Molecular basis of RIG-I activation

The innate immune system represents the first line of defense developed by organisms to fight against infections. Its efficiency strictly depends on its ability to recognize pathogens as foreign, i.e. on its capacity to discriminate between self and non-self molecules. To do so, the cell uses specific receptors such as RIG-I, that is dedicated to the recognition of RNA viruses in the cytoplasm. How does RIG-I discriminate viral RNAs from cellular RNAs? What are the molecular features which are recognized by RIG-I and activate it? What are the strategies set up by viruses to avoid immune recognition? To answer these questions, an extensive study based on synthetic RNAs has been performed. These results were then applied to the model of Influenza virus. Altogether, while providing a functional validation of the structural data obtained by crystalizing this cytoplasmic sensor, this works allowed us to define a new model of RIG-I activation

RIG-I ATPase activity and discrimination of self-RNA versus non-self-RNA.

Many RNA viruses are detected by retinoic acid-inducible gene i (RIG-I), a cytoplasmic sensor that triggers an antiviral response upon binding non-self-RNA that contains a stretch of double-stranded RNA (dsRNA) bearing a base-paired 5' ppp nucleotide. To gain insight into how RIG-I discriminates between self-RNA and non-self-RNA, we used duplexes whose complementary bottom strand contained both ribo- and deoxynucleotides. These duplexes were examined for their binding to RIG-I and their relative abilities to stimulate ATPase activity, to induce RIG-I dimerization on the duplex, and to induce beta interferon (IFN-β) expression. We show that the chemical nature of the bottom strand is not critical for RIG-I binding. However, two key ribonucleotides, at positions 2 and 5 on the bottom strand, are minimally required for the RIG-I ATPase activity, which is necessary but not sufficient for IFN-β stimulation. We find that duplexes with shorter stretches of dsRNA, as model self-RNAs, bind less stably to RIG-I but nevertheless have an enhanced ability to stimulate the ATPase. Moreover, ATPase activity promotes RIG-I recycling on RIG-I/dsRNA complexes. Since pseudo-self-RNAs bind to RIG-I less stably, they are preferentially recycled by ATP hydrolysis that weakens the helicase domain binding of dsRNA. Our results suggest that one function of the ATPase is to restrict RIG-I signaling to its interaction with non-self-RNA. A model of how this discrimination occurs as a function of dsRNA length is presented

Mismatches in Influenza A virus vRNA panhandle prevent RIG-I sensing by impairing RNA/RIG-I complex formation

Influenza virus RNA promoter panhandle structures are believed to be sensed by RIG-I. The occurrence of mismatches in this dsRNA structure raises questions about their effect on innate sensing. Our results suggest that mismatches in vRNA promoters decrease binding to RIG-I in vivo, affecting RNA/RIG-I complex formation, and preventing RIG-I activation. These results can be inferred to apply to other viruses and suggest that mismatches may represent a general viral strategy to escape RIG-I sensing

Influenza A Virus Genetic Tools: From Clinical Sample to Molecular Clone

Implementation of reverse genetics for influenza A virus, that is, the DNA-based generation of infectious viral particles in cell culture, opened new avenues to investigate the function of viral proteins and their interplay with host factors on a molecular level. This powerful technique allows the introduction, depletion, or manipulation of any given sequence in the viral genome, as long as it gives rise to replicating virus progeny. Reverse genetics can be used to generate targeted reassortant viruses by mixing segments of different viral strains, thus providing insight into phenotypes of potentially pandemic viruses arising from natural reassortment. It was further instrumental for the development of novel vaccine strategies, allowing rapid and targeted exchange of viral surface antigens on a well-replicating genetic backbone of cell culture-adapted or cold-adapted/attenuated viral strains. Establishment of reverse genetics and rescue of molecular clones of influenza A virus have been extensively described before. Here we give a detailed stand-alone protocol encompassing clinical sampling of influenza A virus specimens and subsequent plasmid-based genetics to rescue, manipulate, and confirm a fully infectious molecular clone. This protocol is based on the combined techniques and experience of a number of influenza laboratories, which are credited and referenced whenever appropriate

A quantitative model for virus uncoating predicts influenza A infectivity

For virus infection of new host cells, the disassembly of the protective outer protein shell (capsid) is a critical step, but the mechanisms and host-virus interactions underlying the dynamic, active, and regulated uncoating process are largely unknown. Here, we develop an experimentally supported, multiscale kinetics model that elucidates mechanisms of influenza A virus (IAV) uncoating in cells. Biophysical modeling demonstrates that interactions between capsid M1 proteins, host histone deacetylase 6 (HDAC6), and molecular motors can physically break the capsid in a tug-of-war mechanism. Biochemical analysis and biochemical-biophysical modeling identify unanchored ubiquitin chains as essential and allow robust prediction of uncoating efficiency in cells. Remarkably, the different infectivity of two clinical strains can be ascribed to a single amino acid variation in M1 that affects binding to HDAC6. By identifying crucial modules of viral infection kinetics, the mechanisms and models presented here could help formulate novel strategies for broad-range antiviral treatment.ISSN:2666-3864ISSN:2211-124

Lectin genes in the Frankia alni genome

International audienceFrankia alni strain ACN14a's genome was scanned for the presence of determinants involved in interactions with its host plant, Alnus spp. One such determinant type is lectin, proteins that bind speciWcally to sugar motifs. The genome of F. alni was found to contain 7 such lectincoding genes, Wve of which were of the ricinB-type. The proteins coded by these genes contain either only the lectin domain, or also a heat shock protein or a serine-threonine kinase domain upstream. These lectins were found to have several homologs in Streptomyces spp., and a few in other bacterial genomes among which none in Frankia EAN1pec and CcI3 and two in strain EUN1f. One of these F. alni genes, FRAAL0616, was cloned in E. coli, fused with a reporter gene yielding a fusion protein that was found to bind to both root hairs and to bacterial hyphae. This protein was also found to modify the dynamics of nodule formation in A. glutinosa, resulting in a higher number of nodules per root. Its role could thus be to permit binding of microbial cells to root hairs and help symbiosis to occur under conditions of low Frankia cell counts such as in pioneer situations

10 ans d'A&S

Anthropologie & Santé a 10 ans. C'est pour nous l'occasion de rappeler l'histoire et les valeurs de la revue, d’expliciter son fonctionnement et son modèle économique. Nous avons également voulu donner à ce numéro de transition une dimension récréative, affranchie de la rigueur et des codes académiques des revues scientifiques auxquels nous sommes attachées. Ce numéro accueille donc d’autres formes d’écriture de l’anthropologie ou des textes qui abordent des fragments d’expérience de la recherche plus rarement décrits ou analysés dans les articles scientifiques. Leur facture laisse plus de place à la métaphore, à la poésie, à l’humour et à la narration – sans toutefois se départir de la précision des concepts. Anthropologie & Santé is 10 years old. This anniversary is an opportunity for us to recall the journal's history and values, to explain its functioning and its economic model. We also wanted to give this transitional issue a recreational dimension, freed from the rigor and academic codes of the scientific journals to which we are attached. This issue therefore welcomes other forms of writing in anthropology, or texts dealing with fragments of research experience that are more rarely described or analysed in scientific papers. Their writing forms leave room for metaphor, poetry, humour and narratives