalytic complexes have enhanced our understanding of template and primer recognition, VPg uridylylation and rNTP binding and catalysis. In addition, the biochemical and structural analyses of six different FMDV 3D ribavirin resistant mutants provided evidences of three different mechanisms of resistance to this mutagenic nucleoside analogue. Such structural information is providing new insights into the fidelity of RNA replication, and for the design of antiviral compounds. © 2012 Springer Science+Business Media B.V.

Passage of poliovirus (PV) or foot-and-mouth disease virus (FMDV) in the presence of ribavirin (R) selected for viruses with decreased sensitivity to R, which included different mutations in their polymerase (3D): G64S located in the finger subdomain in the case of PV and M296I located within loop β9-α11 at the active site in the case of FMDV. To investigate why disparate substitutions were selected in two closely related 3Ds, we constructed FMDVs with a 3D that included either G62S (the equivalent replacement in FMDV of PV G64S), M296I, or both substitutions. G62S, but not M296I, inflicts upon FMDV a strong selective disadvantage which is partially compensated for by the substitution M296I. The corresponding mutant polymerases, 3D(G62S), 3D(M296I), and 3D(G62S-M296I), were analyzed functionally and structurally. G62S in 3D impairs RNA-binding, polymerization, and R monophosphate incorporation activities. The X-ray structures of the 3D(G62S)-RNA, 3D(M296I)-RNA, and 3D(G62S-M296I)-RNA complexes show that although the two positions are separated by 13.1 Å, the loops where the replacements reside are tightly connected through an extensive network of interactions that reach the polymerase active site. In particular, G62S seems to restrict the flexibility of loop β9-α11 and, as a consequence, the flexibility of the active site and its ability to bind the RNA template. Thus, a localized change in the finger subdomain of 3D may affect the catalytic domain. The results provide a structural interpretation of why different amino acid substitutions were selected to confer R resistance in closely related viruses and reveal a complex network of intra-3D interactions that can affect the recognition of both the RNA template and incoming nucleotide. Copyright © 2010, American Society for Microbiology. All Rights Reserved., Work in Barcelona was supported by grant BIO2008-02556, and work in Madrid was supported by grant BFU2008-02816/BMC from MCINN and by Fundación R. Areces. CIBERehd (Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Work in Barcelona and Madrid was further supported by Proyecto Intramural de Frontera 2000820FO191 (CSIC). X-ray data were collected at ESRF beamlines ID14.1 and ID14.2 (Grenoble, France) within a Block Allocation Group (BAG Barcelona). Financial support was provided by the ESRF. A.A. and C.F.-O. are recipients of I3P and Juan de la Cierva postdoctoral contracts, respectively.

Viral pathogenesis is not alien to the evolutionary history of a virus. Picornaviruses, simply by the fact of sharing a phylogenetic position, need not be associated with similar diseases, reflecting that the nature of the interactions with their host organisms may depend in a subtle manner on minimal genetic change of the virus. Picornaviruses have served to establish core concepts in the understanding of viruses as mutated collectivities and in establishing the relevance of quasispecies for viral pathogenesis. The adaptive potential of RNA viruses is also manifested in the response to selective agents administered to inhibit their replication. High mutation rates result in the almost-systematic selection of viral mutants resistant to antiviral inhibitors, either because resistant mutants are present in mutant spectra or because they are rapidly generated during viral replication. The participation of interfering genomes in virus extinction constitutes the basis of the lethal defection model of virus extinction by enhanced mutagenesis. The initial experiments to test the validity for RNA viruses of the error threshold concept consisted of documenting an adverse effect on viral infectivity as a result of increasing the mutation rate of poliovirus (PV) and vesicular stomatitis virus by chemical mutagens and base and nucleoside analogues added during viral RNA replication. Genetic modifications upon extensive passage of FMDV in BHK- 21 cells included genomes with internal in-frame deletions that were infectious by complementation in the absence of standard, wildtype genomes.

Resistance of viruses to mutagenic agents is an important problem for the development of lethal mutagenesis as an antiviral strategy. Previous studies with RNA viruses have documented that resistance to the mutagenic nucleoside analogue ribavirin (1-b-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is mediated by amino acid substitutions in the viral polymerase that either increase the general template copying fidelity of the enzyme or decrease the incorporation of ribavirin into RNA. Here we describe experiments that show that replication of the important picornavirus pathogen foot- and-mouth disease virus (FMDV) in the presence of increasing concentrations of ribavirin results in the sequential incorporation of three amino acid substitutions (M296I, P44S and P169S) in the viral polymerase (3D). The main biological effect of these substitutions is to attenuate the consequences of the mutagenic activity of ribavirin -by avoiding the biased repertoire of transition mutations produced by this purine analogue-and to maintain the replicative fitness of the virus which is able to escape extinction by ribavirin. This is achieved through alteration of the pairing behavior of ribavirin- triphosphate (RTP), as evidenced by in vitro polymerization assays with purified mutant 3Ds. Comparison of the three- dimensional structure of wild type and mutant polymerases suggests that the amino acid substitutions alter the position of the template RNA in the entry channel of the enzyme, thereby affecting nucleotide recognition. The results provide evidence of a new mechanism of resistance to a mutagenic nucleoside analogue which allows the virus to maintain a balance among mutation types introduced into progeny genomes during replication under strong mutagenic pressure. © 2010 Agudo et al.

RNA-dependent RNA polymerases (RdRPs) play central roles in both transcription and viral genome replication. In picornaviruses, these functions are catalyzed by the virally encoded RdRP, termed 3D. Polymerase 3D also catalyzes the covalent linkage of UMP to a tyrosine on the small protein VPg. Uridylylated VPg then serves as a protein primer for the initiation of RNA synthesis. Seven different crystal structures of foot-and-mouth disease virus (FMDV) 3D catalytic complexes have enhanced our understanding of template and primer recognition, VPg uridylylation, and rNTP binding and catalysis. Such structural information is providing new insights into the fidelity of RNA replication, and for the design of antiviral compounds. © 2009 Elsevier Ltd. All rights reserved., Primary research from Barcelona laboratory described in this review was supported by grant BFU2005-02376/BMC BIO2008-02556. Work in Madrid was supported by grants BFU2008-028/6BMC and Fundación R. Areces. Work in Barcelona and Madrid was further supported by Proyecto Intramural de Frontera 200820FO191(CSIC)

C2 domains are widely-spread protein signaling motifs that in classical PKCs act as Ca2+-binding modules. However, the molecular mechanisms of their targeting process at the plasma membrane remain poorly understood. Here, the crystal structure of PKCα-C2 domain in complex with Ca 2+, 1,2-dihexanoyl-sn-glycero-3-[phospho-L-serine] (PtdSer), and 1,2-diayl-sn-glycero-3-[phosphoinositol-4,5-bisphos-phate] [Ptdlns(4,5)P2] shows that PtdSer binds specifically to the calcium-binding region, whereas Ptdlns(4,5)P2 occupies the concave surface of strands β3 and β4. Strikingly, the structure reveals a Ptdlns(4,5)P2-C2 domain-binding mode in which the aromatic residues Tyr-195 and Trp-245 establish direct interactions with the phosphate moieties of the inositol ring. Mutations that abrogate Tyr-195 and Trp-245 recognition of Ptdlns(4,5)P2 severely impaired the ability of PKCα to localize to the plasma membrane. Notably, these residues are highly conserved among C2 domains of topology I, and a general mechanism of C2 domain-membrane docking mediated by Ptdlns(4,5)P 2 is presented., Work in Murcia was supported by the Fundación Médica Mutua Madrileña, Fundación Ramón Areces, and Fundación Séneca 08700/PI/08 (to S.C.-G.), and Ministerio de Ciencia e Innovación Grants BFU2005-02482 and BFU2008-01010 (to J.G.-F.). Work in Barcelona was supported by Ministerio de Ciencia e Innovación Grants BFU2005-02376/BMC (to N.V.) and BFU2005-08686-C02-01 (to I.F.).Financial support was provided by the European Synchrotron Radiation Facility

RNA virus replication is an error-prone event caused by the low fidelity of viral RNA-dependent RNA polymerases. Replication fidelity can be decreased further by the use of mutagenic ribonucleoside analogs to a point where viral genetic information can no longer be maintained. For foot-and-mouth disease virus, the antiviral analogs ribavirin and 5-fluorouracil have been shown to be mutagenic, contributing to virus extinction through lethal mutagenesis. Here, we report the x-ray structure of four elongation complexes of foot-and-mouth disease virus polymerase 3D obtained in presence of natural substrates, ATP and UTP, or mutagenic nucleotides, ribavirin triphosphate and 5-fluorouridine triphosphate with different RNAs as template-primer molecules. The ability of these complexes to synthesize RNA in crystals allowed us to capture different successive replication events and to define the critical amino acids involved in (i) the recognition and positioning of the incoming nucleotide or analog; (ii) the positioning of the acceptor base of the template strand; and (iii) the positioning of the 3′-OH group of the primer nucleotide during RNA replication. The structures identify key interactions involved in viral RNA replication and provide insights into the molecular basis of the low fidelity of viral RNA polymerases. © 2007 by The National Academy of Sciences of the USA., Work in Barcelona was supported by Grant BFU2005-02376/BMC from the Ministerio de Educación y Ciencia (M.E.C.) (to N.V.) and work in Madrid by Grant BFU2005-00863/BMC from the M.E.C. (to E.D.) and by the Fundación R. Areces. Work in Barcelona and Madrid was further supported by Proyecto Intramural de Frontera (CSIC) (to A.A. and C.F.-O.) and an I3P contract from CSIC (to A.A.). X-ray data were collected at the EMBL protein crystallography beam lines ID14.2 and ID14.4 at European Synchrotron Radiation Facility (ESRF) (Grenoble) within a block allocation group (BAG Barcelona). Financial support was provided by the ESRF

24 pages, 2 figures, 3 tables.-- 17182055 [PubMed].-- PMCID: PMC1855202.-- NIHMSID: NIHMS17626.-- Available online No. 14, 2006, The PhoR/PhoB two-component system is a key regulatory protein network enabling Escherichia coli to respond to inorganic phosphate (Pi) starvation conditions by turning on Pho regulon genes for more efficient Pi uptake and use of alternative phosphorus sources. Under environmental Pi depletion, the response regulator (RR) component, PhoB, is phosphorylated at the receiver domain (RD), a process that requires Mg2+ bound at the active site. Phosphorylation of the RD relieves the inhibition of the PhoB effector domain (ED), a DNA-binding region that binds to Pho regulon promoters to activate transcription. The molecular details of the activation are proposed to involve dimerisation of the RD and a conformational change in the RD detected by the ED. The structure of the PhoB RD shows a symmetrical interaction involving α1, loop β5α5 and N-terminus of α5 elements, also seen in the complex of PhoB RD with Mg2+, in which helix α4 highly increases its flexibility. PhoB RD in complex with Mg2+ and BeF3− (an emulator of the phosphate moiety) undergoes a dramatic conformational change on helix α4 and shows another interaction involving α4, β5 and α5 segments. We have selected a series of constitutively active PhoB mutants (PhoBCA) that are able to turn on the Pho regulon promoters in the absence phosphorylation and, as they cannot be inactivated, should therefore mimic the active RD state of PhoB and its functional oligomerisation. We have analysed the PhoBCA RD crystal structures of two such mutants, Asp53Ala/Tyr102Cys and Asp10Ala/Asp53Glu. Interestingly, both mutants reproduce the homodimeric arrangement through the symmetric interface encountered in the unbound and magnesium-bound wild-type PhoB RD structures. Besides, the mutant RD structures show a modified active site organization as well as changes at helix α4 that correlate with repositioning of surrounding residues, like the active-site events indicator Trp54, putatively redifining the interaction with the ED in the full-length protein., This work was supported by the Spanish Ministerio de Educación y Ciencia (grants BFU2005-512028, GEN2003-20642 and BIO2003-132) and the Generalitat de Catalunya (Centre de Referència en Biotecnologia and grant 2005SGR-00280).

Joana Fort et al., 4F2hc (CD98hc) is a multifunctional type II membrane glycoprotein involved in amino acid transport and cell fusion, adhesion, and transformation. The structure of the ectodomain of human 4F2hc has been solved using monoclinic (Protein Data Bank code 2DH2) and orthorhombic (Protein Data Bank code 2DH3) crystal forms at 2.1 and 2.8 Å, respectively. It is composed of a (βα)8 barrel and an antiparallel β8 sandwich related to bacterial α-glycosidases, although lacking key catalytic residues and consequently catalytic activity. 2DH3 is a dimer with Zn2+ coordination at the interface. Human 4F2hc expressed in several cell types resulted in cell surface and Cys109 disulfide bridge-linked homodimers with major architectural features of the crystal dimer, as demonstrated by cross-linking experiments. 4F2hc has no significant hydrophobic patches at the surface. Monomer and homodimer have a polarized charged surface. The N terminus of the solved structure, including the position of Cys109 residue located four residues apart from the transmembrane domain, is adjacent to the positive face of the ectodomain. This location of theNterminus and the Cys109-intervening disulfide bridge imposes space restrictions sufficient to support a model for electrostatic interaction of the 4F2hc ectodomain with membrane phospholipids. These results provide the first crystal structure of heteromeric amino acid transporters and suggest a dynamic interaction of the 4F2hc ectodomain with the plasma membrane. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc., This work was supported in part by the Spanish Ministry of Science and Education Grant SAF2003-08940, BFU2006-14600, and BIO2005-06753, by the EC Project Grant 502802 EUGINDAT, and by La Marató-TV3

A foot-and-mouth disease virus (FMDV) polymerase (3D) with amino acid replacements G118D, V239M and G373D (triple DMD mutant) was obtained from a molecular clone derived from a virus population treated with ribavirin, in the transition to error catastrophe (virus extinction through lethal mutagenesis). DMD 3D was expressed in Escherichia coli, purified, and its activity compared with that of wild-type enzyme and mutant enzymes with either replacement G118D, G118A or D338A (the latter affecting the catalytic motif YGDD), generated by site-directed mutagenesis. No differences among the enzymes were noted in their interaction with monoclonal antibodies specific for the FMDV polymerase. Mutant enzymes with G118D or G118A showed a 100-fold decrease in polymerization activity relative to wild-type 3D, using poly(A)/oligo(dT)15 and poly(A)/VPg as template-primers, under several reaction conditions. As expected, the activity of 3D with D338A was undetectable (, This work was supported by Grants BMC2001-1823-C02-01 of Comisión Interministerial de Ciencia y Tecnología, 08.2/0015/2001 of Comunidad Autónoma de Madrid and Fundación R. Areces (to E.D.) and Grant BIO2002-00517 of Comisión Interministerial de Ciencia y Tecnología (to N.V.). A.A. is supported by a predoctoral I3P fellowship from Ministerio de Educación y Ciencia. R.A. is supported by a predoctoral fellowship from the Comunidad Autónoma de Madrid. C.F.-O. is supported by a predoctoral fellowship from the Ministerio de Ciencia y Tecnología.

Genome replication in picornaviruses is catalyzed by a virally encoded RNA-dependent RNA polymerase, termed 3D. These viruses also use a small protein primer, named VPg, to initiate RNA replication. The recent explosion of structural information on picornaviral 3D polymerases has provided insights into the initiation of RNA synthesis and chain elongation. Comparing these data with results from previous structural analyses of viral RNA-dependent RNA polymerases that catalyze de novo RNA synthesis sheds light on the different strategies that these viruses use to initiate replication. © 2006 Elsevier Ltd. All rights reserved., Primary research from the Barcelona laboratory described in this review was supported by grant BIO2002-00517. Work in Madrid was supported by grants BMC2001-1823-C02-01, BFU2005-00863/BMC and Fundación R Areces

Genome replication in picornaviruses is catalyzed by a virally encoded RNA-dependent RNA polymerase, termed 3D. The enzyme performs this operation, together with other viral and probably host proteins, in the cytoplasm of their host cells. The crystal structure of the 3D polymerase of foot-and-mouth disease virus, one of the most important animal pathogens, has been determined unliganded and bound to a template-primer RNA decanucleotide. The enzyme folds in the characteristic fingers, palm and thumb subdomains, with the presence of an NH2-terminal segment that encircles the active site. In the complex, several conserved amino acid side chains bind to the template-primer, likely mediating the initiation of RNA synthesis. The structure provides essential information for studies on RNA replication and the design of antiviral compounds., This work was supported in part by Grants BMC2001-1823-C02-01 of Comisión Interministerial de Ciencia y Tecnología, Comunidad Autónoma de Madrid 08.2/0015/2001 and Fundación R. Areces (to E. D.) and Grant BIO2002-00517 of the Comisión Interministerial de Ciencia y Tecnología (to N. V.)

Besides their classical role in alimentary protein degradation, zinc-dependant carboxypeptidases also participate in more selective regulatory processes like prohormone and neuropeptide processing or fibrinolysis inhibition in blood plasma. Human pancreatic procarboxypeptidase B (PCPB) is the prototype for those human exopeptidases that cleave off basic C-terminal residues and are secreted as inactive zymogens. One such protein is thrombin-activatable fibrinolysis inhibitor (TAFI), also known as plasma PCPB, which circulates in human plasma as a zymogen bound to plasminogen. The structure of human pancreatic PCPB displays a 95-residue pro-segment consisting of a globular region with an open-sandwich antiparallel-alpha antiparallel-beta topology and a C-terminal alpha-helix, which connects to the enzyme moiety. The latter is a 309-amino acid residue catalytic domain with alpha/beta hydrolase topology and a preformed active site, which is shielded by the globular domain of the pro-segment. The fold of the proenzyme is similar to previously reported procarboxypeptidase structures, also in that the most variable region is the connecting segment that links both globular moieties. However, the empty active site of human procarboxypeptidase B has two alternate conformations in one of the zinc-binding residues, which account for subtle differences in some of the key residues for substrate binding. The reported crystal structure, refined with data to 1.6A resolution, permits in the absence of an experimental structure, accurate homology modelling of TAFI, which may help to explain its properties.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of one of the most harmful poultry diseases. The IBDV genome encodes five mature proteins; of these, the multifunctional protein VP3 plays an essential role in virus morphogenesis. This protein, which interacts with the structural protein VP2, with the double-stranded RNA genome, and with the virus-encoded, RNA-dependent RNA polymerase, VP1, is involved not only in the formation of the viral capsid, but also in the recruitment of VP1 into the capsid and in the encapsidation of the viral genome. Here, we report the X-ray structure of the central region of VP3, residues 92-220, consisting of two α-helical domains connected by a long and flexible hinge that are organized as a dimer. Unexpectedly, the overall fold of the second VP3 domain shows significant structural similarities with different transcription regulation factors. © 2008 Elsevier Ltd. All rights reserved., This work was supported by grants AGL2003-07189/BIO2006-09407 and BFU2005-02376/BMC from The Ministerio de Educacion y Ciencia to J.F.R. and to N.V., respectively. A.C. was supported by an I3P fellowship from Consejo Superior de Investigaciones Cientificas (CSIC)-Ministerio de Educación y Ciencia, and C.F.-O. was supported by a Proyecto Intramural de Frontera fellowship from CSIC