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]

Identification of potent inhibitors of arenavirus and SARS-CoV-2 exoribonucleases by fluorescence polarization assay

International audienceViral exoribonucleases are uncommon in the world of RNA viruses. To date, they have only been identified in the Arenaviridae and the Coronaviridae families. The exoribonucleases of these viruses play a crucial role in the pathogenicity and interplay with host innate immune response. Moreover, coronaviruses exoribonuclease is also involved in a proofreading mechanism ensuring the genetic stability of the viral genome. Because of their key roles in virus life cycle, they constitute attractive target for drug design.Here we developed a sensitive, robust and reliable fluorescence polarization assay to measure the exoribonuclease activity and its inhibition in vitro. The effectiveness of the method was validated on three different viral exoribonucleases, including SARS-CoV-2, Lymphocytic Choriomeningitis and Machupo viruses. We performed a screening of a focused library consisting of 113 metal chelators. Hit compounds were recovered with an IC50 at micromolar level. We confirmed 3 hits in SARS-CoV-2 infected Vero-E6 cells