Putative Secondary Structure at 5’UTR as a Potential Antiviral Target against SARS-CoV-2

SARS-CoV-2 is an enveloped positive-sense single-stranded RNA coronavirus that causes COVID-19, of which the current outbreak has resulted in a high number of cases and fatalities throughout the world, even vaccine doses are being administered. The aim of this work was to scan the SARS-CoV-2 genome in search for therapeutic targets. We found a sequence in the 5’UTR (NC 045512:74-130), consisting of a typical heptamer next to a structured region that may cause ribosomal frameshifting. The potential biological value of this region is relevant through its low similarity with other viruses, including coronaviruses related to SARS-CoV, and its high sequence conservation within multiple SARS-CoV-2 isolates. We have predicted the secondary structure of the region by means of different bioinformatic tools. We have suggested a most probable secondary structure to proceed with a 3D reconstruction of the structured segment. Finally, we carried out virtual docking on the 3D structure to look for a binding site and then for drug ligands from a database of lead compounds. Several molecules that could be probably administered as oral drugs show promising binding affinity within the structured region, and so it could be possible interfere its potential regulatory role.

Enthalpy–Entropy Compensation in the Promiscuous Interaction of an Intrinsically Disordered Protein with Homologous Protein Partners

Intrinsically disordered proteins (IDPs) can engage in promiscuous interactions with their protein targets; however, it is not clear how this feature is encoded in the primary sequence of the IDPs and to what extent the surface properties and the shape of the binding cavity dictate the binding mode and the final bound conformation. Here we show, using a combination of nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), that the promiscuous interaction of the intrinsically disordered regulatory domain of the mitogen-activated protein kinase kinase MKK4 with p38α and JNK1 is facilitated by folding-upon-binding into two different conformations, despite the high sequence conservation and structural homology between p38α and JNK1. Our results support a model whereby the specific surface properties of JNK1 and p38α dictate the bound conformation of MKK4 and that enthalpy–entropy compensation plays a major role in maintaining comparable binding affinities for MKK4 towards the two kinases.

Structure, mechanism and crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase

There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two “druggable” pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.

The highly conserved FOXJ1 target CFAP161 is dispensable for motile ciliary function in mouse and Xenopus

Cilia are protrusions of the cell surface and composed of hundreds of proteins many of which are evolutionary and functionally well conserved. In cells assembling motile cilia the expression of numerous ciliary components is under the control of the transcription factor FOXJ1. Here, we analyse the evolutionary conserved FOXJ1 target CFAP161 in Xenopus and mouse. In both species Cfap161 expression correlates with the presence of motile cilia and depends on FOXJ1. Tagged CFAP161 localises to the basal bodies of multiciliated cells of the Xenopus larval epidermis, and in mice CFAP161 protein localises to the axoneme. Surprisingly, disruption of the Cfap161 gene in both species did not lead to motile cilia-related phenotypes, which contrasts with the conserved expression in cells carrying motile cilia and high sequence conservation. In mice mutation of Cfap161 stabilised the mutant mRNA making genetic compensation triggered by mRNA decay unlikely. However, genes related to microtubules and cilia, microtubule motor activity and inner dyneins were dysregulated, which might buffer the Cfap161 mutation.

Structural characterization of NORAD reveals a stabilizing role of spacers and two new repeat units.

Long non-coding RNAs (lncRNAs) can perform a variety of key cellular functions by interacting with proteins and other RNAs. Recent studies have shown that the function of lncRNAS are largely mediated by their structures. However, our structural knowledge for most lncRNAS is limited to sequence-based computational predictions. Non-coding RNA activated by DNA damage (NORAD) is an atypical lncRNA due to its abundant expression and high sequence conservation. NORAD regulates genomic stability by interacting with proteins and microRNAs. Previous sequence-based characterization has identified a modular organization of NORAD composed of several NORAD repeat units (NRUs). These units comprise the protein-binding elements and are separated by regular spacers of unknown function. Here, we experimentally determine for the first time the secondary structure of NORAD using the nextPARS approach. Our results suggest that the spacer regions provide structural stability to NRUs. Furthermore, we uncover two previously-unreported NRUs, and determine the core structural motifs conserved across NRUs. Overall, these findings will help to elucidate the function and evolution of NORAD.

Structure, Mechanism and Crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase

The global COVID-19 pandemic is caused by the SARS-CoV-2 virus and has infected over 100 million and caused over 2 million fatalities worldwide at the point of writing. There is currently a lack of effective drugs to treat people infected with SARS-CoV-2. The SARS-CoV-2 Non-structural protein 13 (NSP13) is a superfamily1B helicase that has been identified as a possible target for anti-viral drugs due to its high sequence conservation and essential role in viral replication. In this study we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and the non-hydrolysable ATP analogue (AMP-PNP). Comparisons of these structures reveal details of global and local conformational changes that are induced by nucleotide binding and hydrolysis and provide insights into the helicase mechanism and possible modes of inhibition. Structural analysis reveals two pockets on NSP13 that are classified as "druggable" and include one of the most conserved sites in the entire SARS-CoV-2 proteome. To identify possible starting points for anti-viral drug development we have performed a crystallographic fragment screen against SARS-CoV-2 NSP13 helicase. The fragment screen reveals 65 fragment hits across 52 datasets, with hot spots in pockets predicted to be of functional importance, including the druggable nucleotide and nucleic acid binding sites, opening the way to structure guided development of novel antiviral agents.

A Large-Scale Sequencing-Based Survey of Plasmids in Listeria monocytogenes Reveals Global Dissemination of Plasmids

The food-borne pathogen Listeria monocytogenes is known for its capacity to cope with multiple stress conditions occurring in food and food production environments (FPEs). Plasmids can provide benefits to their host strains, and it is known that various Listeria strains contain plasmids. However, the current understanding of plasmid frequency and function in L. monocytogenes strains remains rather limited. To determine the presence of plasmids among L. monocytogenes strains and their potential contribution to stress survival, a comprehensive dataset was established based on 1,921 published genomes from strains representing 14 L. monocytogenes sequence types (STs). Our results show that an average of 54% of all L. monocytogenes strains in the dataset contained a putative plasmid. The presence of plasmids was highly variable between different STs. While some STs, such as ST1, ST2, and ST4, contained few plasmid-bearing strains (<15% of the strains per ST), other STs, such as ST121, ST5, ST8, ST3, and ST204, possessed a higher proportion of plasmid-bearing strains with plasmids found in >71% of the strains within each ST. Overall, the sizes of plasmids analyzed in this study ranged from 4 to 170 kbp with a median plasmid size of 61 kbp. We also identified two novel groups of putative Listeria plasmids based on the amino acid sequences of the plasmid replication protein, RepA. We show that highly conserved plasmids are shared among Listeria strains which have been isolated from around the world over the last few decades. To investigate the potential roles of plasmids, nine genes related to stress-response were selected for an assessment of their abundance and conservation among L. monocytogenes plasmids. The results demonstrated that these plasmid genes exhibited high sequence conservation but that their presence in plasmids was highly variable. Additionally, we identified a novel transposon, Tn7075, predicted to be involved in mercury-resistance. Here, we provide the largest plasmid survey of L. monocytogenes to date with a comprehensive examination of the distribution of plasmids among L. monocytogenes strains. Our results significantly increase our knowledge about the distribution, composition, and conservation of L. monocytogenes plasmids and suggest that plasmids are likely important for the survival of L. monocytogenes in food and FPEs.

Aberrant Expression of microRNA Clusters in Head and Neck Cancer Development and Progression: Current and Future Translational Impacts

MicroRNAs are small non-coding RNAs known to negative regulate endogenous genes. Some microRNAs have high sequence conservation and localize as clusters in the genome. Their coordination is regulated by simple genetic and epigenetic events mechanism. In cells, single microRNAs can regulate multiple genes and microRNA clusters contain multiple microRNAs. MicroRNAs can be differentially expressed and act as oncogenic or tumor suppressor microRNAs, which are based on the roles of microRNA-regulated genes. It is vital to understand their effects, regulation, and various biological functions under both normal and disease conditions. Head and neck squamous cell carcinomas are some of the leading causes of cancer-related deaths worldwide and are regulated by many factors, including the dysregulation of microRNAs and their clusters. In disease stages, microRNA clusters can potentially control every field of oncogenic function, including growth, proliferation, apoptosis, migration, and intercellular commutation. Furthermore, microRNA clusters are regulated by genetic mutations or translocations, transcription factors, and epigenetic modifications. Additionally, microRNA clusters harbor the potential to act therapeutically against cancer in the future. Here, we review recent advances in microRNA cluster research, especially relative to head and neck cancers, and discuss their regulation and biological functions under pathological conditions as well as translational applications.

DING Proteins Extend to the Extremophilic World

The DING proteins are ubiquitous in the three domains of life, from mesophiles to thermo- and hyperthermophiles. They belong to a family of more than sixty members and have a characteristic N-terminus, DINGGG, which is considered a “signature” of these proteins. Structurally, they share a highly conserved phosphate binding site, and a three dimensional organization resembling the “Venus Flytrap”, both reminding the ones of PstS proteins. They have unusually high sequence conservation, even between distantly related species. Nevertheless despite that the genomes of most of these species have been sequenced, the DING gene has not been reported for all the relative characterized DING proteins. Identity of known DING proteins has been confirmed immunologically and, in some cases, by N-terminal sequence analysis. Only a few of the DING proteins have been purified and biochemically characterized. DING proteins are heterogeneous for their wide range of biological activities and some show different activities not always correlated with each other. Most of them have been originally identified for different biological properties, or rather for binding to phosphate and also to other ligands. Their involvement in pathologies is described. This review is an update of the most recent findings on old and new DING proteins.

Novel genetic constructs for production of recombinant HTLV-1/2 antigens and evaluation of their reactivity to plasma samples from HTLV1-infected patients

HTLV-1 can cause life-threatening diseases for which there are no effective treatments. Prevention of HTLV-1 infection requires massive testing of pregnant women, blood for transfusion and, organs for transplantation as well as safe sex. In this context, serological assays are widely used for monitoring HTLV-1 infections. Despite the necessity of recombinant antigens to compose serological tests, there is little information available on procedures to produce recombinant HTLV1/2 antigens for serological diagnostic purposes. In this work, we tested a series of genetic constructions to select those more amenable for production in bacterial systems. To overcome the constraints to express sections of viral envelope proteins in bacteria, we have used the p24 segment of the gag protein as a scaffold to display the immunogenic regions of gp46 and gp21. Nine recombinant antigenic proteins derived from HTLV-1 and five derived from HTLV-2 were successfully purified. The HTLV-1 antigens showed high efficiency in discriminating HTLV-positive from HTLV-negative samples using ELISA. Interestingly, HTLV-1-positive samples showed a high level of cross-reaction with HTLV-2 antigens. This finding is explained by the high sequence conservation between the structural proteins of these two highly related viruses. In summary, the results presented in this work provide a detailed description of the methods used to produce recombinant HTLV-1 and HTLV-2 antigens and demonstrate that the HTLV-1 antigens show strong potential for serological diagnosis of HTLV1 infections.