Fluorescent probes for monitoring virus fusion kinetics: comparative evaluation of reliability
Autor(a) principal: | |
---|---|
Data de Publicação: | 2002 |
Outros Autores: | , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10316/3881 https://doi.org/10.1016/S0005-2736(01)00457-6 |
Resumo: | Fluorescence assays for viral membrane fusion employ lipidic probes whose kinetics of fluorescence dequenching should mimic the actual kinetics of membrane merging. We examined the fusion of influenza virus with CEM cells, erythrocyte ghosts or liposomes by monitoring the fluorescence dequenching of each one of the three probes, octadecylrhodamine B chloride (R18), N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (Rh-PE), or rac-2,3-dioleoylglycerol ester of rhodamine B (DORh-B), inserted into the virus membrane. Experimental conditions were designed to allow a clear distinction between membrane mixing and non-specific probe transfer. Fluorescence dequenching observed with Rh-PE was much slower than with R18, unless a particular experimental procedure was used. Using liposomes as a target membrane, the kinetics and extent of the decrease in resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) and Rh-PE, initially embedded in the liposome membrane, were matched by that of the dequenching of viral R18, but not of viral Rh-PE. DORh-B was found not to be appropriate to follow membrane merging. Our results indicate that on a time scale of several minutes R18 more accurately reflects the kinetics of membrane fusion. Nevertheless, control experiments should be performed to evaluate non-specific probe transfer of R18 molecules, whose contribution to fluorescence dequenching can become significant after long incubation times. |
id |
RCAP_ba5df9f962394e5681ecddcd59c4b2a6 |
---|---|
oai_identifier_str |
oai:estudogeral.uc.pt:10316/3881 |
network_acronym_str |
RCAP |
network_name_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository_id_str |
7160 |
spelling |
Fluorescent probes for monitoring virus fusion kinetics: comparative evaluation of reliabilityInfluenza virusCEM cellErythrocyte ghostLiposomeMembrane fusionFluorescent probeNonspecific probe transferFluorescence assays for viral membrane fusion employ lipidic probes whose kinetics of fluorescence dequenching should mimic the actual kinetics of membrane merging. We examined the fusion of influenza virus with CEM cells, erythrocyte ghosts or liposomes by monitoring the fluorescence dequenching of each one of the three probes, octadecylrhodamine B chloride (R18), N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (Rh-PE), or rac-2,3-dioleoylglycerol ester of rhodamine B (DORh-B), inserted into the virus membrane. Experimental conditions were designed to allow a clear distinction between membrane mixing and non-specific probe transfer. Fluorescence dequenching observed with Rh-PE was much slower than with R18, unless a particular experimental procedure was used. Using liposomes as a target membrane, the kinetics and extent of the decrease in resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) and Rh-PE, initially embedded in the liposome membrane, were matched by that of the dequenching of viral R18, but not of viral Rh-PE. DORh-B was found not to be appropriate to follow membrane merging. Our results indicate that on a time scale of several minutes R18 more accurately reflects the kinetics of membrane fusion. Nevertheless, control experiments should be performed to evaluate non-specific probe transfer of R18 molecules, whose contribution to fluorescence dequenching can become significant after long incubation times.http://www.sciencedirect.com/science/article/B6T1T-44Y0Y0Y-1/1/b35516dfbf496c530424def0e26ee79b2002info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleaplication/PDFhttp://hdl.handle.net/10316/3881http://hdl.handle.net/10316/3881https://doi.org/10.1016/S0005-2736(01)00457-6engBiochimica et Biophysica Acta (BBA) - Biomembranes. 1561:1 (2002) 65-75Nunes-Correia, IsabelEulálio, AnaNir, ShlomoDüzgünes, NejatRamalho-Santos, JoãoLima, Maria C. Pedroso deinfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2021-09-17T11:05:00Zoai:estudogeral.uc.pt:10316/3881Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T20:55:47.704122Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Fluorescent probes for monitoring virus fusion kinetics: comparative evaluation of reliability |
title |
Fluorescent probes for monitoring virus fusion kinetics: comparative evaluation of reliability |
spellingShingle |
Fluorescent probes for monitoring virus fusion kinetics: comparative evaluation of reliability Nunes-Correia, Isabel Influenza virus CEM cell Erythrocyte ghost Liposome Membrane fusion Fluorescent probe Nonspecific probe transfer |
title_short |
Fluorescent probes for monitoring virus fusion kinetics: comparative evaluation of reliability |
title_full |
Fluorescent probes for monitoring virus fusion kinetics: comparative evaluation of reliability |
title_fullStr |
Fluorescent probes for monitoring virus fusion kinetics: comparative evaluation of reliability |
title_full_unstemmed |
Fluorescent probes for monitoring virus fusion kinetics: comparative evaluation of reliability |
title_sort |
Fluorescent probes for monitoring virus fusion kinetics: comparative evaluation of reliability |
author |
Nunes-Correia, Isabel |
author_facet |
Nunes-Correia, Isabel Eulálio, Ana Nir, Shlomo Düzgünes, Nejat Ramalho-Santos, João Lima, Maria C. Pedroso de |
author_role |
author |
author2 |
Eulálio, Ana Nir, Shlomo Düzgünes, Nejat Ramalho-Santos, João Lima, Maria C. Pedroso de |
author2_role |
author author author author author |
dc.contributor.author.fl_str_mv |
Nunes-Correia, Isabel Eulálio, Ana Nir, Shlomo Düzgünes, Nejat Ramalho-Santos, João Lima, Maria C. Pedroso de |
dc.subject.por.fl_str_mv |
Influenza virus CEM cell Erythrocyte ghost Liposome Membrane fusion Fluorescent probe Nonspecific probe transfer |
topic |
Influenza virus CEM cell Erythrocyte ghost Liposome Membrane fusion Fluorescent probe Nonspecific probe transfer |
description |
Fluorescence assays for viral membrane fusion employ lipidic probes whose kinetics of fluorescence dequenching should mimic the actual kinetics of membrane merging. We examined the fusion of influenza virus with CEM cells, erythrocyte ghosts or liposomes by monitoring the fluorescence dequenching of each one of the three probes, octadecylrhodamine B chloride (R18), N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (Rh-PE), or rac-2,3-dioleoylglycerol ester of rhodamine B (DORh-B), inserted into the virus membrane. Experimental conditions were designed to allow a clear distinction between membrane mixing and non-specific probe transfer. Fluorescence dequenching observed with Rh-PE was much slower than with R18, unless a particular experimental procedure was used. Using liposomes as a target membrane, the kinetics and extent of the decrease in resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) and Rh-PE, initially embedded in the liposome membrane, were matched by that of the dequenching of viral R18, but not of viral Rh-PE. DORh-B was found not to be appropriate to follow membrane merging. Our results indicate that on a time scale of several minutes R18 more accurately reflects the kinetics of membrane fusion. Nevertheless, control experiments should be performed to evaluate non-specific probe transfer of R18 molecules, whose contribution to fluorescence dequenching can become significant after long incubation times. |
publishDate |
2002 |
dc.date.none.fl_str_mv |
2002 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10316/3881 http://hdl.handle.net/10316/3881 https://doi.org/10.1016/S0005-2736(01)00457-6 |
url |
http://hdl.handle.net/10316/3881 https://doi.org/10.1016/S0005-2736(01)00457-6 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Biochimica et Biophysica Acta (BBA) - Biomembranes. 1561:1 (2002) 65-75 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
aplication/PDF |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
repository.mail.fl_str_mv |
|
_version_ |
1799133844252655616 |