Author
Matthijs Raaben
Other affiliations: Utrecht University, Harvard University
Bio: Matthijs Raaben is an academic researcher from Netherlands Cancer Institute. The author has contributed to research in topics: Coronavirus & Viral replication. The author has an hindex of 20, co-authored 34 publications receiving 2870 citations. Previous affiliations of Matthijs Raaben include Utrecht University & Harvard University.
Topics: Coronavirus, Viral replication, Virus, Viral entry, Ebola virus
Papers
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TL;DR: It is shown that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport, which indicates potential antiviral strategies to combat these deadly agents.
Abstract: The extraordinary virulence of the Ebola and Marburg filoviruses has spurred intensive research into the molecular mechanisms by which they multiply and cause disease. Carette et al. use a genome-wide genetic screen in human cells to identify factors required for entry of Ebola virus. The screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann–Pick C1 (NPC1). Cote et al. report the identification of a novel benzylpiperazine adamantane diamide-derived compound that inhibits EboV infection in cell culture, with NPC1 being the target. The unexpected role for the hereditary disease gene NPC1 in Ebola virus infection may facilitate the development of antifilovirus therapeutics. Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available1. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes2. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification3,4,5. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes6, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann–Pick C1 (NPC1)7. Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann–Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.
1,147 citations
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TL;DR: These findings support a model in which GP cleavage by endosomal cysteine proteases unmasks the binding site for NPC1, and GP–NPC1 engagement within lysosomes promotes a late step in entry proximal to viral escape into the host cytoplasm.
Abstract: Ebola and Marburg filoviruses cause deadly outbreaks of haemorrhagic fever. Despite considerable efforts, no essential cellular receptors for filovirus entry have been identified. We showed previously that Niemann-Pick C1 (NPC1), a lysosomal cholesterol transporter, is required for filovirus entry. Here, we demonstrate that NPC1 is a critical filovirus receptor. Human NPC1 fulfills a cardinal property of viral receptors: it confers susceptibility to filovirus infection when expressed in non-permissive reptilian cells. The second luminal domain of NPC1 binds directly and specifically to the viral glycoprotein, GP, and a synthetic single-pass membrane protein containing this domain has viral receptor activity. Purified NPC1 binds only to a cleaved form of GP that is generated within cells during entry, and only viruses containing cleaved GP can utilize a receptor retargeted to the cell surface. Our findings support a model in which GP cleavage by endosomal cysteine proteases unmasks the binding site for NPC1, and GP–NPC1 engagement within lysosomes promotes a late step in entry proximal to viral escape into the host cytoplasm. NPC1 is the first known viral receptor that recognizes its ligand within an intracellular compartment and not at the plasma membrane.
302 citations
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TL;DR: Comprehensive forward genetic screens can be used to define the genetic architecture of a complex disease and point out genes defective in dystroglycanopathies.
Abstract: Glycosylated α-dystroglycan (α-DG) serves as cellular entry receptor for multiple pathogens, and defects in its glycosylation cause hereditary Walker-Warburg syndrome (WWS). At least eight proteins are critical to glycosylate α-DG, but many genes mutated in WWS remain unknown. To identify modifiers of α-DG, we performed a haploid screen for Lassa virus entry, a hemorrhagic fever virus causing thousands of deaths annually that hijacks glycosylated α-DG to enter cells. In complementary screens, we profiled cells for absence of α-DG carbohydrate chains or biochemically related glycans. This revealed virus host factors and a suite of glycosylation units, including all known Walker-Warburg genes and five additional factors critical for the modification of α-DG. Our findings accentuate the complexity of this posttranslational feature and point out genes defective in dystroglycanopathies.
260 citations
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TL;DR: It is shown that to enter a cell, the virus requires a second receptor, this one inside the infected cell, which sheds light on the “enigmatic resistance” of bird cells to Lassa virus, and the resistance of Lamp1-deficient mice to Lasso virus highlights the relevance of this receptor switch in vivo.
Abstract: Lassa virus spreads from a rodent to humans and can lead to lethal hemorrhagic fever Despite its broad tropism, chicken cells were reported 30 years ago to resist infection We found that Lassa virus readily engaged its cell-surface receptor α-dystroglycan in avian cells, but virus entry in susceptible species involved a pH-dependent switch to an intracellular receptor, the lysosome-resident protein LAMP1 Iterative haploid screens revealed that the sialyltransferase ST3GAL4 was required for the interaction of the virus glycoprotein with LAMP1 A single glycosylated residue in LAMP1, present in susceptible species but absent in birds, was essential for interaction with the Lassa virus envelope protein and subsequent infection The resistance of Lamp1-deficient mice to Lassa virus highlights the relevance of this receptor switch in vivo
246 citations
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TL;DR: The results demonstrate that GBF1-mediated ARF1 activation is required for efficient MHV RNA replication and reveal that the early secretory pathway and MHV replication complex formation are closely connected.
Abstract: Coronaviruses induce in infected cells the formation of double membrane vesicles, which are the sites of RNA replication. Not much is known about the formation of these vesicles, although recent observations indicate an important role for the endoplasmic reticulum in the formation of the mouse hepatitis coronavirus (MHV) replication complexes (RCs). We now show that MHV replication is sensitive to brefeldin A (BFA). Consistently, expression of a dominant-negative mutant of ARF1, known to mimic the action of the drug, inhibited MHV infection profoundly. Immunofluorescence analysis and quantitative electron microscopy demonstrated that BFA did not block the formation of RCs per se, but rather reduced their number. MHV RNA replication was not sensitive to BFA in MDCK cells, which are known to express the BFA-resistant guanine nucleotide exchange factor GBF1. Accordingly, individual knockdown of the Golgi-resident targets of BFA by transfection of small interfering RNAs (siRNAs) showed that GBF1, but not BIG1 or BIG2, was critically involved in MHV RNA replication. ARF1, the cellular effector of GBF1, also appeared to be involved in MHV replication, as siRNAs targeting this small GTPase inhibited MHV infection significantly. Collectively, our results demonstrate that GBF1-mediated ARF1 activation is required for efficient MHV RNA replication and reveal that the early secretory pathway and MHV replication complex formation are closely connected.
139 citations
Cited by
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Icahn School of Medicine at Mount Sinai1, University of Michigan2, Pasteur Institute3, University of California, San Francisco4, European Bioinformatics Institute5, University of California, San Diego6, University of North Carolina at Chapel Hill7, Fred Hutchinson Cancer Research Center8, Gladstone Institutes9, Buck Institute for Research on Aging10
TL;DR: A human–SARS-CoV-2 protein interaction map highlights cellular processes that are hijacked by the virus and that can be targeted by existing drugs, including inhibitors of mRNA translation and predicted regulators of the sigma receptors.
Abstract: A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein–protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19. A human–SARS-CoV-2 protein interaction map highlights cellular processes that are hijacked by the virus and that can be targeted by existing drugs, including inhibitors of mRNA translation and predicted regulators of the sigma receptors.
3,319 citations
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TL;DR: Using the bacterial clustered regularly interspaced short palindromic repeats (CRISPR) system, this article constructed a genome-wide single-guide RNA library to screen for genes required for proliferation and survival in a human cancer cell line.
Abstract: Large-scale genetic analysis of lethal phenotypes has elucidated the molecular underpinnings of many biological processes. Using the bacterial clustered regularly interspaced short palindromic repeats (CRISPR) system, we constructed a genome-wide single-guide RNA library to screen for genes required for proliferation and survival in a human cancer cell line. Our screen revealed the set of cell-essential genes, which was validated with an orthogonal gene-trap-based screen and comparison with yeast gene knockouts. This set is enriched for genes that encode components of fundamental pathways, are expressed at high levels, and contain few inactivating polymorphisms in the human population. We also uncovered a large group of uncharacterized genes involved in RNA processing, a number of whose products localize to the nucleolus. Last, screens in additional cell lines showed a high degree of overlap in gene essentiality but also revealed differences specific to each cell line and cancer type that reflect the developmental origin, oncogenic drivers, paralogous gene expression pattern, and chromosomal structure of each line. These results demonstrate the power of CRISPR-based screens and suggest a general strategy for identifying liabilities in cancer cells.
1,371 citations
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Technische Universität München1, German Center for Neurodegenerative Diseases2, University of Helsinki3, University of Göttingen4, Ludwig Maximilian University of Munich5, Life Sciences Institute6, University of Tartu7, National Institute for Health and Welfare8, University of Queensland9, ETH Zurich10, University of California, Santa Barbara11, Discovery Institute12, University of Zurich13
TL;DR: It is found that neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, an effect blocked by a monoclonal blocking antibody against NRP1.
Abstract: The causative agent of coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For many viruses, tissue tropism is determined by the availability of virus receptors and entry cofactors on the surface of host cells. In this study, we found that neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, an effect blocked by a monoclonal blocking antibody against NRP1. A SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells facing the nasal cavity. Our data provide insight into SARS-CoV-2 cell infectivity and define a potential target for antiviral intervention.
1,304 citations
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TL;DR: It is shown that CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle.
Abstract: Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered into lysosomes for degradati...
1,178 citations
01 Nov 2015
TL;DR: A genome-wide single-guide RNA library is constructed to screen for genes required for proliferation and survival in a human cancer cell line and reveals a set of cell-essential genes, which was validated with an orthogonal gene-trap–based screen and comparison with yeast gene knockouts.
Abstract: Large-scale genetic analysis of lethal phenotypes has elucidated the molecular underpinnings of many biological processes. Using the bacterial clustered regularly interspaced short palindromic repeats (CRISPR) system, we constructed a genome-wide single-guide RNA library to screen for genes required for proliferation and survival in a human cancer cell line. Our screen revealed the set of cell-essential genes, which was validated with an orthogonal gene-trap-based screen and comparison with yeast gene knockouts. This set is enriched for genes that encode components of fundamental pathways, are expressed at high levels, and contain few inactivating polymorphisms in the human population. We also uncovered a large group of uncharacterized genes involved in RNA processing, a number of whose products localize to the nucleolus. Last, screens in additional cell lines showed a high degree of overlap in gene essentiality but also revealed differences specific to each cell line and cancer type that reflect the developmental origin, oncogenic drivers, paralogous gene expression pattern, and chromosomal structure of each line. These results demonstrate the power of CRISPR-based screens and suggest a general strategy for identifying liabilities in cancer cells.
1,113 citations