Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here, we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, MERS-CoV, bat coronavirus HKU5 expressing the SARS-CoV-1 spike, and VSV expressing the SARS-CoV-2 spike. We identify known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways including HMGB1 and the SWI/SNF chromatin remodeling complex that are SARS-lineage and pan-coronavirus specific, respectively. We show HMGB1 regulates ACE2 expression and is critical for viral entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. Together this identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS-lineage specific and pan-coronavirus host factors that regulate susceptibility to highly pathogenic coronaviruses.

Highlights d The SARS-CoV-2 genome is probed at single-nucleotide resolution in infected cells d RNA structure prediction reveals an elaborate SARS-CoV-2 genome architecture d Networks of well-folded secondary structure are conserved across b-coronaviruses d Disruption of conserved secondary structures with LNAs inhibits viral growth

Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-β signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis. Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 , represents the greatest public health threat in a century. More than 7,500,000 people have been infected with more than 420,000 deaths globally (1). Novel therapeutics and vaccines are desperately needed. Coronaviruses are enveloped, positive-sense RNA viruses with genomes of approximately 30 kb that exhibit broad host-range among birds and mammals and are typically transmitted via the respiratory route (2, 3). There are four circulating seasonal coronaviruses in humans (NL63, OC43, 229E, and HKU1) and three highly pathogenic zoonotic coronaviruses (SARS-CoV, MERS, and SARS-CoV-2), none of which have effective antiviral drugs or vaccines (4-7).Viral entry, the first stage of the SARS-CoV-2 life cycle, is mediated by the viral spike protein. The receptor binding domain of spike binds to the cell surface receptor angiotensinconverting enzyme 2 (ACE2), a major determinant of host range and cell tropism (8,9). The coronavirus spike protein requires two proteolytic processing steps prior to entry. The first cleavage event occurs at the interface of the S1 and S2 domains of the spike protein (10, 11). This can occur in the producer cell, the extracellular environment, or in the endosome and can be mediated by several proteases including furin and the plasma membrane protease TMPRSS2 (12)(13)(14). A second proteolytic event is required within S2 to expose the viral fusion peptide and enable membrane fusion. This second cleavage event can occur at the target cell plasma membrane by TMPRSS2 or in the endosome by Cathepsin L (14,15). Upon viral membrane fusion, the viral RNA is released into the cytoplasm where it is translated and establishes viral replication and transcription complexes before assembling and budding (16)(17)(18). The host genes that mediate these processes largely remain elusive.Identification of host factors essential for infection is critical to inform mechanisms of COVID-19 pathogenesis, reveal variation in host susceptibility, and identify novel host-directed therapies, which may have efficacy against current and future pand...

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of host factors influencing viral infection is critical to elucidate SARS-CoV-2–host interactions and the progression of Coronavirus disease 2019 (COVID-19). Here, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. We uncovered proviral and antiviral factors across highly interconnected host pathways, including clathrin transport, inflammatory signaling, cell-cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high molecular weight glycoproteins, as a prominent viral restriction network that inhibits SARS-CoV-2 infection in vitro and in murine models. These mucins also inhibit infection of diverse respiratory viruses. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and highlights airway mucins as a host defense mechanism.

Most currently approved strategies for the collection of saliva for COVID-19 diagnostics require specialized tubes containing buffers promoted for the stabilization of SARS-CoV-2 RNA and virus inactivation. Yet many of these are expensive, in limited supply, and not necessarily validated specifically for viral RNA. While saliva is a promising sample type as it can be reliably self-collected for the sensitive detection of SARS-CoV-2, the expense and availability of these collection tubes are prohibitive to mass testing efforts. Therefore, we investigated the stability of SARS-CoV-2 RNA and infectious virus detection from saliva without supplementation. We tested RNA stability over extended periods of time (2-25 days) and at temperatures representing at-home storage and elevated temperatures which might be experienced when cold chain transport may be unavailable. We found SARS-CoV-2 RNA in saliva from infected individuals is stable at 4°C, room temperature (~19°C), and 30°C for prolonged periods and found limited evidence for viral replication in saliva. This work demonstrates that expensive saliva collection options involving RNA stabilization and virus inactivation buffers are not always needed, permitting the use of cheaper collection options. Affordable testing methods are urgently needed to meet current testing demands and for continued surveillance in reopening strategies.