A Diacylglycerol Kinase Inhibitor, R-59-022, Blocks Filovirus Internalization in Host Cells

Filoviruses, such as Ebola virus (EBOV) and Marburg virus, are causative agents of unpredictable outbreaks of severe hemorrhagic fevers in humans and non-human primates. For infection, filoviral particles need to be internalized and delivered to intracellular vesicles containing cathepsin proteases and the viral receptor Niemann-Pick C1. Previous studies have shown that EBOV triggers macropinocytosis of the viral particles in a glycoprotein (GP)-dependent manner, but the molecular events required for filovirus internalization remain mostly unknown. Here we report that the diacylglycerol kinase inhibitor, R-59-022, blocks EBOV GP-mediated entry into Vero cells and bone marrow-derived macrophages. Investigation of the mode of action of the inhibitor revealed that it blocked an early step in entry, more specifically, the internalization of the viral particles via macropinocytosis. Finally, R-59-022 blocked viral entry mediated by a panel of pathogenic filovirus GPs and inhibited growth of replicative Ebola virus. Taken together, our studies suggest that R-59-022 could be used as a tool to investigate macropinocytic uptake of filoviruses and could be a starting point for the development of pan-filoviral therapeutics.

Download Full-text

Ebola virus triggers receptor tyrosine kinase-dependent signaling to promote the delivery of viral particles to entry-conducive intracellular compartments

PLoS Pathogens ◽

10.1371/journal.ppat.1009275 ◽

2021 ◽

Vol 17 (1) ◽

pp. e1009275

Author(s):

Corina M. Stewart ◽

Alexandra Phan ◽

Yuxia Bo ◽

Nicholas D. LeBlond ◽

Tyler K. T. Smith ◽

...

Keyword(s):

Growth Factor ◽

Tyrosine Kinase ◽

Viral Entry ◽

Receptor Tyrosine Kinase ◽

Ebola Virus ◽

Marburg Virus ◽

Host Protein ◽

Signaling Cascades ◽

Viral Glycoprotein ◽

Viral Particles

Filoviruses, such as the Ebola virus (EBOV) and Marburg virus (MARV), are causative agents of sporadic outbreaks of hemorrhagic fevers in humans. To infect cells, filoviruses are internalized via macropinocytosis and traffic through the endosomal pathway where host cathepsin-dependent cleavage of the viral glycoproteins occurs. Subsequently, the cleaved viral glycoprotein interacts with the late endosome/lysosome resident host protein, Niemann-Pick C1 (NPC1). This interaction is hypothesized to trigger viral and host membrane fusion, which results in the delivery of the viral genome into the cytoplasm and subsequent initiation of replication. Some studies suggest that EBOV viral particles activate signaling cascades and host-trafficking factors to promote their localization with host factors that are essential for entry. However, the mechanism through which these activating signals are initiated remains unknown. By screening a kinase inhibitor library, we found that receptor tyrosine kinase inhibitors potently block EBOV and MARV GP-dependent viral entry. Inhibitors of epidermal growth factor receptor (EGFR), tyrosine protein kinase Met (c-Met), and the insulin receptor (InsR)/insulin like growth factor 1 receptor (IGF1R) blocked filoviral GP-mediated entry and prevented growth of replicative EBOV in Vero cells. Furthermore, inhibitors of c-Met and InsR/IGF1R also blocked viral entry in macrophages, the primary targets of EBOV infection. Interestingly, while the c-Met and InsR/IGF1R inhibitors interfered with EBOV trafficking to NPC1, virus delivery to the receptor was not impaired in the presence of the EGFR inhibitor. Instead, we observed that the NPC1 positive compartments were phenotypically altered and rendered incompetent to permit viral entry. Despite their different mechanisms of action, all three RTK inhibitors tested inhibited virus-induced Akt activation, providing a possible explanation for how EBOV may activate signaling pathways during entry. In sum, these studies strongly suggest that receptor tyrosine kinases initiate signaling cascades essential for efficient post-internalization entry steps.

Download Full-text

Phosphatidylserine within the Viral Membrane Enhances Chikungunya Virus Infectivity in a Cell-type Dependent Manner

10.1101/2022.01.14.476428 ◽

2022 ◽

Author(s):

Kerri L Miazgowicz ◽

Judith Mary Reyes Ballista ◽

Marissa D Acciani ◽

Ariana R Jimenez ◽

Ryan S Belloli ◽

...

Keyword(s):

Chikungunya Virus ◽

Intervention Strategy ◽

Host Cells ◽

Virus Infectivity ◽

Dependent Manner ◽

Cell Type ◽

Enveloped Viruses ◽

Outer Leaflet ◽

Viral Particles ◽

In Cells

Chikungunya virus (CHIKV), an alphavirus of the Togaviridae family, is the causative agent of the human disease chikungunya fever (CHIKF), which is characterized by debilitating acute and chronic arthralgia. No licensed vaccines or antivirals exist for CHIKV. Preventing the attachment of viral particles to host cells is an attractive intervention strategy. Viral entry of enveloped viruses from diverse families including Filoviridae and Flaviviridae is mediated or enhanced by phosphatidylserine receptors (PSRs). PSRs facilitate the attachment of enveloped viruses to cells by binding to exposed phosphatidylserine (PS) in the viral lipid membrane - a process termed viral apoptotic mimicry. To investigate the role of viral apoptotic mimicry during CHIKV infection, we produced viral particles with discrete amounts of exposed PS on the virion envelope by exploiting the cellular distribution of phospholipids at the plasma membrane. We found that CHIKV particles containing high outer leaflet PS (produced in cells lacking flippase activity) were more infectious in Vero cells than particles containing low levels of outer leaflet PS (produced in cells lacking scramblase activity). However, the same viral particles were similarly infectious in NIH3T3 and HAP1 cells, suggesting PS levels can influence infectivity only in cells with high levels of PSRs. Interestingly, PS-dependent CHIKV entry was observed in mosquito Aag2 cells, but not C6/36 cells. These data demonstrate that CHIKV entry via viral apoptotic mimicry is cell-type dependent. Furthermore, viral apoptotic mimicry has a mechanistic basis to influence viral dynamics in vivo in both the human and mosquito host.

Download Full-text

Exchange Protein Directly Activated by cAMP Modulates Ebola Virus Uptake into Vascular Endothelial Cells

Viruses ◽

10.3390/v10100563 ◽

2018 ◽

Vol 10 (10) ◽

pp. 563 ◽

Cited By ~ 5

Author(s):

Aleksandra Drelich ◽

Barbara Judy ◽

Xi He ◽

Qing Chang ◽

Shangyi Yu ◽

...

Keyword(s):

Endothelial Cells ◽

Ebola Virus ◽

Vascular Endothelial Cells ◽

Ex Vivo ◽

Marburg Virus ◽

Dependent Manner ◽

Vascular Endothelial ◽

Ultrastructural Studies ◽

Ebov Infection ◽

Exchange Protein

Members of the family Filoviridae, including Ebola virus (EBOV) and Marburg virus (MARV), cause severe hemorrhagic fever in humans and nonhuman primates. Given their high lethality, a comprehensive understanding of filoviral pathogenesis is urgently needed. In the present studies, we revealed that the exchange protein directly activated by cAMP 1 (EPAC1) gene deletion protects vasculature in ex vivo explants from EBOV infection. Importantly, pharmacological inhibition of EPAC1 using EPAC-specific inhibitors (ESIs) mimicked the EPAC1 knockout phenotype in the ex vivo model. ESI treatment dramatically decreased EBOV infectivity in both ex vivo vasculature and in vitro vascular endothelial cells (ECs). Furthermore, postexposure protection of ECs against EBOV infection was conferred using ESIs. Protective efficacy of ESIs in ECs was observed also in MARV infection. Additional studies using a vesicular stomatitis virus pseudotype that expresses EBOV glycoprotein (EGP-VSV) confirmed that ESIs reduced infection in ECs. Ultrastructural studies suggested that ESIs blocked EGP-VSV internalization via inhibition of macropinocytosis. The inactivation of EPAC1 affects the early stage of viral entry after viral binding to the cell surface, but before early endosome formation, in a phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-dependent manner. Our study delineated a new critical role of EPAC1 during EBOV uptake into ECs.

Download Full-text

Membrane Nanoparticles Derived from ACE2-rich Cells Block SARS-CoV-2 Infection

10.1101/2020.08.12.247338 ◽

2020 ◽

Author(s):

Cheng Wang ◽

Shaobo Wang ◽

Yin Chen ◽

Jianqi Zhao ◽

Songling Han ◽

...

Keyword(s):

Viral Entry ◽

Inhibitory Concentration ◽

Cell Metabolism ◽

Host Cells ◽

Dependent Manner ◽

Human Embryonic Kidney ◽

Proximal Tubular Cells ◽

Tubular Cells ◽

Proximal Tubular ◽

Dose Dependent

ABSTRACTThe ongoing COVID-19 epidemic worldwide necessitates the development of novel effective agents against SARS-CoV-2. ACE2 is the main receptor of SARS-CoV-2 S1 protein and mediates viral entry into host cells. Herein, the membrane nanoparticles prepared from ACE2-rich cells are discovered with potent capacity to block SARS-CoV-2 infection. The membrane of human embryonic kidney-239T cell highly expressing ACE2 is screened to prepare nanoparticles. The nanomaterial termed HEK-293T-hACE2 NPs contains 265.1 ng mg−1 of ACE2 on the surface and acts as a bait to trap SARS-CoV-2 S1 in a dose-dependent manner, resulting in reduced recruitment of the viral ligand to host cells. Interestingly, SARS-CoV-2 S1 can translocate to the cytoplasm and affect the cell metabolism, which is also inhibited by HEK-293T-hACE2 NPs. Further studies reveal that HEK-293T-hACE2 NPs can efficiently suppress SARS-CoV-2 S pseudovirions entry into human proximal tubular cells and block viral infection with a low half maximal inhibitory concentration. Additionally, this biocompatible membrane nanomaterial is sufficient to block the adherence of SARS-CoV-2 D614G-S1 mutant to sensitive cells. Our study demonstrates a easy-to-acheive memrbane nano-antagonist for curbing SARS-CoV-2, which enriches the existing antiviral arsenal and provides new possibilities to treat COVID-19. Graphical Table of Contents

Download Full-text

Circulating ACE2-expressing Exosomes Block SARS-CoV-2 Infection as an Innate Antiviral Mechanism

10.1101/2020.12.03.407031 ◽

2020 ◽

Author(s):

Lamiaa El-Shennawy ◽

Andrew D. Hoffmann ◽

Nurmaa K. Dashzeveg ◽

Paul J. Mehl ◽

Zihao Yu ◽

...

Keyword(s):

Viral Entry ◽

Therapeutic Potential ◽

Adaptive Immune Response ◽

Host Cells ◽

Dependent Manner ◽

Angiotensin Converting Enzyme 2 ◽

Healthy Donors ◽

Dose Dependent ◽

Entry Receptor ◽

Antiviral Mechanism

AbstractThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) with innate and adaptive immune response triggered in such patients by viral antigens. Both convalescent plasma and engineered high affinity human monoclonal antibodies have shown therapeutic potential to treat COVID-19. Whether additional antiviral soluble factors exist in peripheral blood remain understudied. Herein, we detected circulating exosomes that express the SARS-CoV-2 viral entry receptor angiotensin-converting enzyme 2 (ACE2) in plasma of both healthy donors and convalescent COVID-19 patients. We demonstrated that exosomal ACE2 competes with cellular ACE2 for neutralization of SARS-CoV-2 infection. ACE2-expressing (ACE2+) exosomes blocked the binding of the viral spike (S) protein RBD to ACE2+ cells in a dose dependent manner, which was 400- to 700-fold more potent than that of vesicle-free recombinant human ACE2 extracellular domain protein (rhACE2). As a consequence, exosomal ACE2 prevented SARS-CoV-2 pseudotype virus tethering and infection of human host cells at a 50-150 fold higher efficacy than rhACE2. A similar antiviral activity of exosomal ACE2 was further demonstrated to block wild-type live SARS-CoV-2 infection. Of note, depletion of ACE2+ exosomes from COVID-19 patient plasma impaired the ability to block SARS-CoV-2 RBD binding to host cells. Our data demonstrate that ACE2+ exosomes can serve as a decoy therapeutic and a possible innate antiviral mechanism to block SARS-CoV-2 infection.

Download Full-text

SARS-CoV-2 infection induces EMT-like molecular changes, including ZEB1-mediated repression of the viral receptor ACE2, in lung cancer models

10.1101/2020.05.28.122291 ◽

2020 ◽

Cited By ~ 6

Author(s):

C. Allison Stewart ◽

Carl M. Gay ◽

Kavya Ramkumar ◽

Kasey R. Cargill ◽

Robert J. Cardnell ◽

...

Keyword(s):

Lung Cancer ◽

Epithelial To Mesenchymal Transition ◽

Surface Proteins ◽

Host Cells ◽

Dependent Manner ◽

Viral Receptor ◽

Mesenchymal Transition ◽

Transcriptional Changes ◽

Infected Cells ◽

Potential Strategy

AbstractCOVID-19 is an infectious disease caused by SARS-CoV-2, which enters host cells via the cell surface proteins ACE2 and TMPRSS2. Using normal and malignant models and tissues from the aerodigestive and respiratory tracts, we investigated the expression and regulation of ACE2 and TMPRSS2. We find that ACE2 expression is restricted to a select population of highly epithelial cells and is repressed by ZEB1, in concert with ZEB1’s established role in promoting epithelial to mesenchymal transition (EMT). Notably, infection of lung cancer cells with SARS-CoV-2 induces metabolic and transcriptional changes consistent with EMT, including upregulation of ZEB1 and AXL, thereby downregulating ACE2 post-infection. This suggests a novel model of SARS-CoV-2 pathogenesis in which infected cells shift toward an increasingly mesenchymal state and lose ACE2 expression, along with its acute respiratory distress syndrome-protective effect, in a ZEB1-dependent manner. AXL-inhibition and ZEB1-reduction, as with bemcentinib, offers a potential strategy to reverse this effect.

Download Full-text

Analysis of Resistance of Ebola Virus Glycoprotein-Driven Entry Against MDL28170, An Inhibitor of Cysteine Cathepsins

Pathogens ◽

10.3390/pathogens8040192 ◽

2019 ◽

Vol 8 (4) ◽

pp. 192 ◽

Cited By ~ 3

Author(s):

Markus Hoffmann ◽

Svenja Victoria Kaufmann ◽

Carina Fischer ◽

Wiebke Maurer ◽

Anna-Sophie Moldenhauer ◽

...

Keyword(s):

Viral Entry ◽

Ebola Virus ◽

Mutational Analysis ◽

Cathepsin L ◽

Cysteine Proteases ◽

Host Cells ◽

Single Amino Acid ◽

Limited Information ◽

Cysteine Cathepsins ◽

Ebov Infection

Ebola virus (EBOV) infection can cause severe and frequently fatal disease in human patients. The EBOV glycoprotein (GP) mediates viral entry into host cells. For this, GP depends on priming by the pH-dependent endolysosomal cysteine proteases cathepsin B (CatB) and, to a lesser degree, cathepsin L (CatL), at least in most cell culture systems. However, there is limited information on whether and how EBOV-GP can acquire resistance to CatB/L inhibitors. Here, we addressed this question using replication-competent vesicular stomatitis virus bearing EBOV-GP. Five passages of this virus in the presence of the CatB/CatL inhibitor MDL28170 were sufficient to select resistant viral variants and sequencing revealed that all GP sequences contained a V37A mutation, which, in the context of native GP, is located in the base of the GP surface unit. In addition, some GP sequences harbored mutation S195R in the receptor-binding domain. Finally, mutational analysis demonstrated that V37A but not S195R conferred resistance against MDL28170 and other CatB/CatL inhibitors. Collectively, a single amino acid substitution in GP is sufficient to confer resistance against CatB/CatL inhibitors, suggesting that usage of CatB/CatL inhibitors for antiviral therapy may rapidly select for resistant viral variants.

Download Full-text

Comprehensive Analysis of Ebola Virus GP1 in Viral Entry

Journal of Virology ◽

10.1128/jvi.79.8.4793-4805.2005 ◽

2005 ◽

Vol 79 (8) ◽

pp. 4793-4805 ◽

Cited By ~ 107

Author(s):

Balaji Manicassamy ◽

Jizhen Wang ◽

Haiqing Jiang ◽

Lijun Rong

Keyword(s):

Viral Entry ◽

Ebola Virus ◽

Binding Pocket ◽

Marburg Virus ◽

Pseudotyped Virus ◽

Viral Glycoprotein ◽

Functional Roles ◽

Critical Residues ◽

And Function ◽

Expression Processing

ABSTRACT Ebola virus infection is initiated by interactions between the viral glycoprotein GP1 and its cognate receptor(s), but little is known about the structure and function of GP1 in viral entry, partly due to the concern about safety when working with the live Ebola virus and the difficulty of manipulating the RNA genome of Ebola virus. In this study, we have used a human immunodeficiency virus-based pseudotyped virus as a surrogate system to dissect the role of Ebola virus GP1 in viral entry. Analysis of more than 100 deletion and amino acid substitution mutants of GP1 with respect to protein expression, processing, viral incorporation, and viral entry has allowed us to map the region of GP1 responsible for viral entry to the N-terminal 150 residues. Furthermore, six amino acids in this region have been identified as critical residues for early events in Ebola virus entry, and among these, three are clustered and are implicated as part of a potential receptor-binding pocket. In addition, substitutions of some 30 residues in GP1 are shown to adversely affect GP1 expression, processing, and viral incorporation, suggesting that these residues are involved in the proper folding and/or overall conformation of GP. Sequence comparison of the GP1 proteins suggests that the majority of the critical residues for GP folding and viral entry identified in Ebola virus GP1 are conserved in Marburg virus. These results provide information for elucidating the structural and functional roles of the filoviral glycoproteins and for developing potential therapeutics to block viral entry.

Download Full-text

Clathrin-Dependent Entry of Severe Acute Respiratory Syndrome Coronavirus into Target Cells Expressing ACE2 with the Cytoplasmic Tail Deleted

Journal of Virology ◽

10.1128/jvi.00253-07 ◽

2007 ◽

Vol 81 (16) ◽

pp. 8722-8729 ◽

Cited By ~ 140

Author(s):

Yuuki Inoue ◽

Nobuyuki Tanaka ◽

Yoshinori Tanaka ◽

Shingo Inoue ◽

Kouichi Morita ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Viral Entry ◽

Cytoplasmic Tail ◽

Host Cells ◽

Target Cells ◽

Dependent Manner ◽

Virus Attachment ◽

Caveolin 1 ◽

Early Endosomes ◽

Interfering Rna

ABSTRACT The penetration of various viruses into host cells is accomplished by hijacking the host endocytosis machinery. In the case of severe acute respiratory syndrome coronavirus (SARS-CoV) infection, viral entry is reported to require a low pH in intracytoplasmic vesicles; however, little is known about how SARS-CoV invades such compartments. Here we demonstrate that SARS-CoV mainly utilizes the clathrin-mediated endocytosis pathway for its entry to target cells by using infectious SARS-CoV, as well as a SARS-CoV pseudovirus packaged in the SARS-CoV envelope. The SARS-CoV entered caveolin-1-negative HepG2 cells, and the entry was significantly inhibited by treatment with chlorpromazine, an inhibitor for clathrin-dependent endocytosis, and by small interfering RNA-mediated gene silencing for the clathrin heavy chain. Furthermore, the SARS-CoV entered COS7 cells transfected with the mutant of ACE2 with the cytoplasmic tail deleted, SARS-CoV receptor, as well as the wild-type ACE2, and their entries were significantly inhibited by treatment with chlorpromazine. In addition, ACE2 translocated into EEA1-positive early endosomes immediately after the virus attachment to ACE2. These results suggest that when SARS-CoV binds ACE2 it is internalized and penetrates early endosomes in a clathrin-dependent manner and that the cytoplasmic tail of ACE2 is not required for the penetration of SARS-CoV.

Download Full-text

Circulating ACE2-expressing Exosomes Block SARS-CoV-2 Infection as an Innate Antiviral Mechanism

10.21203/rs.3.rs-141729/v1 ◽

2021 ◽

Author(s):

Lamiaa El-Shennawy ◽

Andrew Hoffmann ◽

Nurmaa Dashzeveg ◽

Paul Mehl ◽

Zihao Yu ◽

...

Keyword(s):

Viral Entry ◽

Therapeutic Potential ◽

Adaptive Immune Response ◽

Host Cells ◽

Dependent Manner ◽

Angiotensin Converting Enzyme 2 ◽

Healthy Donors ◽

Negative Controls ◽

Dose Dependent ◽

Antiviral Mechanism

Abstract The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) with innate and adaptive immune response triggered in such patients by viral antigens. Both convalescent plasma and engineered high affinity human monoclonal antibodies have shown therapeutic potential to treat COVID-19. Whether additional antiviral soluble factors exist in peripheral blood remain understudied. Herein, we detected circulating exosomes that express the SARS-CoV-2 viral entry receptor angiotensin-converting enzyme 2 (ACE2) in plasma of both healthy donors and convalescent COVID-19 patients. We demonstrated that exosomal ACE2 competes with cellular ACE2 for neutralization of SARS-CoV-2 infection. ACE2-expressing (ACE2+) exosomes, but not the ACE2-negative controls, blocked the binding of the viral spike (S) protein RBD to ACE2+ cells in a dose dependent manner, which was 400- to 700-fold more potent than that of vesicle-free recombinant human ACE2 extracellular domain protein (rhACE2). As a consequence, exosomal ACE2 prevented SARS-CoV-2 pseudotype virus tethering and infection of human host cells at a 50–150 fold higher efficacy than rhACE2. A similar antiviral activity of exosomal ACE2 was further demonstrated to block wild-type live SARS-CoV-2 infection. Of note, depletion of ACE2+ exosomes from COVID-19 patient plasma impaired the ability to block SARS-CoV-2 RBD binding to host cells. Furthermore, a dramatic increase in plasma ACE2+ exosome levels were detected in patients with severe COVID-19 pathogenesis. Our data demonstrate that ACE2+ exosomes can serve as a decoy therapeutic and a possible innate antiviral mechanism to block SARS-CoV-2 infection.

Download Full-text