The severity of the global COVID-19 pandemic, with a high transmission rate, 2.6-4.7% lethality and a huge economic impact, poses an urgent need for efficient medical treatments and vaccines. Currently, there are only non-specific treatments to assist the patients in acute respiratory distress during the inflammatory step following the preliminary infection by SARS-CoV-2. Clinical trials of drug repurposing were quickly launched at the international level. Specific treatments such as the transfusion of plasma from patients who have recovered into infected patients or the use of specific inhibitors of the viral RNA-polymerase complex are promising strategies to block infection. To complete the therapeutic arsenal, we believe that the opportunity of targeting the SARS-CoV-2 genome by RNA therapy should be deeply investigated. In the present paper, we propose to design specific antisense oligonucleotides targeting transcripts encoding viral proteins associated to replication and transcription of SARS-CoV-2, aiming to block infection. We designed antisense oligonucleotides targeting the genomic 5’ untranslated region (5’-UTR), open reading frames 1a and 1b (ORF1a and ORF1b) governing expression of the replicase/transcriptase complex, and the gene N encoding the nucleoprotein that is genome-associated. To maximize the probability of efficiency, we predicted the antisense oligonucleotides by using two design methods: i) conventional antisense oligonucleotides with 100% phosphorothioate modifications (ASO); ii) antisense locked nucleic acids GapmeR. After binding the viral RNA target, the hetero-duplexes antisense oligonucleotide-RNA are cleaved by RNAse H1. Nine potent ASO candidates were found and we selected five of them targeting ORF1a (3), ORF1b (1) and N (1). Nine GapmeR candidates were predicted with excellent properties and we retained four of them targeting 5’-UTR (1), ORF1a (3), ORF1b (1) and N (1). The most potent GapmeR candidate targets the 5’-UTR, a key genomic domain with multiple functions in the viral cycle. By this open publication, we are pleased to share these in silico results with the scientific community in hopes of stimulating innovation in translational research in order to fight the unprecedented COVID-19 pandemic. These antisense oligonucleotide candidates should be now experimentally evaluated.

Respiratory syncytial virus (RSV) RNA synthesis takes place in cytoplasmic viral factories also called inclusion bodies (IBs), which are membrane-less organelles concentrating the viral RNA polymerase complex. IBs assembly is driven by liquid-liquid phase separation promoted by interactions between the viral nucleoprotein N and the phosphoprotein P. We recently demonstrated that cyclopamine (CPM) inhibits RSV multiplication by disorganizing and hardening IBs. Although a single mutation in the viral transcription factor M2-1 induced resistance to CPM, the mechanism of action of CPM still remains to be characterized. Here, using FRAP experiments on reconstituted pseudo-IBs both in cellula and in vitro, we first demonstrated that CPM activity depends on the presence of M2-1 together with N and P. We show that CPM impairs the competition between P and RNA binding to M2-1. As mutations on both P and M2-1 induced resistance against CPM activity, we suggest that CPM may affect the dynamics of the M2-1−P interaction, thereby affecting the relative mobility of proteins contained in RSV IBs. Overall, our results reveal that stabilizing viral protein-protein interactions is an attractive new antiviral approach. They pave the way for the rational chemical optimization of new specific anti-RSV molecules.

Human respiratory syncytial virus (hRSV) is the most common cause of bronchiolitis and pneumonia in new-borns, with all children being infected before the age of two. Reinfections are very common throughout life, and can cause severe respiratory infections in the elderly and immunocompromised adults. Although vaccines and preventive antibodies have recently been licensed for use in specific subpopulations of patients, there is still no therapeutic treatment commonly available for these infections. Here, we investigated the potential antiviral activity of Retro-2.2, a derivative of the cellular retrograde transport inhibitor Retro-2, against hRSV. We show that Retro-2.2 inhibits hRSV replication in cell culture, and impairs the ability of hRSV to form syncytia. Our results suggest that Retro-2.2 treatment affects virus spread by disrupting the trafficking of the viral de novo synthetized F and G glycoproteins to the plasma membrane, leading to a defect in virion morphogenesis. Taken together, our data show that targeting intracellular transport may be an effective strategy against hRSV infection.