Synthetic Lethality-Based Prediction of Anti-SARS-CoV-2 Targets (preprint)

Novel strategies are needed to identify drug targets and treatments for the COVID-19 pandemic. The altered gene expression of virus-infected host cells provides an opportunity to specifically inhibit viral propagation via targeting the synthetic lethal (SL) partners of such altered host genes. Pursuing this antiviral strategy, here we comprehensively analyzed multiple in vitro and in vivo bulk and single-cell RNA-sequencing datasets of SARS-CoV-2 infection to predict clinically relevant candidate antiviral targets that are SL with altered host genes. The predicted SL-based targets are highly enriched for infected cell inhibiting genes reported in four SARS-CoV-2 CRISPR-Cas9 genome-wide genetic screens. Integrating our predictions with the results of these screens, we further selected a focused subset of 26 genes that we experimentally tested in a targeted siRNA screen using human Caco-2 cells. Notably, as predicted, knocking down these targets reduced viral replication and cell viability only under the infected condition without harming non-infected cells. Our results are made publicly available, to facilitate their in vivo testing and further validation.Funding: This research was supported in part by the Intramural Research Program of the National Institutes of Health, NCI, CCR; and used the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov). We acknowledge and thank the National Cancer Institute for providing financial and infrastructural support. This work was also supported by the following grants to the Sanford Burnham Prebys Medical Discovery Institute: DoD: W81XWH-20-1-0270; DHIPC: U19 AI118610; Fluomics/NOSI: U19 AI135972. K.C. and S.S is supported by the NCI-UMD Partnership for Integrative Cancer Research Program.Declaration of Interests: The other authors declare no competing interests.

Functional Landscape of SARS-CoV-2 Cellular Restriction (preprint)

A deficient interferon response to SARS-CoV-2 infection has been implicated as a determinant of severe COVID-19. To identify the molecular effectors that govern interferon control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human interferon stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors that inhibited viral entry, nucleic acid binding proteins that suppressed viral RNA synthesis, and a highly enriched cluster of ER and Golgi-resident ISGs that inhibited viral translation and egress. These included the type II integral membrane protein BST2/tetherin, which was found to impede viral release, and is targeted for immune evasion by SARS-CoV-2 Orf7a protein. Overall, these data define the molecular basis of early innate immune control of viral infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.Funding: This work was supported by the following grants to the Sanford Burnham Prebys Medical Discovery Institute and the Icahn School of medicine at Mount Sinai: DoD: W81XWH-20-10270; DHIPC: U19 AI118610; Fluomics/NOSI: U19 AI135972. This work was also supported by generous philanthropic donations from Dinah Ruch and Susan & James Blair, from the JPB Foundation, the Open Philanthropy Project (research grant 2020-215611 (5384)) and anonymous donors. Additional support has been provided by DARPA grant HR0011-19-2-0020 and by CRIP (Center for research on Influenza Pathogenesis), a NIAID-funded Center of Excellence for Influenza Research and Surveillance (CEIRS, contract # HHSN272201400008C). This work was additionally supported by the following grants to Northwestern University Feinberg School of Medicine: a CTSA supplement to NCATS: UL1 TR002389; a CTSA supplement to NUCATS with the generous support of the Dixon family: UL1 TR001422; and a Cancer Center supplement: P30 CA060553, and the following grant to JG at UC San Diego: NIH grant R37AI081668. This work was also supported by a generous grant from the James B. Pendleton Charitable Trust. Conflict of Interest: The authors declare no competing interests.Ethical Approval: All experiments involving live SARS-CoV-2 followed the approved standard operating procedures of the Biosafety Level 3 facility at the Sanford Burnham Prebys Medical Discovery Institute.

MDA5 Governs the Innate Immune Response to SARS-CoV-2 in Lung Epithelial Cells (preprint)

Recent studies profiling the innate immune signatures in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suggest that cellular responses to viral challenge impact disease severity. Yet, the molecular events that underlie cellular recognition and response to SARS-CoV-2 infection remains to be elucidated. Here, we find that SARS-CoV-2 replication induces a delayed interferon (IFN) response in lung epithelial cells. Through a survey of putative sensors involved in detection of RNA virus infection, we found that MDA5 and LGP2 primarily regulate IFN induction in response to SARS-CoV-2 infection. Additionally, we find that IRF-3, -5, and NF-kB/p65 are the key transcription factors regulating the IFN response during SARS-CoV-2 infection. In summary, these findings provide critical insights into the molecular basis of the innate immune recognition and signaling response to SARS-CoV-2.Funding: This work was supported by the following grants to the Sanford Burnham Prebys Medical Discovery Institute: DoD: W81XWH-20-1-0270; DHIPC: U19 AI118610; Fluomics/NOSI: U19 AI135972, as well as generous philanthropic donations from Dinah Ruch and Susan & James Blair. This work was additionally supported by the following grants to Northwestern University Feinberg School of Medicine: a CTSA supplement to NCATS: UL1 TR002389; a CTSA supplement to NUCATS with the generous support of the Dixon family: UL1 TR001422; and a Cancer Center supplement: P30 CA060553. Development and implementation of iPS cell technology for production of airway epithelial cells was supported by Incubation Program from Office of Society Academia Collaboration for Innovation, Kyoto University. Conflict of Interest: The authors declare no competing interests.

SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2 (preprint)

We show that SARS-CoV-2 spike protein interacts with cell surface heparan sulfate and angiotensin converting enzyme 2 (ACE2) through its Receptor Binding Domain. Docking studies suggest a putative heparin/heparan sulfate-binding site adjacent to the domain that binds to ACE2. In vitro, binding of ACE2 and heparin to spike protein ectodomains occurs independently and a ternary complex can be generated using heparin as a template. Contrary to studies with purified components, spike protein binding to heparan sulfate and ACE2 on cells occurs codependently. Unfractionated heparin, non-anticoagulant heparin, treatment with heparin lyases, and purified lung heparan sulfate potently block spike protein binding and infection by spike protein-pseudotyped virus and SARS-CoV-2 virus. These findings support a model for SARS-CoV-2 infection in which viral attachment and infection involves formation of a complex between heparan sulfate and ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin may represent new therapeutic opportunities.Funding: This work was supported by RAPID grant 2031989 from the National Science Foundation and Project 3 of NIH P01 HL131474 to J.D.E.; The Alfred Benzon Foundation to T.M.C; NIH R01 AI146779 and a Massachusetts Consortium on Pathogenesis Readiness MassCPR grant to A.G.S.; DOD grant W81XWH-20-1-0270 and Fluomics/NOSI U19 AI135972 to S.K.C; a Career Award for Medical Scientists from the Burroughs Wellcome Fund to A.F.C.; Bill and Melinda Gates Foundation grant OPP1170236 to A.B.W.; COVID19 seed funding from the Huck Institutes of the Life Sciences and Penn State start-up funds to J.J.; and T32 training grants GM007753 for B.M.H. and T.C and AI007245 for J.F.; J.P. received funding from the Innovation Fund Denmark and VAR2 Pharmaceuticals.Conflict of Interest: J.D.E. is a co-founder of TEGA Therapeutics. J.D.E. and The Regents of the University of California have licensed a University invention to and have an equity interest in TEGA Therapeutics. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. C.A.G and B.E.T are employees of TEGA Therapeutics.Ethical Approval: The collection of human tissue in this study abided by the Helsinki Principles. This work included postmortem human tissue, collected at the University Hospital, at the University of Copenhagen in Denmark. The patient provided informed consent for the tissue to be used for research purposes. All samples were completely deidentified before transfer to the researchers and this did not need specific IRB approval.