Identification of NPC1 as a novel SARS-CoV-2 intracellular target

AbstractNiemann-Pick type C1 (NPC1) receptor is an endosomal membrane protein that regulates intracellular cholesterol trafficking, which is crucial in the Ebola virus (EBOV) cycle. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the cell by binding of the viral spike (S) protein to the ACE2 receptor. This requires S-protein processing either by the surface transmembrane serine protease TMPRSS2 for plasma membrane fusion or cathepsin L for endosomal entry. Additional host factors are required for viral fusion at endosomes. Here, we report a novel interaction of the SARS-CoV-2 nucleoprotein (N) with the cholesterol transporter NPC1. Moreover, small molecules interfering with NPC1 that inhibit EBOV entry, also inhibited human coronavirus. Our findings suggest an important role for NPC1 in SARS-CoV-2 infection, a common strategy shared with EBOV, and a potential therapeutic target to fight against COVID-19.

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Interfering with Host Proteases in SARS-CoV-2 Entry as a Promising Therapeutic Strategy

Current Medicinal Chemistry ◽

10.2174/0929867328666210526111318 ◽

2021 ◽

Vol 28 ◽

Author(s):

Patrick Müller ◽

Hannah Maus ◽

Stefan Josef Hammerschmidt ◽

Philip Knaff ◽

Volker Mailänder ◽

...

Keyword(s):

Cathepsin L ◽

Cell Entry ◽

New Drugs ◽

Host Cells ◽

S Protein ◽

Cell Receptors ◽

Current State ◽

Causative Drug ◽

Transmembrane Serine Protease ◽

Global Threat

: Due to its fast international spread and substantial mortality, the coronavirus disease COVID-19 evolved to a global threat. Since currently, there is no causative drug against this viral infection available, science is striving for new drugs and approaches to treat the new disease. Studies have shown that the cell entry of coronaviruses into host cells takes place through the binding of the viral spike (S) protein to cell receptors. Priming of the S protein occurs via hydrolysis by different host proteases. The inhibition of these proteases could impair the processing of the S protein, thereby affecting the interaction with the host-cell receptors and preventing virus cell entry. Hence, inhibition of these proteases could be a promising strategy for treatment against SARS-CoV-2. In this review, we discuss the current state of the art of developing inhibitors against the entry proteases furin, the transmembrane serine protease type-II (TMPRSS2), trypsin, and cathepsin L.

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A review of COVID-19: Molecular basis, diagnosis, therapeutics and prevention

Science and Technology Development Journal - Natural Sciences ◽

10.32508/stdjns.v4i3.907 ◽

2020 ◽

Vol 4 (3) ◽

pp. First

Author(s):

Mai T. N. HUYNH ◽

Phuc H. T. NGUYEN ◽

Hieu H. C. PHAN ◽

Nghia T. H. PHAN ◽

Kong H. LE ◽

...

Keyword(s):

Cleavage Site ◽

Vaccine Development ◽

Transmission Rate ◽

Cathepsin L ◽

Virus Transmission ◽

Open Reading Frames ◽

Furin Cleavage ◽

Viral Pathogen ◽

S Protein ◽

Nucleoprotein N

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the confirmed viral pathogen of COVID-19, a pandemic originated from Wuhan, China at the end of 2019. Since then, SARS-CoV-2 has rapidly spread across the globe with over 8 million confirmed cases and more than 430.000 deaths worldwide as of mid-June 2020. Similar to other strains of coronavirus, the envelope of SARS-CoV-2 comprises of three structural proteins: S protein (spike), E protein (envelope) and M glycoprotein (membrane). SARS-CoV-2 capsids are spherical or pleomorphic. Each capsid contains a positive-sense single-stranded RNA (+ssRNA-Class IV-Baltimore) associated with nucleoprotein N. The viral RNA genome is approximately 30 kb in length and contains 14 open reading frames (ORFs). The binding affinity of the viral S protein to the ACE2 (angiotensin-converting enzyme 2) receptor facilitates the attachment of SARS-CoV-2 to human epithelial cells. Upon binding, SARS-CoV-2 spike protein is cleaved and activated by TMPRSS2 (transmembrane protease, serine 2) or by cathepsin L at the cleavage site S2', and also by furin at the cleavage site S1/S2. The furin cleavage motif RR_R is a notable feature, firstly found in SARS-CoV-2 S protein, which may increase virus transmission rate. This feature and many others might result from several evolution events in SARS-CoV-2 genome. These events could occur when coronaviruses, including SARS-CoV-2, spread from one host to another. They can be causative to high virulence and transmission rate of future coronavirus strains, which may require the development of newer vaccine generations. To understand of SARS-CoV-2’s structure, infection mechanism, diagnosis, treatment, and vaccine development strategies, a review of current literature is of highly importance to disease control in Vietnam.

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Reovirus infection is regulated by NPC1 and endosomal cholesterol homeostasis

10.1101/2021.09.27.462002 ◽

2021 ◽

Author(s):

Paula Ortega Gonzalez ◽

Gwen M Taylor ◽

Rohit Jangra ◽

Raquel Tenorio Vela ◽

Isabel Fernandez de Castro Martin ◽

...

Keyword(s):

Ebola Virus ◽

Cholesterol Homeostasis ◽

Endocytic Pathway ◽

Target Cells ◽

Genome Replication ◽

Reovirus Infection ◽

Endosomal Membrane ◽

Late Endosomes ◽

Mammalian Orthoreovirus ◽

Niemann Pick

Cholesterol homeostasis is required for the replication of many viruses, including Ebola virus, hepatitis C virus, and human immunodeficiency virus-1. Niemann-Pick C1 (NPC1) is an endosomal-lysosomal membrane protein involved in cholesterol trafficking from late endosomes and lysosomes to the endoplasmic reticulum. We identified NPC1 in CRISPR and RNA interference screens as a putative host factor for infection by mammalian orthoreovirus (reovirus). Following internalization via clathrin-mediated endocytosis, the reovirus outer capsid is proteolytically removed, the endosomal membrane is disrupted, and the viral core is released into the cytoplasm where viral transcription, genome replication, and assembly take place. We found that reovirus infection is significantly impaired in cells lacking NPC1, but infection is restored by treatment of cells with hydroxypropyl-β-cyclodextrin, which binds and solubilizes cholesterol. Absence of NPC1 did not dampen infection by infectious subvirion particles, which are reovirus disassembly intermediates that bypass the endocytic pathway for infection of target cells. NPC1 is not required for reovirus attachment to the plasma membrane, internalization into cells, or uncoating within endosomes. Instead, NPC1 is required for delivery of transcriptionally active reovirus core particles into the cytoplasm. These findings suggest that cholesterol homeostasis, ensured by NPC1 transport activity, is required for reovirus penetration into the cytoplasm, pointing to a new function for NPC1 and cholesterol homeostasis in viral infection.

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Structure of human Niemann–Pick C1 protein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1607795113 ◽

2016 ◽

Vol 113 (29) ◽

pp. 8212-8217 ◽

Cited By ~ 77

Author(s):

Xiaochun Li ◽

Jiawei Wang ◽

Elias Coutavas ◽

Hang Shi ◽

Qi Hao ◽

...

Keyword(s):

Ebola Virus ◽

Cholesterol Biosynthesis ◽

Regulatory Proteins ◽

Endosomal Membrane ◽

Transmembrane Segments ◽

Niemann Pick Disease ◽

Type C ◽

Niemann Pick ◽

Ebola Virus Infection ◽

Pick Disease

Niemann–Pick C1 protein (NPC1) is a late-endosomal membrane protein involved in trafficking of LDL-derived cholesterol, Niemann–Pick disease type C, and Ebola virus infection. NPC1 contains 13 transmembrane segments (TMs), five of which are thought to represent a “sterol-sensing domain” (SSD). Although present also in other key regulatory proteins of cholesterol biosynthesis, uptake, and signaling, the structure and mechanism of action of the SSD are unknown. Here we report a crystal structure of a large fragment of human NPC1 at 3.6 Å resolution, which reveals internal twofold pseudosymmetry along TM 2–13 and two structurally homologous domains that protrude 60 Å into the endosomal lumen. Strikingly, NPC1's SSD forms a cavity that is accessible from both the luminal bilayer leaflet and the endosomal lumen; computational modeling suggests that this cavity is large enough to accommodate one cholesterol molecule. We propose a model for NPC1 function in cholesterol sensing and transport.

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Identification of Niemann-Pick C1 protein as a potential novel SARS-CoV-2 intracellular target

Antiviral Research ◽

10.1016/j.antiviral.2021.105167 ◽

2021 ◽

pp. 105167

Author(s):

Isabel Garcia-Dorival ◽

Miguel Ángel Cuesta-Geijo ◽

Lucía Barrado-Gil ◽

Inmaculada Galindo ◽

Urtzi Garaigorta ◽

...

Keyword(s):

Intracellular Target ◽

Niemann Pick

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Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells

Journal of Virology ◽

10.1128/jvi.00683-18 ◽

2018 ◽

Vol 92 (19) ◽

Cited By ~ 30

Author(s):

Shutoku Matsuyama ◽

Kazuya Shirato ◽

Miyuki Kawase ◽

Yutaka Terada ◽

Kengo Kawachi ◽

...

Keyword(s):

Middle East ◽

Viral Entry ◽

Cathepsin L ◽

Inhibitory Effect ◽

Middle East Respiratory Syndrome ◽

Cell Entry ◽

Target Cells ◽

Furin Cleavage ◽

S Protein ◽

Entry Assay

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host cellular proteases to enter cells. A previous report shows that furin, which is distributed mainly in the Golgi apparatus and cycled to the cell surface and endosomes, proteolytically activates the MERS-CoV spike (S) protein following receptor binding to mediate fusion between the viral and cellular membranes. In this study, we reexamined furin usage by MERS-CoV using a real-time PCR-based virus cell entry assay after inhibition of cellular proteases. We found that the furin inhibitor dec-RVKR-CMK blocked entry of MERS-CoV harboring an S protein lacking furin cleavage sites; it even blocked entry into furin-deficient LoVo cells. In addition, dec-RVKR-CMK inhibited not only the enzymatic activity of furin but also those of cathepsin L, cathepsin B, trypsin, papain, and TMPRSS2. Furthermore, a virus cell entry assay and a cell-cell fusion assay provided no evidence that the S protein was activated by exogenous furin. Therefore, we conclude that furin does not play a role in entry of MERS-CoV into cells and that the inhibitory effect of dec-RVKR-CMK is specific for TMPRSS2 and cathepsin L rather than furin. IMPORTANCE Previous studies using the furin inhibitor dec-RVKR-CMK suggest that MERS-CoV utilizes a cellular protease, furin, to activate viral glycoproteins during cell entry. However, we found that dec-RVKR-CMK inhibits not only furin but also other proteases. Furthermore, we found no evidence that MERS-CoV uses furin. These findings suggest that previous studies in the virology field based on dec-RVKR-CMK should be reexamined carefully. Here we describe appropriate experiments that can be used to assess the effect of protease inhibitors on virus cell entry.

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Structure of the Ebola virus envelope protein MPER/TM domain and its interaction with the fusion loop explains their fusion activity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1708052114 ◽

2017 ◽

Vol 114 (38) ◽

pp. E7987-E7996 ◽

Cited By ~ 31

Author(s):

Jinwoo Lee ◽

David A. Nyenhuis ◽

Elizabeth A. Nelson ◽

David S. Cafiso ◽

Judith M. White ◽

...

Keyword(s):

Membrane Fusion ◽

Viral Entry ◽

Ebola Virus ◽

Rna Virus ◽

Hemorrhagic Fever ◽

Virus Envelope ◽

Tm Domain ◽

Virus Envelope Protein ◽

Niemann Pick ◽

Fusion Loop

Ebolavirus (EBOV), an enveloped filamentous RNA virus causing severe hemorrhagic fever, enters cells by macropinocytosis and membrane fusion in a late endosomal compartment. Fusion is mediated by the EBOV envelope glycoprotein GP, which consists of subunits GP1 and GP2. GP1 binds to cellular receptors, including Niemann-Pick C1 (NPC1) protein, and GP2 is responsible for low pH-induced membrane fusion. Proteolytic cleavage and NPC1 binding at endosomal pH lead to conformational rearrangements of GP2 that include exposing the hydrophobic fusion loop (FL) for insertion into the cellular target membrane and forming a six-helix bundle structure. Although major portions of the GP2 structure have been solved in pre- and postfusion states and although current models place the transmembrane (TM) and FL domains of GP2 in close proximity at critical steps of membrane fusion, their structures in membrane environments, and especially interactions between them, have not yet been characterized. Here, we present the structure of the membrane proximal external region (MPER) connected to the TM domain: i.e., the missing parts of the EBOV GP2 structure. The structure, solved by solution NMR and EPR spectroscopy in membrane-mimetic environments, consists of a helix-turn-helix architecture that is independent of pH. Moreover, the MPER region is shown to interact in the membrane interface with the previously determined structure of the EBOV FL through several critical aromatic residues. Mutation of aromatic and neighboring residues in both binding partners decreases fusion and viral entry, highlighting the functional importance of the MPER/TM–FL interaction in EBOV entry and fusion.

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COVID-19: a fatal case of acute liver failure associated with SARS-CoV-2 infection in pre-existing liver cirrhosis

BMC Infectious Diseases ◽

10.1186/s12879-021-06605-7 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Jana Ihlow ◽

Alexander Seelhoff ◽

Victor M. Corman ◽

Achim D. Gruber ◽

Simon Dökel ◽

...

Keyword(s):

Liver Failure ◽

Acute Liver Failure ◽

Liver Damage ◽

Cathepsin L ◽

Fatal Case ◽

Clinical Signs ◽

Angiotensin Converting Enzyme 2 ◽

Severe Liver Damage ◽

Severe Jaundice ◽

Transmembrane Serine Protease

Abstract Background The detection of severe acute respiratory syndrome coronavirus (SARS-CoV-2) is challenging, particularly in post-mortem human tissues. However, there is increasing evidence for viral SARS-CoV-2 manifestation in non-respiratory tissues. In this context, it is a current matter of debate, whether SARS-CoV-2 shows hepatotropism. Case presentation Here, we report a case of an 88-year-old women with massive SARS-CoV-2 viremia, severe jaundice and clinical signs of an acute hepatitis, who died within a few days from an acute liver failure without showing any clinical signs of pneumonia. Autopsy revealed a severe chronic and acute liver damage with bile duct infestation by SARS-CoV-2 that was accompanied by higher expressions of angiotensin-converting enzyme-2 (ACE2), Cathepsin L and transmembrane serine protease 2 (TMPRSS2). Conclusion Our findings indicate an enhanced biliary susceptibility to viral infection with SARS-CoV-2, that might have resulted from pre-existing severe liver damage. Furthermore, our findings emphasize the differential diagnosis of coronavirus disease 2019 (COVID-19)-associated liver failure in the clinical setting of an inexplicable jaundice.

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The Anticoagulant Nafamostat Potently Inhibits SARS-CoV-2 S Protein-Mediated Fusion in a Cell Fusion Assay System and Viral Infection In Vitro in a Cell-Type-Dependent Manner

Viruses ◽

10.3390/v12060629 ◽

2020 ◽

Vol 12 (6) ◽

pp. 629 ◽

Cited By ~ 36

Author(s):

Mizuki Yamamoto ◽

Maki Kiso ◽

Yuko Sakai-Tagawa ◽

Kiyoko Iwatsuki-Horimoto ◽

Masaki Imai ◽

...

Keyword(s):

Cell Fusion ◽

Assay System ◽

Dependent Manner ◽

Cell Type ◽

S Protein ◽

Candidate Drug ◽

A Cell ◽

Transmembrane Serine Protease ◽

Nafamostat Mesylate

Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 μM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat’s safety, make it a likely candidate drug to treat COVID-19.

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siRNA-Mediated Simultaneous Regulation of the Cellular Innate Immune Response and Human Respiratory Syncytial Virus Replication

Biomolecules ◽

10.3390/biom9050165 ◽

2019 ◽

Vol 9 (5) ◽

pp. 165 ◽

Cited By ~ 4

Author(s):

María Martín-Vicente ◽

Salvador Resino ◽

Isidoro Martínez

Keyword(s):

Immune Response ◽

Respiratory Syncytial Virus ◽

Virus Replication ◽

Innate Immune Response ◽

Human Respiratory Syncytial Virus ◽

Innate Immune ◽

Viral Fusion ◽

Small Interfering Rnas ◽

Syncytial Virus ◽

Nucleoprotein N

Human respiratory syncytial virus (HRSV) infection is a common cause of severe lower respiratory tract diseases such as bronchiolitis and pneumonia. Both virus replication and the associated inflammatory immune response are believed to be behind these pathologies. So far, no vaccine or effective treatment is available for this viral infection. With the aim of finding new strategies to counteract HRSV replication and modulate the immune response, specific small interfering RNAs (siRNAs) were generated targeting the mRNA coding for the viral fusion (F) protein or nucleoprotein (N), or for two proteins involved in intracellular immune signaling, which are named tripartite motif-containing protein 25 (TRIM25) and retinoic acid-inducible gene-I (RIG-I). Furthermore, two additional bispecific siRNAs were designed that silenced F and TRIM25 (TRIM25/HRSV-F) or N and RIG-I (RIG-I/HRSV-N) simultaneously. All siRNAs targeting N or F, but not those silencing TRIM25 or RIG-I alone, significantly reduced viral titers. However, while siRNAs targeting F inhibited only the expression of the F mRNA and protein, the siRNAs targeting N led to a general inhibition of viral mRNA and protein expression. The N-targeting siRNAs also induced a drastic decrease in the expression of genes of the innate immune response. These results show that both virus replication and the early innate immune response can be regulated by targeting distinct viral products with siRNAs, which may be related to the different role of each protein in the life cycle of the virus.

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