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Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome (1, 2) . The frameshift stimulation element (FSE) of the SARS-CoV-2 genome is required for balanced expression of essential viral proteins and is highly conserved, making it a potential candidate for antiviral targeting by small molecules and oligonucleotides (3–6) . To aid global efforts focusing on SARS-CoV-2 frameshifting, we report exploratory results from frameshifting and cellular replication experiments with locked nucleic acid (LNA) antisense oligonucleotides (ASOs), which support the FSE as a therapeutic target but highlight difficulties in achieving strong inactivation. To understand current limitations, we applied cryogenic electron microscopy (cryo-EM) and the Ribosolve (7) pipeline to determine a three-dimensional structure of the SARS-CoV-2 FSE, validated through an RNA nanostructure tagging method. This is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date. The tertiary structure model, defined to an estimated accuracy of 5.9 Å, presents a topologically complex fold in which the 5′ end threads through a ring formed inside a three-stem pseudoknot. Our results suggest an updated model for SARS-CoV-2 frameshifting as well as binding sites that may be targeted by next generation ASOs and small molecules.

Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome1, 2. The frameshift stimulation element (FSE) of the SARS-CoV-2 genome is required for balanced expression of essential viral proteins and is highly conserved, making it a potential candidate for antiviral targeting by small molecules and oligonucleotides3–6. To aid global efforts focusing on SARS-CoV-2 frameshifting, we report exploratory results from frameshifting and cellular replication experiments with locked nucleic acid (LNA) antisense oligonucleotides (ASOs), which support the FSE as a therapeutic target but highlight difficulties in achieving strong inactivation. To understand current limitations, we applied cryogenic electron microscopy (cryo-EM) and the Ribosolve7 pipeline to determine a three-dimensional structure of the SARS-CoV-2 FSE, validated through an RNA nanostructure tagging method. This is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date. The tertiary structure model, defined to an estimated accuracy of 5.9 Å, presents a topologically complex fold in which the 5′ end threads through a ring formed inside a three-stem pseudoknot. Our results suggest an updated model for SARS-CoV-2 frameshifting as well as binding sites that may be targeted by next generation ASOs and small molecules.

Authors thank the iBio platform (UMR Ecologie Microbienne, Universite Lyon 1) for bioinformatics processor resources and support, and Sylvie Barraud for support on chemical data analyses. They also thank OTHU (Field Observatory in Urban Hydrology) of ZABR (Zone Atelier Bassin du Rhone), the Grand Lyon Metropolis and Rhone-Mediterranean-Corsica Water Agency for technical and administrative supports.; International audience; Urban activities generate surface deposits over impervious surfaces that can represent ecological and health hazards. Bacteriome genetic structures of deposits washed off during rainfall events, over an urban industrial watershed, were inferred from 16S rRNA gene (rrs) sequences generated by high throughput sequencing. Deposits were sampled over a 4 year-period from a detention basin (DB). Major shifts, matching key management practices, in the structure of these urban bacteriomes, were recorded. Correlation analyses of rrs similarities between samples and their respective concentrations in chemical pollutants, markers of human fecal contaminations (HF183) and antimicrobial resistances (integrons), were performed. Harsher environmental constraints building up in the older deposits led to an increase number of rrs reads from extremophiles such as Acidibacter and Haliangium. Deposits accumulating in the decantation pit of the DB showed an increase in rrs reads from warm blooded intestinal tract bacteria such as Bacteroides and Prevotella. This enrichment matched higher concentrations of Bacteroides HF183 genotypes normally restricted to humans. Bacteriomes of urban deposits appeared good indicators of human-driven environmental changes. Their composition was found representative of their origin. Soil particles and rain appeared to be major contributors of the inferred bacterial taxa recovered from recent deposits.

Influenza A virus’s (IAV’s) frequent genetic changes challenge vaccine strategies and engender resistance to current drugs. We sought to identify conserved and essential RNA secondary structures within IAV’s genome that are predicted to have greater constraints on mutation in response to therapeutic targeting. We identified and genetically validated an RNA structure (packaging stem–loop 2 (PSL2)) that mediates in vitro packaging and in vivo disease and is conserved across all known IAV isolates. A PSL2-targeting locked nucleic acid (LNA), administered 3 d after, or 14 d before, a lethal IAV inoculum provided 100% survival in mice, led to the development of strong immunity to rechallenge with a tenfold lethal inoculum, evaded attempts to select for resistance and retained full potency against neuraminidase inhibitor-resistant virus. Use of an analogous approach to target SARS-CoV-2, prophylactic administration of LNAs specific for highly conserved RNA structures in the viral genome, protected hamsters from efficient transmission of the SARS-CoV-2 USA_WA1/2020 variant. These findings highlight the potential applicability of this approach to any virus of interest via a process we term ‘programmable antivirals’, with implications for antiviral prophylaxis and post-exposure therapy.

Background. Parkinson's disease (PD) is a disorder characterized by dopaminergic neuron programmed cell death. The etiology of PD remains uncertain-some cases are due to selected genes associated with familial heredity, others are due to environmental exposure to toxic components, but over 90% of cases have a sporadic origin. No cardia are Actinobacteria that can cause human diseases like nocardiosis. This illness can lead to lung infection or central nervous system(CNS) invasion in both immunocompromised and immunocompetent individuals. The main species involved in CNS are N. farcinica, N. nova, N. brasiliensis and N. cyriacigeorgica. Some studies have highlighted the ability of N. cyriacigeorgica to induce Parkinson's disease-like symptoms in animals. Actinobacteria are known to produce a large variety of secondary metabolites, some of which can be neurotoxic. We hypothesized that neurotoxic secondary metabolite production and the onset of PD-like symptoms in animals could be linked. Methods. Here we used a method to screen bacteria that could induce dopaminergic neurodegeneration before performing mouse experiments. Results. The nematode Caenorhabditis elegans allowed us to demonstrate that Nocardia strains belonging to N. cyriacigeorgica and N. farcinica species can induce dopaminergic neurodegeneration. Strains of interest involved with the nematodes in neurodegenerative disorders were then injected in mice. Infected mice had behavioral disorders that may be related to neuronal damage, thus confirming the ability of Nocardia strains to induce neurodegeneration. These behavioral disorders were induced by N. cyriacigeorgica species (N. cyriacigeorgica GUH-2 and N. cyriacigeorgica 44484) and N. farcinica 10152. Discussion. We conclude that C. elegans is a good model for detecting Nocardia strains involved in neurodegeneration. This model allowed us to detect bacteria with high neurodegenerative effects and which should be studied in mice to characterize the induced behavioral disorders and bacterial dissemination.