Biomolecular condensates have emerged as an important subcellular organizing principle 1 . Replication of many viruses, including human respiratory syncytial virus (RSV), occurs in virus-induced compartments called inclusion bodies (IBs) or viroplasm 2,3 . IBs of negative-strand RNA viruses were recently shown to be biomolecular condensates that form through phase separation 4,5 . Here we report that the steroidal alkaloid cyclopamine and its chemical analogue A3E inhibit RSV replication by disorganizing and hardening IB condensates. The actions of cyclopamine and A3E were blocked by a point mutation in the RSV transcription factor M2-1. IB disorganization occurred within minutes, which suggests that these molecules directly act on the liquid properties of the IBs. A3E and cyclopamine inhibit RSV in the lungs of infected mice and are condensate-targeting drug-like small molecules that have in vivo activity. Our data show that condensate-hardening drugs may enable the pharmacological modulation of not only many previously undruggable targets in viral replication but also transcription factors at cancer-driving super-enhancers 6 .RSV is a major cause of respiratory illness in young children, the older people and individuals who are immunocompromised worldwide 7,8 . Currently, multiple targets are pursued for the development of a safe and effective therapy to treat RSV infections 9 .In infected cells, RSV induces the formation of cytoplasmic IBs, in which nucleoprotein (N), phosphoprotein (P), polymerase L, the transcription factor M2-1 and viral genomic RNA are concentrated. We recently demonstrated that IBs are 'viral factories' in which viral RNA synthesis occurs 3 . The morphology of IBs suggests that they are condensates formed by liquid-liquid phase separation (LLPS). A recent study showed that N and P were sufficient to drive the formation of pseudo-IB condensates through LLPS in vitro, both in cells and in biochemical assays 10 . However, these N-P pseudo-IB condensates are not functional, as they do not shelter RNA synthesis and do not reflect the complexity of IBs in virus-infected cells, which have multiple compartments. Strikingly similar in size and phase organization to the nucleolus condensate 11 , RSV IBs are multiphasic and contain a sub-compartment called the IB-associated granule (IBAG), which is composed of newly synthesized viral mRNA and M2-1 3,12 . Condensates have emerged as an important subcellular organizing principle 1 . An important question in anti-viral drug developmentand medicinal chemistry more generally-is whether these condensates are druggable. In principle, a drug that dissolved or hardened would prevent viral replication. Neither mechanism has yet been reported. Chemical analogues without hedgehog antagonismWe previously identified the hedgehog (HH) pathway antagonist cyclopamine (CPM) as a potent inhibitor of RSV replication 13 . Inhibition of Sonic hedgehog (SHH) signalling is an unwanted feature of CPM as an RSV inhibitor. On the basis of the binding model of the Smoothe...

Hepatitis C virus (HCV) has no animal reservoir, infecting only humans. To investigate species barrier determinants limiting infection of rodents, murine liver complementary DNA library screening was performed, identifying transmembrane proteins Cd302 and Cr1l as potent restrictors of HCV propagation. Combined ectopic expression in human hepatoma cells impeded HCV uptake and cooperatively mediated transcriptional dysregulation of a noncanonical program of immunity genes. Murine hepatocyte expression of both factors was constitutive and not interferon inducible, while differences in liver expression and the ability to restrict HCV were observed between the murine orthologs and their human counterparts. Genetic ablation of endogenous Cd302 expression in human HCV entry factor transgenic mice increased hepatocyte permissiveness for an adapted HCV strain and dysregulated expression of metabolic process and host defense genes. These findings highlight human-mouse differences in liver-intrinsic antiviral immunity and facilitate the development of next-generation murine models for preclinical testing of HCV vaccine candidates.

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Human respiratory syncytial virus (hRSV) infection is a leading cause of severe respiratory tract infections. Effective, directly acting antivirals against hRSV are not available.

Adjusting the protecting group strategy, from an alkyl ether to a bidentate ketal at the carbohydrate backbone of uridine, facilitates a switchable diastereoselective α- or β-C4′/C5′-spirocyclopropanation. Using these spirocyclopropanated nucleosides as key intermediates, we synthesized a variety of C4′-methylated d-ribose and l-lyxose-configured uridine derivatives by a base-mediated ring-opening of the spirocyclopropanol moiety. Investigations of antiviral activity against the human respiratory syncytial virus were carried out for selected derivatives, showing moderate activity.

Libraries composed of licensed drugs represent a vast repertoire of molecules modulating physiologic processes in humans, thus providing unique opportunities for discovery of host targeting antivirals. We interrogated the ReFRAME repurposing library with 12,993 molecules for broad-spectrum coronavirus antivirals and discovered 134 compounds inhibiting an alphacoronavirus, mapping to 59 molecular target categories. Dominant targets included the 5-hydroxytryptamine receptor and dopamine receptor and cyclin-dependent kinase inhibitors. Counter-screening with SARS-CoV-2 and validation in primary cells identified Phortress, an aryl hydrocarbon receptor (AHR) ligand, Bardoxolone and Omaveloxolone, two nuclear factor, erythroid 2 like 2 (NFE2L2) activators as inhibitors of both alpha- and betacoronaviruses. The landscape of coronavirus targeting molecules provides important information for the development of broad-spectrum antivirals reinforcing pandemic preparedness.