There is an urgent need for antiviral agents that treat SARS-CoV-2 infection. We screened a library of 1,900 clinically safe drugs against OC43, a human beta-coronavirus that causes the common cold and evaluated the top hits against SARS-CoV-2. Twenty drugs significantly inhibited replication of both viruses in vitro. Eight of these drugs inhibited the activity of the SARS-CoV-2 main protease, 3CLpro, with the most potent being masitinib, an orally bioavailable tyrosine kinase inhibitor. X-ray crystallography and biochemistry show that masitinib acts as a competitive inhibitor of 3CLpro. Mice infected with SARS-CoV-2 and then treated with masitinib showed >200-fold reduction in viral titers in the lungs and nose, as well as reduced lung inflammation. Masitinib was also effective in vitro against all tested variants of concern (B.1.1.7, B.1.351 and P.1).

The human polyoma viruses JCV and BKV establish asymptomatic persistent infection in 65%-90% of humans but can cause severe illness under immunosuppressive conditions. The mechanisms by which these viruses evade immune recognition are unknown. Here we show that a viral miRNA identical in sequence between JCV and BKV targets the stress-induced ligand ULBP3, which is a protein recognized by the killer receptor NKG2D. Consequently, viral miRNA-mediated ULBP3 downregulation results in reduced NKG2D-mediated killing of virus-infected cells by natural killer (NK) cells. Importantly, when the activity of the viral miRNA was inhibited during infection, NK cells killed the infected cells more efficiently. Because NKG2D is also expressed by various T cell subsets, we propose that JCV and BKV use an identical miRNA that targets ULBP3 to escape detection by both the innate and adaptive immune systems, explaining how these viruses remain latent without being eliminated by the immune system.

Viral infection is usually studied at the population level by averaging over millions of cells. However, infection at the single-cell level is highly heterogeneous, with most infected cells giving rise to no or few viral progeny while some cells produce thousands. Analysis of Herpes Simplex virus 1 (HSV-1) infection by population-averaged measurements has taught us a lot about the course of viral infection, but has also produced contradictory results, such as the concurrent activation and inhibition of type I interferon signaling during infection. Here, we combine live-cell imaging and single-cell RNA sequencing to characterize viral and host transcriptional heterogeneity during HSV-1 infection of primary human cells. We find extreme variability in the level of viral gene expression among individually infected cells and show that these cells cluster into transcriptionally distinct sub-populations. We find that anti-viral signaling is initiated in a rare group of abortively infected cells, while highly infected cells undergo cellular reprogramming to an embryonic-like transcriptional state. This reprogramming involves the recruitment of β-catenin to the host nucleus and viral replication compartments, and is required for late viral gene expression and progeny production. These findings uncover the transcriptional differences in cells with variable infection outcomes and shed new light on the manipulation of host pathways by HSV-1.

9Viral infection is usually studied at the population level by averaging over millions of cells. However, 10 infection at the single-cell level is highly heterogeneous. Here, we combine live-cell imaging and single-11 cell RNA sequencing to characterize viral and host transcriptional heterogeneity during HSV-1 infection of 12 primary human cells. We find extreme variability in the level of viral gene expression among individually 13 infected cells and show that they cluster into transcriptionally distinct sub-populations. We find that anti-14 viral signaling is initiated in a rare group of abortively infected cells, while highly infected cells undergo 15 cellular reprogramming to an embryonic-like transcriptional state. This reprogramming involves the 16 recruitment of beta-catenin to the host nucleus and viral replication compartments and is required for late 17 viral gene expression and progeny production. These findings uncover the transcriptional differences in 18 cells with variable infection outcomes and shed new light on the manipulation of host pathways by HSV-19 1. 20 21 24 throughout the host life with occasional reactivation. Here, we focus on the lytic part of the virus life cycle. 57While lytic infection is usually asymptomatic, in some cases -particularly in immune-compromised 58 individuals and infants -it can results in life threatening conditions such as meningitis and encephalitis. 59To initiate infection, HSV-1 must bind to its receptors, enter the cytoplasm, travel to the nuclear pore and 60 inject its linear double-stranded DNA into the host nucleus (Kobiler et al., 2012). Once in the nucleus, viral 61 gene expression proceeds in a temporal cascade of three classes of viral genes: immediate-early, early and 62 late Roizman, 1974, 1975;Harkness et al., 2014). DNA replication occurs in sub-nuclear 63 structures, called replication compartments (RCs), that aggregate the seven essential replication proteins as 64 well as other viral and host proteins (de Bruyn Kops and Knipe, 1988;Liptak et al., 1996; Weller and Coen, 65 2012;Dembowski and DeLuca, 2015;Dembowski et al., 2017; Reyes et al., 2017; Dembowski and DeLuca, 66 2018). ICP4 is the major viral trans-activator and is required for viral infection to progress beyond the point 67 of immediate-early gene expression. Upon viral DNA replication, ICP4 is predominantly localized in the 68 RCs, with some diffuse nuclear and cytoplasmic localization (Knipe et al., 1987;Zhu and Schaffer, 1995). 69Several studies applied high-throughput technologies to analyze the cellular response to HSV-1 infection 70 at the population level. RNA sequencing revealed a widespread deregulation of host transcription, including 71

Extended Data Fig. 5 | Comparison of the drug screening results using different variations of the network proximity-based screening methods. (a) Network proximity-based drug screening using directed human protein-protein interactome vs. undirected human protein-protein interactome. (b) Network proximity-based drug screening using degree preserved edge shuffling vs. degree preserved node shuffling. PCC, Pearson correlation coefficient.