The structure-function relationship of HIV-1 Vif protein and its regulation

Bibliographic Details
Main Author: Couto, Carla
Publication Date: 2008
Language: eng
Source: Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
Download full: http://hdl.handle.net/10451/275
Summary: Tese de doutoramento em Farmácia (Microbiologia), apresentada à Universidade de Lisboa através da Faculdade de Farmácia, 2008
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spelling The structure-function relationship of HIV-1 Vif protein and its regulationMicrobiologiaTeses de doutoramento - 2008Tese de doutoramento em Farmácia (Microbiologia), apresentada à Universidade de Lisboa através da Faculdade de Farmácia, 2008One of the critical determinants of HIV-1 infectivity is the capacity of the virus to infect new cells. The viral Vif protein is one of the critical factors involved in the increase infectivity of the virus. Vif protein is required for HIV to replicate in some human cell types (termed non-permissive' cells), but not others ( permissive' cells). The reason for this is the expression, in non-permissive cells, of a potent antiretroviral enzyme: APOBEC3G (A3G) and its paralog APOBEC3F (A3F). Each of these enzymes contains two RNA-binding motifs and incorporates into assembling HIV-1 capsids where they cause lethal dC-to-dU hypermutations in the single-stranded viral DNA that transiently forms during reverse transcription. One of the mechanisms that Vif uses to overcome this viral block is by reducing A3G/A3F intracellular expression levels recruiting it for polyubiquitination and proteasomal degradation in the 26S proteasome. In order to understand how A3G half-life can influence its antiviral activity, both at the level of viral encapsidation and catalytic activity in the cell, we decided to model the intracellular half-life of A3G by targeting it to the N-end rule pathway. This system allowed us to show that A3G variants with different stabilities are capable to be targeted for proteasomal degradation in a Vif-independent manner. Our results confirmed the importance of A3G steady-state expression for the maintenance of its antiretroviral activity indicating that in order to counteract A3G activity, Vif has to act at the very early stages of A3G life-time, probably within the first 13 minutes after its synthesis, in order to impede its incorporation into virions. In addition, we show that active deamination in the cytoplasm does not seem to be important for antiviral activity of A3G and deamination activity does not increase with the half-life of A3G. Identification of protein biding partners between Vif and A3G proteins is one of the major challenges for the development of new antiviral drugs, as disruption of A3G-Vif interaction is predicted to stimulate natural antiviral infectivity. In this work, we used two different approaches to study the interaction of Vif with A3G. One strategy was based on an oligomerization-assisted interaction whereby fragments of Vif and A3G were fused to independently folding and interacting domains of homodimerizing GCN4 leucine zipper-forming sequences. This allowed us to show that the C-terminal region of Vif alone is not sufficient to induce A3G degradation and that binding of Vif to A3G must involve an additional co-factor that is probably linked to the N-terminus of Vif and may be essential for the interaction and consequent degradation of A3G. The other strategy involved a Protein Complementation Assay (PCA). We produced several mutants of both Vif and A3G bearing mutations on key residues that are thought to be crucial for the interaction between these two proteins. By using this strategy we were able to show that some mutations in A3G protein that had been described as being involved in the interaction with HIV-1 Gag, were also important for the interaction with Vif. As a result, we were able to achieve a quantitative assessment of HIV-1 Vif-A3G interactions, which allowed us to do a fine dissection of the regions involved in this interaction. Finally, we found that a region in Vif (DRMR) may be important for the selection of the APOBEC protein to inactivate. By studying the interaction of Vif with A2 protein we reached the conclusion that an additional co-factor may be required in order to A2 to be able to exert antiviral action. Therefore, we provided supporting information for a new direction of the study of Vif-A3 problematic regarding the involvement of anadditional co-factor.Fundação para a Ciência e Tecnologia (FCT), (SFRH/BD/18805/2004)Gonçalves, João, 1967-Repositório da Universidade de LisboaCouto, Carla2010-06-16T09:46:35Z20082008-01-01T00:00:00Zdoctoral thesisinfo:eu-repo/semantics/publishedVersionapplication/pdfapplication/pdfapplication/pdftext/xmlhttp://hdl.handle.net/10451/275enginfo:eu-repo/semantics/openAccessreponame:Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)instname:FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologiainstacron:RCAAP2025-03-17T12:34:54Zoai:repositorio.ulisboa.pt:10451/275Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireinfo@rcaap.ptopendoar:https://opendoar.ac.uk/repository/71602025-05-29T02:23:29.768765Repositórios Científicos de Acesso Aberto de Portugal (RCAAP) - FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologiafalse
dc.title.none.fl_str_mv The structure-function relationship of HIV-1 Vif protein and its regulation
title The structure-function relationship of HIV-1 Vif protein and its regulation
spellingShingle The structure-function relationship of HIV-1 Vif protein and its regulation
Couto, Carla
Microbiologia
Teses de doutoramento - 2008
title_short The structure-function relationship of HIV-1 Vif protein and its regulation
title_full The structure-function relationship of HIV-1 Vif protein and its regulation
title_fullStr The structure-function relationship of HIV-1 Vif protein and its regulation
title_full_unstemmed The structure-function relationship of HIV-1 Vif protein and its regulation
title_sort The structure-function relationship of HIV-1 Vif protein and its regulation
author Couto, Carla
author_facet Couto, Carla
author_role author
dc.contributor.none.fl_str_mv Gonçalves, João, 1967-
Repositório da Universidade de Lisboa
dc.contributor.author.fl_str_mv Couto, Carla
dc.subject.por.fl_str_mv Microbiologia
Teses de doutoramento - 2008
topic Microbiologia
Teses de doutoramento - 2008
description Tese de doutoramento em Farmácia (Microbiologia), apresentada à Universidade de Lisboa através da Faculdade de Farmácia, 2008
publishDate 2008
dc.date.none.fl_str_mv 2008
2008-01-01T00:00:00Z
2010-06-16T09:46:35Z
dc.type.driver.fl_str_mv doctoral thesis
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10451/275
url http://hdl.handle.net/10451/275
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
text/xml
dc.source.none.fl_str_mv reponame:Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
instname:FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologia
instacron:RCAAP
instname_str FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologia
instacron_str RCAAP
institution RCAAP
reponame_str Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
collection Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
repository.name.fl_str_mv Repositórios Científicos de Acesso Aberto de Portugal (RCAAP) - FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologia
repository.mail.fl_str_mv info@rcaap.pt
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