Characterisation and application of an ebola virus glycoprotein pseudotyped influenza virus (E-S-Flu)

<p>Ebola virus (EBOV) is a single-stranded RNA virus from the Filoviridae family. It is a biosafety level 4 pathogen, and infection causes fatal haemorrhagic fever in humans, yet no approved vaccines or therapeutics are available.</p> <p>We describe a new EBOV surrogate, E-S-FLU,...

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Main Author: Xiao, HJ
Other Authors: Townsend, A
Format: Thesis
Language:English
Published: 2019
Subjects:
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author Xiao, HJ
author2 Townsend, A
author_facet Townsend, A
Xiao, HJ
author_sort Xiao, HJ
collection OXFORD
description <p>Ebola virus (EBOV) is a single-stranded RNA virus from the Filoviridae family. It is a biosafety level 4 pathogen, and infection causes fatal haemorrhagic fever in humans, yet no approved vaccines or therapeutics are available.</p> <p>We describe a new EBOV surrogate, E-S-FLU, a single-cycle Influenza virus pseudotyped (coated) with the EBOV glycoprotein (GP). E-S-FLU does not contain any genetic information from EBOV, and encodes enhanced green fluorescence protein as a reporter that replaces the Influenza virus hemagglutinin. It can thus be handled in biosafety level 1 containment for laboratory analysis. We show that E-S-FLU mimics wild-type EBOV during the process of cell entry. Infection of cells by E-S-FLU was dependent on the Niemann-Pick type C1 protein, the wellcharacterised receptor for EBOV entry at the late endosomal/lysosomal membrane. In addition, known small molecule entry inhibitors that blocked wildtype EBOV infection in vitro blocked E-S-FLU with similar potency.</p> <p>We employed E-S-FLU to screen a library of 1,280 pharmacologically active compounds for inhibition of virus entry. We identified 215 inhibitory compounds with very dispersed targets. The majority of the inhibitors shared a cationic amphiphilic feature, and this had the strongest correlation with specific inhibition of E-S-FLU infection, regardless of the pharmacological group of the drugs.</p> <p>The two-pore channels 1 and 2 (TPC1/2), and acid sphingomyelinase (ASM), both localised in the lysosomal membrane, have been proposed as candidate drug targets. To test this, we knocked out the corresponding genes from HeLa cells using the CRISPR/Cas 9 technique. We showed that EBOV-GP dependent infection by E-S-FLU was not affected in TPC1/2 or ASM deficient HeLa cells, and concluded that these proteins are not required for EBOV infection.</p> <p>Our results suggest that the most effective cationic amphiphilic drugs act by concentrating in the lysosome, and by binding and destabilising the GP, and intercalating in the lysosomal membrane, may interfere directly with the fusion function of the EBOV-GP.</p>
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spelling oxford-uuid:6c6432e0-385b-4f51-ba37-35df3d26303d2024-02-22T09:53:43ZCharacterisation and application of an ebola virus glycoprotein pseudotyped influenza virus (E-S-Flu)Thesishttp://purl.org/coar/resource_type/c_db06uuid:6c6432e0-385b-4f51-ba37-35df3d26303dDrug ScreeningVirologyImmunologyEnglishHyrax Deposit2019Xiao, HJTownsend, A<p>Ebola virus (EBOV) is a single-stranded RNA virus from the Filoviridae family. It is a biosafety level 4 pathogen, and infection causes fatal haemorrhagic fever in humans, yet no approved vaccines or therapeutics are available.</p> <p>We describe a new EBOV surrogate, E-S-FLU, a single-cycle Influenza virus pseudotyped (coated) with the EBOV glycoprotein (GP). E-S-FLU does not contain any genetic information from EBOV, and encodes enhanced green fluorescence protein as a reporter that replaces the Influenza virus hemagglutinin. It can thus be handled in biosafety level 1 containment for laboratory analysis. We show that E-S-FLU mimics wild-type EBOV during the process of cell entry. Infection of cells by E-S-FLU was dependent on the Niemann-Pick type C1 protein, the wellcharacterised receptor for EBOV entry at the late endosomal/lysosomal membrane. In addition, known small molecule entry inhibitors that blocked wildtype EBOV infection in vitro blocked E-S-FLU with similar potency.</p> <p>We employed E-S-FLU to screen a library of 1,280 pharmacologically active compounds for inhibition of virus entry. We identified 215 inhibitory compounds with very dispersed targets. The majority of the inhibitors shared a cationic amphiphilic feature, and this had the strongest correlation with specific inhibition of E-S-FLU infection, regardless of the pharmacological group of the drugs.</p> <p>The two-pore channels 1 and 2 (TPC1/2), and acid sphingomyelinase (ASM), both localised in the lysosomal membrane, have been proposed as candidate drug targets. To test this, we knocked out the corresponding genes from HeLa cells using the CRISPR/Cas 9 technique. We showed that EBOV-GP dependent infection by E-S-FLU was not affected in TPC1/2 or ASM deficient HeLa cells, and concluded that these proteins are not required for EBOV infection.</p> <p>Our results suggest that the most effective cationic amphiphilic drugs act by concentrating in the lysosome, and by binding and destabilising the GP, and intercalating in the lysosomal membrane, may interfere directly with the fusion function of the EBOV-GP.</p>
spellingShingle Drug Screening
Virology
Immunology
Xiao, HJ
Characterisation and application of an ebola virus glycoprotein pseudotyped influenza virus (E-S-Flu)
title Characterisation and application of an ebola virus glycoprotein pseudotyped influenza virus (E-S-Flu)
title_full Characterisation and application of an ebola virus glycoprotein pseudotyped influenza virus (E-S-Flu)
title_fullStr Characterisation and application of an ebola virus glycoprotein pseudotyped influenza virus (E-S-Flu)
title_full_unstemmed Characterisation and application of an ebola virus glycoprotein pseudotyped influenza virus (E-S-Flu)
title_short Characterisation and application of an ebola virus glycoprotein pseudotyped influenza virus (E-S-Flu)
title_sort characterisation and application of an ebola virus glycoprotein pseudotyped influenza virus e s flu
topic Drug Screening
Virology
Immunology
work_keys_str_mv AT xiaohj characterisationandapplicationofanebolavirusglycoproteinpseudotypedinfluenzavirusesflu