Inhibition of Ebola virus entry by a C-peptide targeted to endosomes.
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ABSTRACT:
SUBMITTER: Miller EH
PROVIDER: S-EPMC3091195 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:UnlabelledFiloviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to develop prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors, which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs, including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step following the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy.ImportanceInfection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epidemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors that are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. Our results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy.
| S-EPMC4577884 | biostudies-literature
Project description:Transcriptomic analyses showed that Ebola virus delta peptide triggers damage response and cell survival pathways. Delta peptide also downregulates the expression of transporters and exchangers that mediate absorption of ions, sugars, and small chain fatty acids.
2021-12-01 | GSE189787 | GEO
Project description:Zaire ebolavirus (ZEBOV), a highly pathogenic zoonotic virus, poses serious public health, ecological and potential bioterrorism threats. Currently no specific therapy or vaccine is available. Virus entry is an attractive target for therapeutic intervention. However, current knowledge of the ZEBOV entry mechanism is limited. While it is known that ZEBOV enters cells through endocytosis, which of the cellular endocytic mechanisms used remains unclear. Previous studies have produced differing outcomes, indicating potential involvement of multiple routes but many of these studies were performed using noninfectious surrogate systems such as pseudotyped retroviral particles, which may not accurately recapitulate the entry characteristics of the morphologically distinct wild type virus. Here we used replication-competent infectious ZEBOV as well as morphologically similar virus-like particles in specific infection and entry assays to demonstrate that in HEK293T and Vero cells internalization of ZEBOV is independent of clathrin, caveolae, and dynamin. Instead the uptake mechanism has features of macropinocytosis. The binding of virus to cells appears to directly stimulate fluid phase uptake as well as localized actin polymerization. Inhibition of key regulators of macropinocytosis including Pak1 and CtBP/BARS as well as treatment with the drug EIPA, which affects macropinosome formation, resulted in significant reduction in ZEBOV entry and infection. It is also shown that following internalization, the virus enters the endolysosomal pathway and is trafficked through early and late endosomes, but the exact site of membrane fusion and nucleocapsid penetration in the cytoplasm remains unclear. This study identifies the route for ZEBOV entry and identifies the key cellular factors required for the uptake of this filamentous virus. The findings greatly expand our understanding of the ZEBOV entry mechanism that can be applied to development of new therapeutics as well as provide potential insight into the trafficking and entry mechanism of other filoviruses.
| S-EPMC2940741 | biostudies-literature
Project description:Ebolaviruses constitute a public health threat, particularly in Central and Western Africa. Host cell factors required for spread of ebolaviruses may serve as targets for antiviral intervention. Lectins, TAM receptor tyrosine kinases (Tyro3, Axl, Mer), T cell immunoglobulin and mucin domain (TIM) proteins, integrins, and Niemann-Pick C1 (NPC1) have been reported to promote entry of ebolaviruses into certain cellular systems. However, the factors used by ebolaviruses to invade macrophages, major viral targets, are poorly defined. Here, we show that mannose-specific lectins, TIM-1 and Axl augment entry into certain cell lines but do not contribute to Ebola virus (EBOV)-glycoprotein (GP)-driven transduction of macrophages. In contrast, expression of Mer, integrin ?V, and NPC1 was required for efficient GP-mediated transduction and EBOV infection of macrophages. These results define cellular factors hijacked by EBOV for entry into macrophages and, considering that Mer and integrin ?V promote phagocytosis of apoptotic cells, support the concept that EBOV relies on apoptotic mimicry to invade target cells.
| S-EPMC4564540 | biostudies-other
Project description:Multiple cell surface molecules including TAM receptors (TYRO3, AXL, and MERTK), a family of tyrosine kinase receptors, can serve as attachment receptors for Ebola virus (EBOV) entry into cells. The interaction of these receptors with EBOV particles is believed to trigger the initial internalization events that lead to macropinocytosis. However, the details of how these interactions lead to EBOV internalization have yet to be elucidated. Here, we screened receptor tyrosine kinase (RTK) inhibitors for anti-EBOV activity by using our previously established biologically contained Ebola virus that lacks the VP30 gene (EBOVΔVP30) and identified several RTKs, including human epidermal growth factor receptor 2 (HER2), as potential targets of anti-EBOV inhibitors and as novel host factors that have a role in EBOV infection. Of these identified RTKs, it was only HER2 whose knockdown by siRNAs impaired EBOVΔVP30-induced AKT1 phosphorylation, an event that is required for AKT1 activation and subsequent macropinocytosis. Stable expression of HER2 resulted in constitutive activation of AKT1, resulting in the enhancement of EBOVΔVP30 growth, EBOV GP-mediated entry, and macropinocytosis. Moreover, we found that HER2 interacts with the TAM receptors, and in particular forms a complex with TYRO3 and EBOVΔVP30 particles on the cell surface. Interestingly, HER2 was required for EBOVΔVP30-induced TYRO3 and AKT1 activation, but the other TAM receptors (TYRO3 and MERTK) were not essential for EBOVΔVP30-induced HER2 and AKT1 activation. Our findings demonstrate that HER2 plays an important role in EBOV entry and provide novel insights for the development of therapeutics against the virus.
| S-EPMC7556532 | biostudies-literature
Project description:Concerns about the possible use of Variola virus, the causative agent of smallpox, as a weapon for bioterrorism have led to renewed efforts to identify new antivirals against orthopoxviruses. We identified a peptide, EB, which inhibited infection by Vaccinia virus with an EC(50) of 15 microM. A control peptide, EBX, identical in composition to EB but differing in sequence, was inactive (EC50>200 microM), indicating sequence specificity. The inhibition was reversed upon removal of the peptide, and EB treatment had no effect on the physical integrity of virus particles as determined by electron microscopy. Viral adsorption was unaffected by the presence of EB, and the addition of EB post-entry had no effect on viral titers or on early gene expression. The addition of EB post-adsorption resulted in the inhibition of beta-galactosidase expression from an early viral promoter with an EC(50) of 45 microM. A significant reduction in virus entry was detected in the presence of the peptide when the number of viral cores released into the cytoplasm was quantified. Electron microscopy indicated that 88% of the virions remained on the surface of cells in the presence of EB, compared to 37% in the control (p<0.001). EB also blocked fusion-from-within, suggesting that virus infection is inhibited at the fusion step. Analysis of EB derivatives suggested that peptide length may be important for the activity of EB. The EB peptide is, to our knowledge, the first known small molecule inhibitor of Vaccinia virus entry.
| S-EPMC2923446 | biostudies-literature
Project description:After binding to its cell surface receptor ganglioside GM1, simian virus 40 (SV40) is endocytosed by lipid raft-mediated endocytosis and slowly transported to the endoplasmic reticulum, where partial uncoating occurs. We analyzed the intracellular pathway taken by the virus in HeLa and CV-1 cells by using a targeted small interfering RNA (siRNA) silencing screen, electron microscopy, and live-cell imaging as well as by testing a variety of cellular inhibitors and other perturbants. We found that the virus entered early endosomes, late endosomes, and probably endolysosomes before reaching the endoplasmic reticulum and that this pathway was part of the infectious route. The virus was especially sensitive to a variety of perturbations that inhibited endosome acidification and maturation. Contrary to our previous models, which postulated the passage of the virus through caveolin-rich organelles that we called caveosomes, we conclude that SV40 depends on the classical endocytic pathway for infectious entry.
| S-EPMC3126231 | biostudies-literature
Project description:BackgroundThe current Ebola virus (EBOV) outbreak has highlighted the troubling absence of available antivirals or vaccines to treat infected patients and stop the spread of EBOV. The EBOV glycoprotein (GP) plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a potential target for the development of anti-EBOV drugs. We report the identification of 2 novel EBOV inhibitors targeting viral entry.MethodsTo identify small molecule inhibitors of EBOV entry, we carried out a cell-based high-throughput screening using human immunodeficiency virus-based pseudotyped viruses expressing EBOV-GP. Two compounds were identified, and mechanism-of-action studies were performed using immunoflourescence, AlphaLISA, and enzymatic assays for cathepsin B inhibition.ResultsWe report the identification of 2 novel entry inhibitors. These inhibitors (1) inhibit EBOV infection (50% inhibitory concentration, approximately 0.28 and approximately 10 µmol/L) at a late stage of entry, (2) induce Niemann-Pick C phenotype, and (3) inhibit GP-Niemann-Pick C1 (NPC1) protein interaction.ConclusionsWe have identified 2 novel EBOV inhibitors, MBX2254 and MBX2270, that can serve as starting points for the development of an anti-EBOV therapeutic agent. Our findings also highlight the importance of NPC1-GP interaction in EBOV entry and the attractiveness of NPC1 as an antifiloviral therapeutic target.
| S-EPMC4564548 | biostudies-literature
Project description: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.
| S-EPMC1069533 | biostudies-literature
Project description:The Ebolaviruses are members of the family Filoviridae ("filoviruses") and cause severe hemhorragic fever with human case fatality rates as high as 90%. Infection requires attachment of the viral particle to cells and triggering of membrane fusion between the host and viral membranes, a process that occurs in the host endosome and is facilitated by the envelope glycoprotein (GP). One potential strategy for therapeutic intervention is the development of agents (antibodies, peptides, and small molecules) that can interfere with viral entry aspects such as attachment, uptake, priming, or membrane fusion. This paper highlights recent developments in the discovery and evaluation of therapeutic entry inhibitors and identifies opportunities moving forward.
| S-EPMC4426353 | biostudies-literature