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).

In late 2019 a human coronavirus, now known as SARS-CoV-2, emerged, likely from a zoonotic reservoir. This virus causes COVID-19 disease, has infected millions of people, and has led to hundreds of thousands of deaths across the globe. While the best interventions to control and ultimately stop the pandemic are prophylactic vaccines, antiviral therapeutics are important to limit morbidity and mortality in those already infected. At this time, only one FDA approved anti-SARS-CoV-2 antiviral drug, remdesivir, is available and unfortunately, its efficacy appears to be limited. Thus, the identification of new and efficacious antivirals is of highest importance. In order to facilitate rapid drug discovery, flexible, sensitive, and high-throughput screening methods are required. With respect to drug targets, most attention is focused on either the viral RNA-dependent RNA polymerase or the main viral protease, 3CLpro. 3CLpro is an attractive target for antiviral therapeutics as it is essential for processing newly translated viral proteins, and the viral lifecycle cannot be completed without protease activity. In this work, we present a new assay to identify inhibitors of the SARS-CoV-2 main protease, 3CLpro. Our reporter is based on a GFP-derived protein that only fluoresces after cleavage by 3CLpro. This experimentally optimized reporter assay allows for antiviral drug screening in human cell culture at biosafety level-2 (BSL2) with high-throughput compatible protocols. Using this screening approach in combination with existing drug libraries may lead to the rapid identification of novel antivirals to suppress SARS-CoV-2 replication and spread. IMPORTANCE The COVID-19 pandemic has already led to more than 700,000 deaths and innumerable changes to daily life worldwide. Along with development of a vaccine, identification of effective antivirals to treat infected patients is of the highest importance. However, rapid drug discovery requires efficient methods to identify novel compounds that can inhibit the virus. In this work, we present a method for identifying inhibitors of the SARS-CoV-2 main protease, 3CLpro. This reporter-based assay allows for antiviral drug screening in human cell culture at biosafety level-2 (BSL2) with high-throughput compatible sample processing and analysis. This assay may help identify novel antivirals to control the COVID-19 pandemic.

There is an urgent need for anti-viral agents that treat SARS-CoV-2 infection. The shortest path to clinical use is repurposing of drugs that have an established safety profile in humans. Here, we first screened a library of 1,900 clinically safe drugs for inhibiting replication of OC43, a human beta-coronavirus that causes the common-cold and is a relative of SARS-CoV-2, and identified 108 effective drugs. We further evaluated the top 26 hits and determined their ability to inhibit SARS-CoV-2, as well as other pathogenic RNA viruses. 20 of the 26 drugs significantly inhibited SARS-CoV-2 replication in human lung cells (A549 epithelial cell line), with EC50 values ranging from 0.1 to 8 micromolar. We investigated the mechanism of action for these and found that masitinib, a drug originally developed as a tyrosine-kinase inhibitor for cancer treatment, strongly inhibited the activity of the SARS-CoV-2 main protease 3CLpro. X-ray crystallography revealed that masitinib directly binds to the active site of 3CLpro, thereby blocking its enzymatic activity. Mastinib also inhibited the related viral protease of picornaviruses and blocked picornaviruses replication. Thus, our results show that masitinib has broad anti-viral activity against two distinct beta-coronaviruses and multiple picornaviruses that cause human disease and is a strong candidate for clinical trials to treat SARS-CoV-2 infection.

Type I interferon (IFN) signaling in fetal tissues causes developmental abnormalities and fetal demise. Although pathogens that infect fetal tissues can induce birth defects through the local production of type I IFN, it remains unknown why systemic IFN generated during maternal infections only rarely causes fetal developmental defects. Here, we report that activation of the guanine nucleotide–binding protein–coupled estrogen receptor 1 (GPER1) during pregnancy is both necessary and sufficient to suppress IFN signaling and does so disproportionately in reproductive and fetal tissues. Inactivation of GPER1 in mice halted fetal development and promoted fetal demise, but only in the context of maternal inflammation. Thus, GPER1 is a central regulator of IFN signaling during pregnancy that allows dynamic antiviral responses in maternal tissues while also preserving fetal health.

The type I interferon (IFN) response is an important component of the innate immune response to viral infection. Precise control of IFN responses is critical because insufficient expression of IFN-stimulated genes (ISGs) can lead to a failure to restrict viral spread, whereas excessive ISG activation can result in IFN-related pathologies. Although both positive and negative regulatory factors control the magnitude and duration of IFN signaling, it is also appreciated that several ISGs regulate aspects of the IFN response themselves. In this study, we performed a CRISPR activation screen to identify previously unknown regulators of the type I IFN response. We identified the strongly induced ISG encoding ETS variant transcription factor 7 (ETV7) as a negative regulator of the type I IFN response. However, ETV7 did not uniformly suppress ISG transcription. Instead, ETV7 preferentially targeted a subset of antiviral ISGs that were particularly important for IFN-mediated control of influenza viruses. Together, our data assign a function for ETV7 as an IFN response regulator and also identify ETV7 as a potential therapeutic target to increase innate antiviral responses and enhance IFN-based antiviral therapies.

word count: 242 22 Text word count: 3,127 23 SARS-CoV-2 3CL pro reporter assay 2 ABSTRACT 24In late 2019 a human coronavirus, now known as SARS-CoV-2, emerged, likely from a zoonotic 25 reservoir. This virus causes COVID-19 disease, has infected millions of people, and has led to 26 hundreds of thousands of deaths across the globe. While the best interventions to control and 27 ultimately stop the pandemic are prophylactic vaccines, antiviral therapeutics are important to limit 28 morbidity and mortality in those already infected. At this time, only one FDA approved anti-29 SARS-CoV-2 antiviral drug, remdesivir, is available and unfortunately, its efficacy appears to be 30 limited. Thus, the identification of new and efficacious antivirals is of highest importance. In order 31 to facilitate rapid drug discovery, flexible, sensitive, and high-throughput screening methods are 32 required. With respect to drug targets, most attention is focused on either the viral RNA-dependent 33 RNA polymerase or the main viral protease, 3CL pro . 3CL pro is an attractive target for antiviral 34 therapeutics as it is essential for processing newly translated viral proteins, and the viral lifecycle 35 cannot be completed without protease activity. In this work, we present a new assay to identify 36 inhibitors of the SARS-CoV-2 main protease, 3CL pro . Our reporter is based on a GFP-derived 37 protein that only fluoresces after cleavage by 3CL pro . This experimentally optimized reporter assay 38 allows for antiviral drug screening in human cell culture at biosafety level-2 (BSL2) with high-39 throughput compatible protocols. Using this screening approach in combination with existing drug 40 libraries may lead to the rapid identification of novel antivirals to suppress SARS-CoV-2 41 replication and spread. 42 43 44 SARS-CoV-2 3CL pro reporter assay

The productive replication of human influenza viruses is almost exclusively restricted to cells in the respiratory tract. However, a key aspect of the host response to viral infection is the production of inflammatory cytokines and chemokines that are not similarly tissue restricted. As such, circulating inflammatory mediators, as well as the resulting activated immune cells, can induce damage throughout the body, particularly in individuals with underlying conditions. As a result, more holistic experimental approaches are required to fully understand the pathogenesis and scope of influenza virus‐induced disease. This review summarizes what is known about some of the most well‐appreciated nonrespiratory tract sites of influenza virus‐induced disease, including neurological, cardiovascular, gastrointestinal, muscular and fetal developmental phenotypes. In the context of this discussion, we describe the in vivo experimental systems currently being used to study nonrespiratory symptoms. Finally, we highlight important future questions and potential models that can be used for a more complete understanding of influenza virus‐induced disease.

42The type I interferon (IFN) response is an important component of the innate immune 43 response to viral infection. Precise control of interferon responses is critical, as insufficient 44 levels of interferon-stimulated genes (ISGs) can lead to a failure to restrict viral spread, 45 while excessive ISG activation results in interferon-related pathologies. While both 46 positive and negative regulatory factors can control the magnitude and duration of IFN 47Significance 60Interferons (IFNs) were first described in 1957 and are now known to be critical for 61 restriction of viruses. Still, our understanding of the complex web of interactions that 62 underlie IFN responses remains incomplete. In particular, negative regulation of interferon 63 responses has received disproportionately less study. In this work, we performed a 64 genome-wide overexpression screen for factors capable of suppressing IFN response 65 signaling. We identified a DNA binding transcription factor (ETV7) that, after induction by 66 interferon, acts to suppress a subset of IFN-stimulated genes required for control of 67 influenza viruses. Our work highlights the importance of understanding negative IFN 68 signaling not only with respect to the magnitude and duration of the response, but also 69 the specificity of its antiviral effects. 70