La protéine L du virus Ébola porte une nouvelle activité enzymatique impliquée dans la méthylation interne des ARN

International audienceL’infection par le virus Ébola conduit à des fièvres hémorragiques souvent fatales. Depuis sa découverte en 1976 en République Démocratique du Congo, plus d’une vingtaine d’épidémies d’Ébola ont été recensées en Afrique. La plus importante a eu lieu en 2015-2016, entraînant le décès de plus de 11 000 personnes. En raison du risque de réémergence de ce virus, et en l’absence de vaccins et d’agents thérapeutiques approuvés, la recherche de médicaments ciblant le virus Ébola est une priorité pour l’Organisation mondiale de la santé. Le virus Ébola est un virus enveloppé possédant un ARN génomique simple brin de polarité négative et appartient à l’ordre les Mononegavirales

The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade

International audienceIn 2019, a new coronavirus (2019-nCoV) infecting Humans has emerged in Wuhan, China. Its genome has been sequenced and the genomic information promptly released. Despite a high similarity with the genome sequence of SARS-CoV and SARS-like CoVs, we identified a peculiar furin-like cleavage site in the Spike protein of the 2019-nCoV, lacking in the other SARS-like CoVs. In this article, we discuss the possible functional consequences of this cleavage site in the viral cycle, pathogenicity and its potential implication in the development of antivirals

Rhamnolipids Are Virulence Factors That Promote Early Infiltration of Primary Human Airway Epithelia by Pseudomonas aeruginosa

The opportunistic bacterium Pseudomonas aeruginosa causes chronic respiratory infections in cystic fibrosis and immunocompromised individuals. Bacterial adherence to the basolateral domain of the host cells and internalization are thought to participate in P. aeruginosa pathogenicity. However, the mechanism by which the pathogen initially modulates the paracellular permeability of polarized respiratory epithelia remains to be understood. To investigate this mechanism, we have searched for virulence factors secreted by P. aeruginosa that affect the structure of human airway epithelium in the early stages of infection. We have found that only bacterial strains secreting rhamnolipids were efficient in modulating the barrier function of an in vitro-reconstituted human respiratory epithelium, irrespective of their release of elastase and lipopolysaccharide. In contrast to previous reports, we document that P. aeruginosa was not internalized by epithelial cells. We further report that purified rhamnolipids, applied on the surfaces of the epithelia, were sufficient to functionally disrupt the epithelia and to promote the paracellular invasion of rhamnolipid-deficient P. aeruginosa. The mechanism involves the incorporation of rhamnolipids within the host cell membrane, leading to tight-junction alterations. The study provides direct evidence for a hitherto unknown mechanism whereby the junction-dependent barrier of the respiratory epithelium is selectively altered by rhamnolipids

Structure and Sequence Requirements for RNA Capping at the Venezuelan Equine Encephalitis Virus RNA 5′ End

International audienceVenezuelian enquine encephalitis virus (VEEV) is a reemerging arthropod-borne virus causing encephalitis in humans and domesticated animals. VEEV possesses a positive single-stranded RNA genome capped at its 5′ end. The capping process is performed by the nonstructural protein nsP1, which bears methyl and guanylyltransferase activities. The capping reaction starts with the methylation of GTP. The generated m7GTP is complexed to the enzyme to form an m7GMP-nsP1 covalent intermediate. The m7GMP is then transferred onto the 5′-diphosphate end of the viral RNA. Here, we explore the specificities of the acceptor substrate in terms of length, RNA secondary structure, and/or sequence. Any diphosphate nucleosides but GDP can serve as acceptors of the m7GMP to yield m7GpppA, m7GpppC, or m7GpppU. We show that capping is more efficient on small RNA molecules, whereas RNAs longer than 130 nucleotides are barely capped by the enzyme. The structure and sequence of the short, conserved stem-loop, downstream to the cap, is an essential regulatory element for the capping process

Drugs against SARS‐CoV ‐2: What do we know about their mode of action?

International audienceThe health emergency caused by the recent Covid-19 pandemic highlights the need to identify effective treatments against the virus causing this disease (SARS-CoV-2). The first clinical trials have been testing repurposed drugs that show promising anti-SARS-CoV-2 effects in cultured cells. Although more than 2400 clinical trials are already under way, the actual number of tested compounds is still limited to approximately 20, alone or in combination. In addition, knowledge on their mode of action (MoA) is currently insufficient. Their first results reveal some inconsistencies and contradictory results and suggest that cohort size and quality of the control arm are two key issues for obtaining rigorous and conclusive results. Moreover, the observed discrepancies might also result from differences in the clinical inclusion criteria, including the possibility of early treatment that may be essential for therapy efficacy in patients with Covid-19. Importantly, efforts should also be made to test new compounds with a documented MoA against SARS-CoV-2 in clinical trials. Successful treatment will probably be based on multitherapies with antiviral compounds that target different steps of the virus life cycle. Moreover, a multidisciplinary approach that combines artificial intelligence, compound docking, and robust in vitro and in vivo assays will accelerate the development of new antiviral molecules. Finally, large retrospective studies on hospitalized patients are needed to evaluate the different treatments with robust statistical tools and to identify the best treatment for each Covid-19 stage. This review describes different candidate antiviral strategies for Covid-19, by focusing on their mechanism of action

The C-Terminal Domain of the Sudan Ebolavirus L Protein Is Essential for RNA Binding and Methylation

International audienceThe large (L) protein of Ebola virus is a key protein for virus replication. Its N-terminal region harbors the RNA-dependent RNA polymerase activity, and its C terminus contains a cap assembling line composed of a capping domain and a methyltransferase domain (MTase) followed by a C-terminal domain (CTD) of unknown function. The L protein MTase catalyzes methylation at the 2'-O and N-7 positions of the cap structures. In addition, the MTase of Ebola virus can induce capin-dependent internal adenosine 2'-O-methylation. In this work, we investigated the CTD role in the regulation of the cap-dependent and cap-independent MTase activities of the L protein. We found that the CTD, which is enriched in basic amino acids, plays a key role in RNA binding and in turn regulates the different MTase activities. We demonstrated that the mutation of CTD residues modulates specifically the different MTase activities. Altogether, our results highlight the pivotal role of the L protein CTD in the control of viral RNA methylation, which is critical for Ebola virus replication and escape from the innate response in infected cells.IMPORTANCE Ebola virus infects human and nonhuman primates, causing severe infections that are often fatal. The epidemics, in West and Central Africa, emphasize the urgent need to develop antiviral therapies. The Ebola virus large protein (L), which is the central protein for viral RNA replication/transcription, harbors a methyltransferase domain followed by a C-terminal domain of unknown function. We show that the C-terminal domain regulates the L protein methyltransferase activities and consequently participates in viral replication and escape of the host innate immunity

First insights into the structural features of Ebola virus methyltransferase activities

International audienceThe Ebola virus is a deadly human pathogen responsible for several outbreaks in Africa. Its genome encodes the 'large' L protein, an essential enzyme that has polymerase, capping and methyltransferase activities. The methyltransferase activity leads to RNA co-transcriptional modifications at the N7 position of the cap structure and at the 2-O position of the first transcribed nucleotide. Unlike other Mononegavirales viruses, the Ebola virus methyltransferase also catalyses 2-O-methylation of adenosines located within the RNA sequences. Herein, we report the crystal structure at 1.8Å resolution of the Ebola virus methyltransferase domain bound to a fragment of a camelid single-chain antibody. We identified structural determinants and key amino acids specifically involved in the internal adenosine-2-Omethylation from cap-related methylations. These results provide the first high resolution structure of an ebolavirus L protein domain, and the framework to investigate the effects of epitranscriptomic modifications and to design possible antiviral drugs against the Filoviridae family

Design, Synthesis and Discovery of N,N'-Carbazoyl-aryl-urea Inhibitors of Zika NS5 Methyltransferase and Virus Replication

The recent outbreaks of Zika virus (ZIKV) infection worldwide make the discovery of novel antivirals against flaviviruses a research priority. This work describes the identification of novel inhibitors of ZIKV through a structure-based virtual screening approach using the ZIKV NS5-MTase. A novel series of molecules with a carbazoyl-aryl-urea structure has been discovered and a library of analogues has been synthesized. The new compounds inhibit ZIKV MTase with IC50 between 23-48 μM. In addition, carbazoyl-aryl-ureas also proved to inhibit ZIKV replication activity at micromolar concentration.status: publishe