Crystal structures of Lymphocytic choriomeningitis virus endonuclease domain complexed with diketo-acid ligands

International audienceThe Arenaviridae family, together with the Bunyaviridae and Orthomyxoviridae families, is one of the three negative-stranded RNA viral families that encode an endonuclease in their genome. The endonuclease domain is at the N-terminus of the L protein, a multifunctional protein that includes the RNA-dependent RNA polymerase. The synthesis of mRNA in arenaviruses is a process that is primed by capped nucleotides that are 'stolen' from the cellular mRNA by the endonuclease domain in cooperation with other domains of the L protein. This molecular mechanism has been demonstrated previously for the endonuclease of the prototype Lymphocytic choriomeningitis virus (LCMV). However, the mode of action of this enzyme is not fully understood as the original structure did not contain catalytic metal ions. The pivotal role played by the cap-snatching process in the life cycle of the virus and the highly conserved nature of the endonuclease domain make it a target of choice for the development of novel antiviral therapies. Here, the binding affinities of two diketo-acid (DKA) compounds (DPBA and L-742,001) for the endonuclease domain of LCMV were evaluated using biophysical methods. X-ray structures of the LCMV endonuclease domain with catalytic ions in complex with these two compounds were determined, and their efficacies were assessed in an in vitro endonuclease-activity assay. Based on these data and computational simulation, two new DKAs were synthesized. The LCMV endonuclease domain exhibits a good affinity for these DKAs, making them a good starting point for the design of arenavirus endonuclease inhibitors. In addition to providing the first example of an X-ray structure of an arenavirus endonuclease incorporating a ligand, this study provides a proof of concept that the design of optimized inhibitors against the arenavirus endonuclease is possible

Caractérisation de nucléases d'Arenaviridae et développement d'inhibiteurs

Mon projet a porté sur la caractérisation du mécanisme moléculaire des enzymes d'arenavirus (une 3'-5' exoribonucléase et une endonuclease) impliquées dans l'inhibition de la réponse innée IFN de type I et dans le vole de coiffe respectivement, et le développement d'une stratégie thérapeutique basée sur leur structures. Premièrement, j'ai résolu deux structures cristallographiques à haute résolution du domaine exoribonucléases du virus Mopeia (NP-exo MOPV) -un homologue du virus Lassa pathogène- en complexe avec deux ions différents. Ensuite, j'ai effectué une caractérisation fonctionnelle de l’activité exoribonucléase 3'-5' codée par ce domaine. Une corrélation entre la structure et la fonction de NP-exo MOPV démontre que; L’activité exoribonucléase 3'-5' est conservée chez les arenavirus pathogènes ainsi que chez les non-pathogènes. J'ai démontré pour la première fois que l'exoribonucléase est capable d'exciser un ARN misapparié, suggérant ainsi une potentielle activité de correction d'erreur par cette enzyme. Avec la structure de NP-exo MOPV, j'ai développé une stratégie in silico pour identifier des inhibiteurs potentiels spécifiques contre son activité et un inhibiteur a était identifié.En parallèle, nous avons résolu deux structures cristallographiques du domaine de l'endonuclease du virus de la LCMV en complexe avec deux ions catalytiques et deux composés appartenant a la famille des diketo. En résumé, ce travail éclaircit le rôle des exoribonucléases de la famille d'Arenaviridae allant de l’évasion de l'immunité innée à son implication directe dans la réplication. Il ouvre également la voie au développement des inhibiteurs contre ces nucléases.My PhD work focused on the characterization of the molecular mechanism of two arenavirus enzymes - a 3'-5' exoribonuclease and an endonuclease - implicated in type I IFN suppression and mRNA cap-snatching respectively and the design of a structure based-drug strategy against them. First I solved two high resolution crystal structures of the exoribonuclease domain of Mopeia virus (NP-exo MOPV) -a non pathogenic homologue of the highly pathogenic Lassa virus- in complex with different metal ions. Next I performed an in depth functional characterization of the 3'-5' exoribonuclease activity encoded by this domain. By correlating the structure and function of NP-exo MOPV, I showed that; the 3'-5' exoribonuclease activity is conserved in pathogenic as well as in non-pathogenic arenaviruses. Also, I showed for the first time that this enzyme is able to excise a mismatched RNA suggesting that, arenaviruses might posses a mechanism to limit error incorporation by the RdR polymerase during replication. Using the crystal structure of NP-exo MOPV I designed a structure-based strategy to identify potential inhibitors specific for the 3'-5' exoribonuclease activity and have identified a potential inhibitor.Alongside, we solved two crystal structures of the endonuclease domain of LCMV in complex with two catalytic ions and two compounds belonging to the diketo family.In conclusion, this work has a deep implication extending the role of the Arenaviridae exoribonuclease from innate immunity evasion to direct implication in replication. It also paves the way for the development of inhibitors against these arenavirus nucleases

Arenaviridae exoribonuclease presents genomic RNA edition capacity.

The Arenaviridae is a large family of viruses causing both acute and persistent infections and causing significant public health concerns in afflicted regions. A “trademark” of infection is the quick and efficient immuno-suppression mediated in part by a 3’-5’ RNA exonuclease domain (ExoN) of the Nucleoprotein (NP). Mopeia virus, the eastern African counterpart of Lassa virus, carries such ExoN domain, but does not suppress the host innate immunity. We have recently reported the crystal structure of the Mopeia virus ExoN domain, which presents a conserved fold and active site. In the present study, we show that the ExoN activity rules out a direct link between ExoN activity and alteration of the host innate immunity. We found that the Arenavirus ExoN, however, is able to excise mis-incorporated bases present at the 3’-end of double stranded RNA. ExoN(-) arenaviruses cultured in cells dampened in innate immunity still replicated in spite of a significant reduction in the viral charge over several passages. The remaining ExoN(-) virus population showed an increased base substitution rate on a narrow nucleotide spectrum, linking the ExoN activity to genome editing. Since, the Arenavirus ExoN belongs to the same nuclease family as that of the nsp14 coronavirus ExoN; which has been recently shown to promote viral RNA synthesis proofreading; we propose that Arenavirus ExoN is involved in a “limited RNA editing” mechanism mainly controlled by structural constraints and a low mutational/fitness ratio

Rate of viral load change and adherence of HIV adult patients treated with Efavirenz or Nevirapine antiretroviral regimens at 24 and 48 weeks in Yaoundé, Cameroon: a longitudinal cohort study

Abstract Background HIV-load decrease and suppression over time is associated with consistent adherence to antiretroviral therapy (ART). Our study aimed to evaluate the difference in viral load and adherence of patients treated with a combination of either Tenofovir (TDF), Lamivudine (3TC) and Efavirenz (EFV) or TDF / Zidovudine (AZT), 3TC and Nevirapine (NVP) regimens at 24 and 48 weeks. Methods A longitudinal study was conducted from May 2016 to June 2017 among 256 HIV infected adult patients who were enrolled at two approved treatment hospitals in Yaoundé, before the start of first-line ART. Whole blood samples were collected using standard operating procedures. HIV-loads were determined by a quantitative RealTime PCR assay. Adherence was evaluated by pharmacy refill data records. Statistical analyses were performed using the PRISM 5.0 software. Results Off the 256 HIV infected patients enrolled, 180 (70%) patients completed the study and 76 (30%) patients were lost to follow-up. The success rate in achieving viral load < 40 copies/ml was 1.8 times higher with the EFV regimen at 24 weeks and was 1.2 times higher in the NVP regimen at 48 weeks. At 48 weeks the treatment failure rate was 12.0 and 40.0% in patients on EFV and the NVP regimen, respectively. The rate of adherence varied in both ART based regimens with 84.0 to 74.0% for EFV and 65.5 to 62.5% for NVP, at 24 and 48 weeks respectively. Conclusion In our study and setting, the rate of viral load decrease was higher in the NVP based regimen than with the EFV regimen. The adherence rate to ART was higher in the EFV regimen, compared to the NVP regimen. This adds to evidence that the EFV regimen is the preferred ART combination for non-nucleoside reverse transcriptase inhibitors (NNRTIs)

Reinforced cementitious panels evaluated

Aussi disponible en fran\ue7ais: \uc9valuation des panneaux cimentaires renforc\ue9sPeer reviewed: NoNRC publication: Ye

Structural characterisation and inhibition of Arenavirus replication complex elements : assembly, function and inhibition of embedded nucleases

Arenaviruses, belongs to a family of emerging enveloped segmented and ambisens RNA viruses associated withneurological and hemorrhagic diseases in humans. Arenavirus transcription and genome replication are cytoplasmic ensured by aribonucleoproteine replicase complex NP-L. After penetration, L protein initiates transcription to produce NP and L mRNAs[ 1].The priming of transcription is the result of a cap-snatching mechanism ensured by an endonuclease domain associated to the Lpolymerase. As the concentration of NP in the cell increases, genome segments are replicated, to produce full-length copies(cRNA). cRNAs are now templates for transcription of GPC mRNA (from the S segment) and Z mRNA (from the L segment).The NP caries an exonuclease in charge of clearing out from the cytoplasm dsRNA triggering innate immunity response. Bothnucleases have a similar two metal ion catalytic mechanism, with the particularity of transitioning ion brought by the RNAsubstrate. Any alteration of the remaining ion impairs greatly theses activities[2]. We present a global study aiming to characterizethe assembly of the NP[3], through flexible domains[4], a step critical for vRNApackaging and the polsitioning of L for vRNAreplication, as well as using a combined approach of biophysical screening, crystallography and in silico docking, identifyingactive compounds against both nucleases[5]. Crystal structures of the nucleases domain complexed with several compounds wereobtained[67]. By developing specific compounds to alter both transcription and innate immunity shadowing, our strategy is togive the cell a fighting chance to clear the infection. Combining structure, enzymology, rational synthesis, hit-To-leadoptimization, in cellula evaluation, and screening methods, we are presenting the results of a 2nd generation of molecules pavingthe way to the design of a 3rd generation increasing specificity towards Arenaviral nucleases in the context of the replicationcomplex[8]

Spatiotemporal circulation of influenza viruses in 5 African countries during 2008-2009: a collaborative study of the Institut Pasteur International Network.

International audienceBACKGROUND: Although recent work has described the spatiotemporal diffusion of influenza viruses worldwide, comprehensive data on spatiotemporal patterns of influenza from the African continent and Madagascar are still lacking. METHODS: National Influenza Centers from 5 countries-Cameroon, Côte d'Ivoire, Madagascar, Niger, and Senegal--collected specimens from patients presenting with influenza-like illness who visited sentinel surveillance clinics during a 2-year period (2008-2009). Isolates were genetically and antigenically characterized. RESULTS: Overall, 8312 specimens were tested. Seasonal influenza A virus subtypes H1N1 and H3N2 and influenza B viruses were detected in 329, 689, and 148 specimens, respectively. In 2009, pandemic influenza A virus subtype H1N1 was detected in Madagascar most commonly (98.5% of cases). Influenza activity was either significant year-round or occurred during a specific period of the year in the African countries we evaluated. CONCLUSIONS: Our results demonstrate that, from Madagascar to Senegal, the epidemiologic and virologic characteristics of influenza viruses are diverse in terms of spatiotemporal circulation of the different virus types, subtypes, and strains. Our data highlight the importance of country-specific surveillance and of data and virus sharing, and they provide a rational basis to aid policy makers to develop strategies, such as vaccination at the right moment and with the right formulation, aimed at reducing the disease burden in Africa and Madagascar