The COVID-19 (Coronavirus disease-2019) pandemic, caused by the SARS-CoV-2 coronavirus, is a significant threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and MERS-CoV. Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analysis for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 Orf9b, an interaction we structurally characterized using cryo-EM. Combining genetically-validated host factors with both COVID-19 patient genetic data and medical billing records identified important molecular mechanisms and potential drug treatments that merit further molecular and clinical study.

In late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly described and effective procedures that enable the propagation and quantification of infectious virus. As work with the virus is recommended to be performed at biosafety level 3, validated methods to effectively inactivate the virus to enable the safe study of RNA, DNA, and protein from infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell lines and to measure virus infectivity by plaque assay using either agarose or microcrystalline cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat.

In late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-9 CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has 10 been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 11 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease 12 that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly 13 described and effective procedures that enable the propagation and quantification of infectious 14 virus. Because work with the virus is recommended to be performed at biosafety level 3, validated 15 methods to effectively inactivate the virus to enable safe study of RNA, DNA and protein from 16 infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell 17 lines and to measure virus infectivity by plaque assay using either agarose or microcrystalline 18 cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate 19 effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat. 20 cells to characterize viral genome sequences, monitor viral gene expression and genome replication, 43 and to characterize host responses to infection. Removal of intact, virus-infected cells is critical for 44 studies involving microscopy aimed at understanding the SARS-CoV-2:host interplay at the cellular 45 level, and also for high-throughput analysis of the progression of viral replication in response to 46 antivirals. Whole inactivated virus and viral proteins are needed for the development of inactivated 47 whole-virus vaccine preparations and also as a source of antigen for immunoassays. 48To help address these needs and to facilitate SARS-CoV-2 research efforts, we describe here 49 methods for the propagation of SARS-CoV-2 in multiple cell lines. We have also determined a more 50 efficient method for quantifying virus by plaque assay and have developed a SARS-CoV-2-specific 51 focus forming assay which can enhance throughput of assays requiring quantification of viral titers. 52Additionally, we describe validation if methods for the inactivation of SARS-CoV-2 through the use 53 of TRIzol, 10% neutral buffered formalin, beta-propiolactone, and heat. Taken together, the data 54 presented here will serve to provide researchers with a helpful basis of information to aid in their 55 work on SARS-CoV-2. 56 57 2. Materials and Methods 58 2.1 Cells and Virus 59 60 Vero E6 (ATCC# CRL-1586), Calu-3 (ATCC# HTB-55), Caco-2 (ATCC# HTB-37), Huh7, A549 61 (ATCC# CCL-185), and 293T cells were maintained in DMEM (Corning) supplemented with 10% heat 62 inactivated fetal bovine serum (FBS; GIBCO). Cells were kept in a 37°C, 5% CO2 incubator without 63 antibiotics or antimycotics. SARS-CoV-2, strain USA_WA1/2020, was obtained from the World 64 Reference Collection for Emerging Viruses and Arboviruses at the University of Texas Medical 65 Branch-Galveston. 6...

Coronaviruses rely on host membranes for entry, establishment of replication centers, and egress. Compounds targeting cellular membrane biology and lipid biosynthetic pathways have previously shown promise as antivirals and are actively being pursued as treatments for other conditions. Here, we test small molecule inhibitors that target the PI3 kinase VPS34 or fatty acid metabolism for anti-SARS-CoV-2 activity. Our studies determine that compounds targeting VPS34 are potent SARS-CoV-2 inhibitors. Mechanistic studies with compounds targeting multiple steps up- and downstream of fatty acid synthase (FASN) identify the importance of triacylglycerol production and protein palmitoylation as requirements for efficient viral RNA synthesis and infectious virus production. Further, FASN knockout results in significantly impaired SARS-CoV-2 replication that can be rescued with fatty acid supplementation. Together, these studies clarify roles for VPS34 and fatty acid metabolism in SARS-CoV-2 replication and identify promising avenues for the development of countermeasures against SARS-CoV-2.

Summary The influenza nonstructural protein 1 (NS1) plays a critical role in antagonizing the innate immune response to infection. One interaction that facilitates this function is between NS1 and RIG-I, one of the main sensors of influenza virus infection. While NS1 and RIG-I are known to interact, it is currently unclear whether this interaction is direct or if it is mediated by other biomolecules. In the present study, we demonstrate a direct, strain dependent interaction between the NS1 RNA binding domain (NS1RBD) of the influenza A/Brevig Mission/1918 H1N1 (1918H1N1) virus and the second CARD domain of RIG-I. Solving the solution structure of the 1918H1N1 NS1RBD revealed features in a functionally novel region that may facilitate the observed interaction. The biophysical and structural data herein suggest a possible mechanism by which strain specific differences in NS1 modulate influenza virulence.