Centro Nacional de Biotecnología

The present outbreak of a coronavirus-associated acute respiratory disease called coronavirus disease 19 (COVID-19) is the third documented spillover of an animal coronavirus to humans in only two decades that has resulted in a major epidemic. The Coronaviridae Study Group (CSG) of the International Committee on Taxonomy of Viruses, which is responsible for developing the classification of viruses and taxon nomenclature of the family Coronaviridae, has assessed the placement of the human pathogen, tentatively named 2019-nCoV, within the Coronaviridae. Based on phylogeny, taxonomy and established practice, the CSG recognizes this virus as forming a sister clade to the prototype human and bat severe acute respiratory syndrome coronaviruses (SARS-CoVs) of the species Severe acute respiratory syndrome-related coronavirus, and designates it as SARS-CoV-2. In order to facilitate communication, the CSG proposes to use the following naming convention for individual isolates: SARS-CoV-2/host/location/isolate/date. While the full spectrum of clinical manifestations associated with SARS-CoV-2 infections in humans remains to be determined, the independent zoonotic transmission of SARS-CoV and SARS-CoV-2 highlights the need for studying viruses at the species level to complement research focused on individual pathogenic viruses of immediate significance. This will improve our understanding of virus–host interactions in an ever-changing environment and enhance our preparedness for future outbreaks.

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Work in our laboratory is supported by grants BIO2008-00090 from the Spanish Ministry of Science and Innovation and KBBE-227258 (BIOHYPO), HEALTH-F3-2011-282004 (EVOTAR), and HEALTH-F3-2010-241476 (PAR) from European Union.

Exosomes are released by most cells to the extracellular environment and are involved in cell-to-cell communication. Exosomes contain specific repertoires of mRNAs, microRNAs (miRNAs) and other non-coding RNAs that can be functionally transferred to recipient cells. However, the mechanisms that control the specific loading of RNA species into exosomes remain unknown. Here we describe sequence motifs present in miRNAs that control their localization into exosomes. The protein heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) specifically binds exosomal miRNAs through the recognition of these motifs and controls their loading into exosomes. Moreover, hnRNPA2B1 in exosomes is sumoylated, and sumoylation controls the binding of hnRNPA2B1 to miRNAs. The loading of miRNAs into exosomes can be modulated by mutagenesis of the identified motifs or changes in hnRNPA2B1 expression levels. These findings identify hnRNPA2B1 as a key player in miRNA sorting into exosomes and provide potential tools for the packaging of selected regulatory RNAs into exosomes and their use in biomedical applications. Cells secrete micro-RNAs by packaging them into exosomes; however, the mechanisms by which this packaging occurs are unclear. Here, the authors identify a sequence motif that confers exosomal targeting to micro-RNAs and identify a ribonucleoprotein complex that plays a role in this process.

The large majority of antibiotics currently used for treating infections and the antibiotic resistance genes acquired by human pathogens each have an environmental origin. Recent work indicates that the function of these elements in their environmental reservoirs may be very distinct from the "weapon-shield" role they play in clinical settings. Changes in natural ecosystems, including the release of large amounts of antimicrobials, might alter the population dynamics of microorganisms, including selection of resistance, with consequences for human health that are difficult to predict.

Analysis of the genomes of four patients with chronic lymphocytic leukaemia, and validation in more than 300 patients, has identified four genes — NOTCH1, MYD88, XPO1 and KLHL6 — that are recurrently mutated in the condition. Mutations in NOTCH1, MYD88 and XPO1 are thought to contribute to the clinical evolution of the disease. Evidence that NOTCH1 and MYD88 mutations are activating events highlights them as potential therapeutic targets. Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution1,2. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes3,4. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer.

Abstract The present outbreak of lower respiratory tract infections, including respiratory distress syndrome, is the third spillover, in only two decades, of an animal coronavirus to humans resulting in a major epidemic. Here, the Coronavirus Study Group (CSG) of the International Committee on Taxonomy of Viruses, which is responsible for developing the official classification of viruses and taxa naming (taxonomy) of the Coronaviridae family, assessed the novelty of the human pathogen tentatively named 2019-nCoV. Based on phylogeny, taxonomy and established practice, the CSG formally recognizes this virus as a sister to severe acute respiratory syndrome coronaviruses (SARS-CoVs) of the species Severe acute respiratory syndrome-related coronavirus and designates it as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To facilitate communication, the CSG further proposes to use the following naming convention for individual isolates: SARS-CoV-2/Isolate/Host/Date/Location. The spectrum of clinical manifestations associated with SARS-CoV-2 infections in humans remains to be determined. The independent zoonotic transmission of SARS-CoV and SARS-CoV-2 highlights the need for studying the entire (virus) species to complement research focused on individual pathogenic viruses of immediate significance. This research will improve our understanding of virus-host interactions in an ever-changing environment and enhance our preparedness for future outbreaks.

Cryo-EM maps are valuable sources of information for protein structure modeling. However, due to the loss of contrast at high frequencies, they generally need to be post-processed to improve their interpretability. Most popular approaches, based on global B-factor correction, suffer from limitations. For instance, they ignore the heterogeneity in the map local quality that reconstructions tend to exhibit. Aiming to overcome these problems, we present DeepEMhancer, a deep learning approach designed to perform automatic post-processing of cryo-EM maps. Trained on a dataset of pairs of experimental maps and maps sharpened using their respective atomic models, DeepEMhancer has learned how to post-process experimental maps performing masking-like and sharpening-like operations in a single step. DeepEMhancer was evaluated on a testing set of 20 different experimental maps, showing its ability to reduce noise levels and obtain more detailed versions of the experimental maps. Additionally, we illustrated the benefits of DeepEMhancer on the structure of the SARS-CoV-2 RNA polymerase.

Cross-talk between ethylene and jasmonate signaling pathways determines the activation of a set of defense responses against pathogens and herbivores. However, the molecular mechanisms that underlie this cross-talk are poorly understood. Here, we show that ethylene and jasmonate pathways converge in the transcriptional activation of ETHYLENE RESPONSE FACTOR1 (ERF1), which encodes a transcription factor that regulates the expression of pathogen response genes that prevent disease progression. The expression of ERF1 can be activated rapidly by ethylene or jasmonate and can be activated synergistically by both hormones. In addition, both signaling pathways are required simultaneously to activate ERF1, because mutations that block any of them prevent ERF1 induction by any of these hormones either alone or in combination. Furthermore, 35S:ERF1 expression can rescue the defense response defects of coi1 (coronative insensitive1) and ein2 (ethylene insensitive2); therefore, it is a likely downstream component of both ethylene and jasmonate signaling pathways. Transcriptome analysis in Col;35S:ERF1 transgenic plants and ethylene/jasmonate-treated wild-type plants further supports the notion that ERF1 regulates in vivo the expression of a large number of genes responsive to both ethylene and jasmonate. These results suggest that ERF1 acts downstream of the intersection between ethylene and jasmonate pathways and suggest that this transcription factor is a key element in the integration of both signals for the regulation of defense response genes.

Plants have evolved a number of adaptive responses to cope with growth in conditions of limited phosphate (Pi) supply involving biochemical, metabolic, and developmental changes. We prepared an EMS-mutagenized M(2) population of an Arabidopsis thaliana transgenic line harboring a reporter gene specifically responsive to Pi starvation (AtIPS1::GUS), and screened for mutants altered in Pi starvation regulation. One of the mutants, phr1 (phosphate starvation response 1), displayed reduced response of AtIPS1::GUS to Pi starvation, and also had a broad range of Pi starvation responses impaired, including the responsiveness of various other Pi starvation-induced genes and metabolic responses, such as the increase in anthocyanin accumulation. PHR1 was positionally cloned and shown be related to the PHOSPHORUS STARVATION RESPONSE 1 (PSR1) gene from Chlamydomonas reinhardtii. A GFP::PHR1 protein fusion was localized in the nucleus independently of Pi status, as is the case for PSR1. PHR1 is expressed in Pi sufficient conditions and, in contrast to PSR1, is only weakly responsive to Pi starvation. PHR1, PSR1, and other members of the protein family share a MYB domain and a predicted coiled-coil (CC) domain, defining a subtype within the MYB superfamily, the MYB-CC family. Therefore, PHR1 was found to bind as a dimer to an imperfect palindromic sequence. PHR1-binding sequences are present in the promoter of Pi starvation-responsive structural genes, indicating that this protein acts downstream in the Pi starvation signaling pathway.

In spite of the importance of jasmonates (JAs) as plant growth and stress regulators, the molecular components of their signaling pathway remain largely unknown. By means of a genetic screen that exploits the cross talk between ethylene (ET) and JAs, we describe the identification of several new loci involved in JA signaling and the characterization and positional cloning of one of them, JASMONATE-INSENSITIVE1 (JAI1/JIN1). JIN1 encodes AtMYC2, a nuclear-localized basic helix-loop-helix-leucine zipper transcription factor, whose expression is rapidly upregulated by JA, in a CORONATINE INSENSITIVE1-dependent manner. Gain-of-function experiments confirmed the relevance of AtMYC2 in the activation of JA signaling. AtMYC2 differentially regulates the expression of two groups of JA-induced genes. The first group includes genes involved in defense responses against pathogens and is repressed by AtMYC2. Consistently, jin1 mutants show increased resistance to necrotrophic pathogens. The second group, integrated by genes involved in JA-mediated systemic responses to wounding, is activated by AtMYC2. Conversely, Ethylene-Response-Factor1 (ERF1) positively regulates the expression of the first group of genes and represses the second. These results highlight the existence of two branches in the JA signaling pathway, antagonistically regulated by AtMYC2 and ERF1, that are coincident with the alternative responses activated by JA and ET to two different sets of stresses, namely pathogen attack and wounding.

During the summer of 2012, in Jeddah, Saudi Arabia, a hitherto unknown coronavirus (CoV) was isolated from the sputum of a patient with acute pneumonia and renal failure (1, 2). The isolate was provisionally called human coronavirus Erasmus Medical Center (EMC) (3). Shortly thereafter, in September 2012, the same type of virus, named human coronavirus England 1, was recovered from a patient with severe respiratory illness who had been transferred from the Gulf region of the Middle East to London, United Kingdom (4) (GenBank accession no. KC164505.2). The onset of the new disease was traced back to an even earlier time point. Already in April 2012, a cluster of pneumonia cases in health care workers had occurred in an intensive care unit of a hospital in Zarqa, Jordan (5). Two persons died, both of whom were confirmed to have been infected with the novel coronavirus through a retrospective analysis of stored samples (6). These findings met with considerable concern. Although the number of laboratory-confirmed cases is limited (34 as of 12 May 2013), the morbidity and mortality of the infection is alarming, as is its uncanny resemblance—at least in its clinical features—to severe acute respiratory syndrome (SARS). While in a small minority of the known cases the patients developed mild disease, most patients presented with a severe acute respiratory condition requiring hospitalization; the mortality rate is approximately 60% (7).

Ustilago maydis is an important fungal pathogen of maize, causing corn smut. It is well adapted to its host and proliferates in living plant tissue without inducing a defence response. The genome sequence of U. maydis has now been determined, the first for a biotrophic plant parasite. Several gene clusters that encode secreted proteins of unknown function were identified: genome-wide expression analysis shows that the clustered genes are upregulated during disease. Mutations in these gene clusters frequently affect virulence, ranging from complete loss of pathogenicity to hypervirulence. Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant–microbe interactions1. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development2. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no ‘true’ virulence factors3 had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.

Jasmonates (JAs) trigger an important transcriptional reprogramming of plant cells to modulate both basal development and stress responses. In spite of the importance of transcriptional regulation, only one transcription factor (TF), the Arabidopsis thaliana basic helix-loop-helix MYC2, has been described so far as a direct target of JAZ repressors. By means of yeast two-hybrid screening and tandem affinity purification strategies, we identified two previously unknown targets of JAZ repressors, the TFs MYC3 and MYC4, phylogenetically closely related to MYC2. We show that MYC3 and MYC4 interact in vitro and in vivo with JAZ repressors and also form homo- and heterodimers with MYC2 and among themselves. They both are nuclear proteins that bind DNA with sequence specificity similar to that of MYC2. Loss-of-function mutations in any of these two TFs impair full responsiveness to JA and enhance the JA insensitivity of myc2 mutants. Moreover, the triple mutant myc2 myc3 myc4 is as impaired as coi1-1 in the activation of several, but not all, JA-mediated responses such as the defense against bacterial pathogens and insect herbivory. Our results show that MYC3 and MYC4 are activators of JA-regulated programs that act additively with MYC2 to regulate specifically different subsets of the JA-dependent transcriptional response.

The non-structural (NS1) protein of influenza A viruses is a non-essential virulence factor that has multiple accessory functions during viral infection. In recent years, the major role ascribed to NS1 has been its inhibition of host immune responses, especially the limitation of both interferon (IFN) production and the antiviral effects of IFN-induced proteins, such as dsRNA-dependent protein kinase R (PKR) and 2'5'-oligoadenylate synthetase (OAS)/RNase L. However, it is clear that NS1 also acts directly to modulate other important aspects of the virus replication cycle, including viral RNA replication, viral protein synthesis, and general host-cell physiology. Here, we review the current literature on this remarkably multifunctional viral protein. In the first part of this article, we summarize the basic biochemistry of NS1, in particular its synthesis, structure, and intracellular localization. We then discuss the various roles NS1 has in regulating viral replication mechanisms, host innate/adaptive immune responses, and cellular signalling pathways. We focus on the NS1-RNA and NS1-protein interactions that are fundamental to these processes, and highlight apparent strain-specific ways in which different NS1 proteins may act. In this regard, the contributions of certain NS1 functions to the pathogenicity of human and animal influenza A viruses are also discussed. Finally, we outline practical applications that future studies on NS1 may lead to, including the rational design and manufacture of influenza vaccines, the development of novel antiviral drugs, and the use of oncolytic influenza A viruses as potential anti-cancer agents.

Oncogenic Ras transforms immortal rodent cells to a tumorigenic state, in part, by constitutively transmitting mitogenic signals through the mitogen-activated protein kinase (MAPK) cascade. In primary cells, Ras is initially mitogenic but eventually induces premature senescence involving the p53 and p16(INK4a) tumor suppressors. Constitutive activation of MEK (a component of the MAPK cascade) induces both p53 and p16, and is required for Ras-induced senescence of normal human fibroblasts. Furthermore, activated MEK permanently arrests primary murine fibroblasts but forces uncontrolled mitogenesis and transformation in cells lacking either p53 or INK4a. The precisely opposite response of normal and immortalized cells to constitutive activation of the MAPK cascade implies that premature senescence acts as a fail-safe mechanism to limit the transforming potential of excessive Ras mitogenic signaling. Consequently, constitutive MAPK signaling activates p53 and p16 as tumor suppressors.

Considerable progress has been made in identifying the targets of plant microRNAs, many of which regulate the stability or translation of mRNAs that encode transcription factors involved in development. In most cases, it is unknown, however, which immediate transcriptional targets mediate downstream effects of the microRNA-regulated transcription factors. We identified a new process controlled by the miR319-regulated clade of TCP (TEOSINTE BRANCHED/CYCLOIDEA/PCF) transcription factor genes. In contrast to other miRNA targets, several of which modulate hormone responses, TCPs control biosynthesis of the hormone jasmonic acid. Furthermore, we demonstrate a previously unrecognized effect of TCPs on leaf senescence, a process in which jasmonic acid has been proposed to be a critical regulator. We propose that miR319-controlled TCP transcription factors coordinate two sequential processes in leaf development: leaf growth, which they negatively regulate, and leaf senescence, which they positively regulate.

Shoot branching patterns depend on a key developmental decision: whether axillary buds grow out to give a branch or whether they remain dormant in the axils of leaves. This decision is controlled by endogenous and environmental stimuli mediated by hormonal signals. Although genes involved in the long-distance signaling of this process have been identified, the genes responding inside the buds to cause growth arrest remained unknown in Arabidopsis thaliana. Here, we describe an Arabidopsis gene encoding a TCP transcription factor closely related to teosinte branched1 (tb1) from maize (Zea mays), BRANCHED1 (BRC1), which represents a key point at which signals controlling branching are integrated within axillary buds. BRC1 is expressed in developing buds, where it arrests bud development. BRC1 downregulation leads to branch outgrowth. BRC1 responds to developmental and environmental stimuli controlling branching and mediates the response to these stimuli. Mutant and expression analyses suggest that BRC1 is downstream of the MORE AXILLARY GROWTH pathway and that it is required for auxin-induced apical dominance. Therefore, BRC1 acts inside the buds as an integrator of signals controlling bud outgrowth and translates them into a response of cell growth arrest. The conservation of BRC1/tb1 function among distantly related angiosperm species suggests that a single ancestral mechanism of branching control integration evolved before the radiation of flowering plants.

Analyses of Arabidopsis thaliana defense response to the damping-off oomycete pathogen Pythium irregulare show that resistance to P. irregulare requires a multicomponent defense strategy. Penetration represents a first layer, as indicated by the susceptibility of pen2 mutants, followed by recognition, likely mediated by ERECTA receptor-like kinases. Subsequent signaling of inducible defenses is predominantly mediated by jasmonic acid (JA), with insensitive coi1 mutants showing extreme susceptibility. In contrast with the generally accepted roles of ethylene and salicylic acid cooperating with or antagonizing, respectively, JA in the activation of defenses against necrotrophs, both are required to prevent disease progression, although much less so than JA. Meta-analysis of transcriptome profiles confirmed the predominant role of JA in activation of P. irregulare-induced defenses and uncovered abscisic acid (ABA) as an important regulator of defense gene expression. Analysis of cis-regulatory sequences also revealed an unexpected overrepresentation of ABA response elements in promoters of P. irregulare-responsive genes. Subsequent infections of ABA-related and callose-deficient mutants confirmed the importance of ABA in defense, acting partly through an undescribed mechanism. The results support a model for ABA affecting JA biosynthesis in the activation of defenses against this oomycete.