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Dissertations / Theses on the topic 'HIV-1 Nef'

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1

Chen, Nan. "The role of HIV-1 Nef gene in HIV-mediated pathogenesis." Thesis, University of Oxford, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.404289.

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2

Rahim, Mir Munir Ahmed 1975. "Pathogenesis of HIV-1 nef in adult mice." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=115698.

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Development of a suitable animal model of AIDS is much needed in AIDS research to study infection and pathogenesis as well as to evaluate methods of prevention and treatment of HIV infection. Small animals such as rodents are attractive candidates for AIDS research due to the availability of various inbred and genetically engineered strains, extensive knowledge or their immune system, especially in mice, and the relative ease of breeding and maintaining animal colonies. Transgenic small animal models carrying entire HIV genome or selected genes have been instrumental to understand functions of HIV genes in vivo and their role in HIV pathogenesis. The type of cells in which HIV genes are expressed seems to be an import prerequisite for the study of HIV gene functions in transgenic mice. Mice constitutively expressing the entire HIV-1 genome or HIV-1 nef gene in CD4 + T cells and in the cells of macrophage/dendritic lineage develop an AIDS-like disease very similar to AIDS disease in humans. Similarly, expression of Nef in adult mice, using inducible system, results in the AIDS-like disease. This disease is characterized by thymic atrophy, impaired thymocyte maturation, loss of CD4+ T cells, increased activation and turnover of T cells, which can occur in the absence of lymphypenia, and non-lymphoid organ disease involving the lungs and kidneys. Susceptibility of adult mice to the pathological effects of Nef suggests that the AIDS-like disease in the constitutively expressing Nef Tg mice is not due to developmental defects caused by early expression of Nef. This model highlights the important role of Nef in HIV-1 pathogenesis. The high similarity in the disease in these Tg mice with human AIDS strongly suggest that these mice are a relevant model to study AIDS. This study further evidence that mouse cells can support functions of Nef and these Tg mice represent a unique model to study Nef functions in vivo in the context of the primary immune system. Moreover, the inducible Nef Tg model has given us the ability to control the level and time of expression of Nef which was impossible to do in the previously reported constitutive Nef Tg mouse models. These mice will be useful to study immune reconstitution since Nef expression can be turned off after withdrawal from dox.
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3

Chuah, Lay Khim Marinee. "Role of nef in HIV-1 gene regulation." Doctoral thesis, Universite Libre de Bruxelles, 1992. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/212893.

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4

Craig, Heather Marie. "Alterations of intracellular protein sorting by HIV-1 nef /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2000. http://wwwlib.umi.com/cr/ucsd/fullcit?p9970642.

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5

Jennelle, Lucas Trent. "Contribution of Calnexin to HIV-1 Nef effects on ABCA1." Thesis, The George Washington University, 2013. http://pqdtopen.proquest.com/#viewpdf?dispub=3557581.

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HIV-infected patients are at increased risk of developing atherosclerosis, in part due to an altered HDL profile exacerbated by downmodulation and impairment of ATP-Binding Cassette Transporter A1 (ABCA1) activity by the HIV-1 protein Nef. Nef has been shown to increase delivery of cholesterol to lipid rafts, sites of viral assembly and egress, by inhibition of ABCA1 cholesterol efflux functionality and reduction of ABCA1 protein levels through lysosomal degradation. Important mechanistic details of ABCA1 inactivation and degradation by Nef, and whether these two processes are intimately linked or separable are still to be defined. The studies presented here were designed to identify cellular co-factors for ABCA1-mediated cholesterol efflux that may be targeted by Nef to achieve ABCA1 inactivation. In these studies, a novel cellular factor, the ER-resident lectin chaperone calnexin, was shown to be involved in a physical interaction with ABCA1 that is disrupted by Nef. Nef was found to bind and redistribute calnexin and reduce binding and co-localization of ABCA1 with calnexin. In vitro knockdown of calnexin via RNAi reproduced several previously described biochemical effects of Nef, including redistribution of ABCA1, increased ABCA1 membrane localization, and reduced ABCA1 recycling. Importantly, knockdown of calnexin also resulted in reduced ABCA1-mediated cholesterol efflux, but without the Nef-mediated reduction in ABCA1 protein levels, suggesting that Nef utilizes a bipartite mechanism to inactivate and degrade ABCA1 and that these functions may be separable. Despite the lack of effect of calnexin knockdown on ABCA1 protein levels, interference with the ABCA1-calnexin interaction was critical for Nef-mediated functional impairment of ABCA1. This was shown with a Nef mutant defective in interaction with calnexin which was incapable of preventing ABCA1-calnexin interaction and was also defective in impairing ABCA1-mediated cholesterol efflux activity. Thus, these studies identified a novel mechanism by which HIV-1 Nef impairs functional activity of cholesterol transporter ABCA1 by blocking its interaction with calnexin. Calnexin acts as an ABCA1 functional chaperone, limiting total and cell surface ABCA1 expression while increasing ABCA1-mediated cholesterol efflux. Combined with the demonstration that Nef increases delivery of ABCA1 to lysosomes, these results suggest the Nef-mediated impairment of ABCA1 function involves reduced interaction with calnexin followed by delivery of ABCA1 to lysosomes for degradation.

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6

Dai, Weiwei. "SERINC5: Its Sensitivity to Nef and Restriction of HIV-1." eScholarship@UMMS, 2018. https://escholarship.umassmed.edu/gsbs_diss/984.

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The accessory protein Nef of human immunodeficiency virus type 1 (HIV-1) has long been known to enhance the infectivity of HIV-1 progeny virions. The multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 were recently identified as novel antiviral proteins that restrict HIV-1 infectivity. Nef enhances HIV-1 infectivity by removing SERINCs from the plasma membrane, which prevents their incorporation into progeny HIV-1 virions. To exploit this potent intrinsic antiretroviral factor for potential therapy development, it is critical to explore the determinants in SERINC5 that govern its downregulation by Nef and its restriction on HIV-1 infectivity. Here I report that the ability to inhibit HIV-1 infectivity is conserved among vertebrate SERINC5 proteins, whereas the sensitivity to downregulation by Nef is not. However, a Nef-resistant SERINC5 became Nef-sensitive when its intracellular loop 4 (ICL4) was replaced by that of Nef-sensitive human SERINC5. Conversely, human SERINC5 became resistant to Nef when its ICL4 was replaced by that of a Nef-resistant SERINC5. In general, ICL4 regions from SERINCs that exhibited resistance to a given Nef conferred resistance to the same Nef when transferred to a sensitive SERINC, and vice versa. I demonstrate that human SERINC5 can be modified to restrict HIV-1 infectivity even in the presence of Nef. Moreover, by generating chimeras between SERINC5 and SERINC2, which does not exhibit antiretroviral activity, I demonstrate that SERINC5’s inhibitory function, unlike the sensitivity to Nef, requires the participation of more than one region. Helix 4 and extracellular loop 5 (ECL5) of SERINC5 are both required for the potent restriction of HIV-1 infectivity. In contrast, a large amino-terminal portion of SERINC5 is not required for its antiretroviral activity of SERINC5. The determinants in ECL5 disperse throughout the loop. Furthermore, the ECL5 of SERINC5 is a hotspot region that determines the Env-dependent antiretroviral activity of SERINC5.
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7

Ramalho, Eduardo Dias. "Variabilidade genética de NEF em amostras de HIV-1 circulantes." reponame:Repositório Institucional da UnB, 2006. http://repositorio.unb.br/handle/10482/2319.

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Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2006.
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Em 2005, cerca de 40 milhões de pessoas viviam com HIV/AIDS no mundo. Desse total, aproximadamente 600 mil residiam no Brasil. Em vista do elevado percentual de infecções pelo HIV-1, o desenvolvimento de vacinas contra esse vírus é uma das prioridades no que se refere à saúde pública mundial. Entretanto, apesar dos estudos sobre esse vírus, pouco se conhece sobre a importância da sua diversidade genética para a eficácia de potenciais vacinas. Assim, conhecer a variabilidade do HIV-1 em diferentes partes do mundo, bem como o seu perfil antigênico, pode ser de fundamental importância para a aplicação dessas. Uma das proteínas mais imunogênicas do HIV-1 é Nef, que desempenha diversas funções importantes durante a infecção viral in vivo, como, por exemplo, aumentar a infectividade e a replicação viral e modular negativamente proteínas presentes na superfície celular. Por essas características, Nef pode ter papel importante na imunização contra o vírus. O presente trabalho teve como objetivo principal analisar amostras de uma série histórica de isolados do HIV-1 circulantes no Distrito Federal e no estado de Rondônia e, desse modo, contribuir para a caracterização desses vírus, pela descrição da variabilidade do gene nef. Além disso, foram avaliados motivos e resíduos importantes para as atividades biológicas de Nef. Para isso, 33 amostras, 17 do Distrito Federal e 16 de Rondônia, foram amplificadas por nested-PCR para o gene nef e seqüenciadas. Todas foram caracterizadas como pertencentes ao subtipo B. Pela dedução da seqüência de aminoácido foi possível observar que duas amostras apresentaram códons de parada prematuros, devido a mutações que levaram a mundança na fase de leitura do gene. Os motivos e resíduos associados às atividades biológicas de Nef também foram analisados. Os sítios de miristoilação, fosforilação por PKA e reconhecimento de Nef pela protease viral encontraram-se conservados em 100%, 66,6% e 69,7% das amostras, respectivamente. Em relação à modulação de CD4 por Nef, 36,4% das amostras apresentaram todos os motivos e resíduos conservados. Os motivos e resíduos associados à modulação negativa de MHC de classe I estiveram conservados em 57,6% das amostras analisadas, enquanto 54,5% dos isolados apresentaram os motivos envolvidos na modulação de MHC de classe II. Quanto à modulação de CD1d, 45,4% das amostras apresentaram a totalidade de motivos e resíduos associados a essa atividade conservados, enquanto aqueles associados à modulação de CD28 estiveram conservados em 81,8%. Enquanto 93,9% das amostras apresentaram o motivo e o resíduo associados à sinalização para apoptose celular conservados. Em relação às alterações em vias de sinalização celular, 63,6% das amostras apresentaram os motivos e resíduos responsáveis pela interação de Nef com domínios SH3 conservados, enquanto 84,8% mostraram-se conservadas para aqueles necessários para a interação de Nef com proteínas Pak. Quanto aos epitopos de Nef reconhecidos por linfócitos T CD4, 18 dos 29 já descritos na literatura foram observados, sendo que o mais freqüente foi encontrado em 75,8% das amostras. Em relação aos epitopos de Nef reconhecidos por linfócitos T citotóxicos considerados ótimos, o mais freqüente foi observado em 84,8% das amostras. Pelo fato de Nef ser uma das regiões imunodominantes do HIV-1, bem como ser uma proteína da fase precoce da infecção, vacinas que utilizem suas porções antigênicas encontram-se em desenvolvimento. Dessa forma, a caracterização da variabilidade do gene nef pode ser de grande importância para o desenvolvimento de estratégias de utilização de potenciais vacinas anti-HIV-1 futuramente no Distrito Federal e em Rondônia. __________________________________________________________________________________________ ABSTRACT
Nearly forty million people around the world were living with HIV/AIDS in 2005, about six hundred thousand in Brazil. Thus, the development of vaccines against this virus is a world public health priority. However, despite several studies concerning this virus, little is known about the relevance of its genetic variability to the efficacy of potential vaccines. Consequently, knowing the HIV-1 variability in different parts of the world, and also its antigenic profile, may be of high importance for the applicability of those vaccines. Nef is one of the most immunogenic proteins of HIV-1, it performs many important functions during the viral infection in vivo, such as increase the viral infectivity and replication, and downmodulate proteins of the cell surface. For these characteristics, Nef may have an important role in the immunization against the virus. The present study had as main objective the analysis of samples from a historical series of isolates of HIV-1 circulating in Federal District and Rondônia, and the contribution to the characterization of this virus, by the description of the variability of the nef gene. Additionally, the conservation of important motifs and residues related to the biological activities of Nef were also analyzed. With this purpose, thirty-three samples, seventeen from Federal District and sixteen from Rondônia, were amplified by nested-PCR for the nef gene and sequenced. All of them clustered with subtype B references isolates in the philogenetic analysis. The motifs and residues associated to Nef biological activities were also analyzed. The miristoilation, fosforilation by PKA and recognition of Nef by the viral protease sites were conserved in 100%, 66,6% and 69,7% of the samples, respectively. Considering the CD4 modulation by Nef, 36,4% of the samples presented all the motifs and residues conserved. 57,6% and 54,5% of the samples had all motifs and residues associated to the down-modulation of MHC class I and to the modulation of MHC class II conserved, respectively. Considering the downmodulation of CD1d, 45,4% of the samples had all the motifs and residues associated to this activity, while 81,8% had those associated to the modulation of CD28. 93,9% of the samples had the motif and the residue associated to the signalization of cell apoptosis conserved. Considering Nef effects on cell signalization pathways, 63,6% of the samples presented the motifs and residues responsible for the interaction of Nef with SH3 domains, while 84,8% had those necessary to the interaction of Nef with Pak proteins. Considering the Nef epitopes recognized by T CD4 lymphocytes eighteen of the twenty-nine previously described were observed, the most frequent one occurred in 75,8% of the samples. Considering the Nef epitopes recognized by T cytotoxic lymphocytes, the most frequent one was observed in 84,8% of the samples. Because Nef is one of the most immunodominant regions of HIV-1 virus, as well one early phase protein, vaccines that use its antigenic regions are in development. In this way, the characterization of the variability of the nef gene may be of high importance to the development of utilization strategies for the potential anti-HIV-1 vaccines in Federal District and Rondônia in the future.
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8

Chaudhuri, Rittik. "The mechanism of HIV-1 Nef-mediated downregulation of CD4." Thesis, University of Cambridge, 2010. https://www.repository.cam.ac.uk/handle/1810/224775.

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Nef, an accessory protein of HIV-1, is a critical determinant of viral pathogenicity. The pathogenic effects of Nef are in large part dependent on its ability to decrease the amount of CD4 on the surface of infected cells. Early studies suggested that Nef induces downregulation by linking the cytosolic tail of CD4 to components of the host-cell protein-trafficking machinery. However, the specific sorting pathway that Nef uses to modulate CD4 expression remained uncertain. According to one model, Nef was thought to interfere with the transport of newly synthesized CD4 from the TGN to the cell-surface. Another model claimed that Nef facilitated the removal of CD4 from the plasma membrane. The primary goal of this thesis was to determine which of these models was correct. To accomplish this objective, a novel Nef-CD4 system was developed in Drosophila S2 cells. Nef was not only able to downregulate human CD4 in S2 cells, but it did so in a manner that was phenotypically indistinguishable from its activity in human cells. An RNAi screen targeting protein-trafficking genes in S2 cells revealed a requirement for clathrin and the clathrin-associated, plasma membrane-localized AP-2 complex in the Nef-mediated downregulation of CD4. In contrast, depletion of the related AP-1 and AP-3 complexes, which direct transport from the TGN and endosomes, had no effect. The requirement for AP-2 was subsequently confirmed in a human cell line. Yeast three-hybrid and GST pull-down assays were then used to demonstrate a robust, direct interaction between Nef and AP-2. This interaction was found to depend on a [D/E]xxxL[L/I]-type dileucine motif, located in the C-terminal loop of Nef, that is essential for CD4 downregulation. While mapping the binding site of AP-2 on Nef, a second determinant of interaction in the C-terminal loop was identified. Mutation of this motif, which conforms to a consensus [D/E]D diacidic sequence, prevented Nef from binding to AP-2 and down-regulating CD4. However, the same mutations did not affect the ability of Nef to interact with either AP-1 or AP-3, providing further evidence that these complexes are not required for the modulation of CD4 expression. Additional experiments indicated that the Nef diacidic motif most likely binds to a basic patch on AP-2 α-adaptin that is not present in the homologous AP-1 γ and AP-3 δ subunits. As with the Nef diluecine and diacidic motifs, the α-adaptin basic patch was shown to be necessary for CD4 downregulation. Moreover, all three of these motifs were needed for the cooperative assembly of a CD4-Nef-AP-2 tripartite complex, which was observed here for the first time using a yeast four-hybrid system. The data in this thesis uniformly support an endocytic model of Nef-mediated CD4 downregulation. Indeed, there is now strong evidence that Nef simultaneously binds CD4 and AP-2, thereby connecting the receptor to the cellular endocytic machinery and promoting its rapid internalization from the plasma membrane. In addition, the identification of novel motifs required for this process has provided new insights on endocytosis, and may facilitate the development of pharmacological inhibitors of Nef function.
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9

Chang, Alex Hongsheng. "Intracellular inhibition of immune dysfunction induced by HIV-1 Nef protein." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0017/NQ56524.pdf.

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10

Cosma, Antonio. "Use of the regulatory protein Nef for vaccination against HIV-1." Diss., kostenfrei, 2008. http://edoc.ub.uni-muenchen.de/8329/.

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11

Mangasarian, Aram A. "The HIV-1 Nef protein is a receptor specific sorting adaptor /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1997. http://wwwlib.umi.com/cr/ucsd/fullcit?p9814548.

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12

Lubben, Nienke Brenya. "Mechanism of HIV-1 Nef-induced down regulation of MHC I." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611876.

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13

Gondim, Marcos Vinícius Pereira. "Mecanismos moleculares das proteínas acessórias NEF e VPU relacionados à patogênese do HIV-1." reponame:Repositório Institucional da UnB, 2015. http://dx.doi.org/10.26512/2015.03.T.18243.

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Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Patologia Molecular, 2015.
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A modulação de CD4 é uma das principais características da infecção por HIV e Nef desempenha um papel importante neste processo. Foi observada uma clara relação entre a degradação do receptor viral CD4, com o aumento da infecciosidade e a replicação viral, sugerindo a participação deste fenômeno na patogênese e progressão da infecção. No entanto, os mecanismos envolvidos na internalização e degradação de CD4 mediada por Nef não são totalmente compreendidas e diferentes proteínas celulares têm sido apontadas como importantes parceiros celulares de Nef. Nef agiria na superfície celular como um conector da cauda citoplasmática da molécula CD4, com membros do complexo adaptador heterotetramérico de clatrina, redirecionando a molécula CD4 para vesículas endocíticas. Uma bomba de prótons vacuolar (V-ATPase) e a proteína Epsn15 foram envolvidas no aumento da força de ligação Nef e a subunidade μ2 de AP-2. Com a finalidade de evitar a reciclagem de CD4 para a superfície celular, uma segunda conexão entre CD4 e a proteína β-COP é estabelecida por Nef, o que permitiria direcionar o receptor para degradação lisossômica. Adicionalmente, duas proteínas foram também envolvidas no mecanismo a tioesterase humana II (hTE-II/AcoT8) e a dinamina 2 (Dyn2). Entretanto experimentos para validar a relevância fisiológica dessas proteínas não foram conclusivos. Neste trabalho, pretendemos determinar a relevância fisiológica das proteínas: AP1, AP2, AP3, VH1 ATPase vacuolar, β-COP e AcoT8, na modulação de CD4 mediada por Nef, em linhas de células T e linfócitos T CD4+ de sangue periférico, infectados pelo HIV-1 . Primeiramente, análises de FRET-FACS entre alelos de Nef e as proteínas celulares apresentaram diferentes níveis de interação. O receptor CD4 mostrou variáveis níveis de interação com os alelos de Nef, NA7, NL4.3 e Mac239 (39%, 15% e 13%, respectivamente) e uma forte interação com as proteínas Ap2, β-COP (50,4%, 32% respectivamente). Por outro lado os alelos de Nef mostraram interagir principalmente com Ap2, β-COP e AcoT8, este ultimo apenas com o alelo NA7. Experimentos de microscopia confocal confirmaram os resultados obtidos por FACS-FRET mostrando em alguns casos alterações claras na localização subcelular destas proteínas. Finalmente, o silenciamento da expressão endógena das proteínas celulares na linha de célula T SupT-1 e em linfócitos primários CD4+ mostrou a proteína AP2 como sendo necessária e suficiente para a modulação de CD4 mediada por Nef.
Down-modulation of CD4 is one of the hallmarks of HIV infection and Nef plays a major role in this process. Has been observed a clear relation between the CD4 viral receptor degradation with the infectivity and viral replication, which suggest the high relevance of this mechanism during the pathology and infection progress. However, the exact mechanisms governing Nef mediated internalization and degradation of CD4, are incompletely understood and different cellular proteins have been suggested as key players. Nef would act on the cell surface as a connector in cytoplasmic tail of CD4 molecule with members of heterotetrameric clathrin adapter complex, redirecting the CD4 molecule for endocytic vesicles. Vacuolar proton pump (V-ATPase) and Epsn15 protein would be involved enhancing the strength binding of Nef on subunit μ2 of AP-2. In order to avoid recycling of CD4 to the cell surface, a second connection between CD4 and β-COP is established by Nef protein, which would direct the receptor to lysosomal degradation. Additionally, two proteins were also involved in the mechanism human thioesterase II (hTEII/AoT8) and dynamin 2 (DYN2). However experiments to validate the physiological relevance of these proteins were not conclusive. In this work, we aim to address the physiological relevance of AP1, AP2, AP3, VH1 vacuolar ATPase, β-COP and AcoT8 for Nef mediated down-modulation of CD4 receptor in T-cell lines, and infected primary isolated CD4 positive cells. First, FRET-FACS analyses with different Nef HIV-1 strains showed different levels of interaction. The receptor CD4 shown variable level of interaction with Nef alleles; NA7, NL4.3 and Mac239 (39%, 15% e 13%, respectively) and a strong interaction with Ap2, β-COP (50,4%, 32% respectively). On other hand, the alleles shown interacts mainly with Ap2, β-COP, and AcoT8 (only with NA7 alleles). Confocal microscopy experiments confirmed FACS-FRET results showing clear changes in subcellular localization of these proteins. Finally, endogenous cellular proteins knock-down experiments by RNA interference in transduced SupT1 and primary lymphocyte CD4+ cells showed AP2 protein as being necessary and sufficient for Nef CD4 down-modulation.
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Coutinho, Junior Raimundo. "Reatividade de linfócitos T de indivíduos infectados pelo HIV-1 contra epitopos GAG e NEF de diferentes subtipos HIV-1." reponame:Repositório Institucional da FIOCRUZ, 2008. https://www.arca.fiocruz.br/handle/icict/4266.

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Made available in DSpace on 2012-08-03T18:29:13Z (GMT). No. of bitstreams: 1 Raimundo Coutinho Junior. Reatividade de Linfócitos T de indivíduos infectados pelo HIV-1 contra epitopos GAG e NEF de diferentes subtipos HIV-1. Dissertação Mestrado - CPqGM - 2008.pdf: 883674 bytes, checksum: e99dbac32085a623e129289408e7f7d6 (MD5) Previous issue date: 2008
Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil
O Vírus da imunodeficiência humana é o agente causador da AIDS. Apesar de existir tratamento para a infecção do HIV-1, este não leva a cura dos pacientes. A identificação de epitopos de proteínas virais do HIV-1 reconhecidos por linfócitos T pode ser eficiente para o desenvolvimento de uma vacina. Neste trabalho foi avaliado o padrão da resposta imune de indivíduos infectados pelo HIV -1 frente às proteínas GAG e NEF de diferentes subtipos do HIV-1. Foi utilizado PBMC de pacientes infectados pelo HIV-1 sob o uso de terapia antiretroviral. Foram utilizados 23 pools de peptídeos do HIV-1 na concentração de 2μg/mL derivados dos subtipos brasileiros BBR, C, F e do isolado de referencia B (HXB2), em ensaio de ELISPOT. Todos os pacientes responderam a pelo menos um dos pools de peptídeos. Destes, 41% responderam para os pools de proteínas do isolado HXB2 e 49% para os pools de proteínas do subtipo brasileiro BBR, C e F. Isoladamente, as maiores freqüências de respondedores foram para os pools de proteínas do isolado HXB2 NEF 3 (68%) e HXB2 NEF 2 (63%). Da mesma forma, os pools de peptídeos das seqüências brasileiras BBR, C e F que apresentaram as maiores freqüências de respondedores foram NEF F (87%), NEF C (62%), GAG F1 (62%). A magnitude da resposta foi em média de 2174 SFCX106 PBMC para os pools do isolado HXB2 e de 1331 SFCX106 PBMC para os pools dos subtipos brasileiros. O pool que apresentou a maior magnitude da resposta foi GAG p24-1 do isolado HXB2 com 3290 SFCX106 PBMC. Estes resultados demonstram que os pacientes infectados pelo HIV-1 respondem aos pools de peptídeos das proteínas GAG e NEF do isolado HXB2 e dos subtipos brasileiros BBR, C e F.
The human immunodeficiency virus is the etiological agent of AIDS. The antiretroviral treatment is efficient, however the cure is not reach. The identification of T cell epitopes from HIV-1 proteins could be an efficient approach for development of an HIV-1 vaccine. In this work, we evaluated the immune responses of HIV-1 infected individuals to GAG and NEF HIV-1 proteins from different virus subtypes. Pools of peptides of proteins from Brazilians HIV-1 subtypes BBR, C, F sequences and from HIV-1 subtype B isolate (HXB2) were tested by ELISPOT assay. All tested pools were recognized. PBMC of 41% of patients recognized subtype HXB2 pools and 49% recognized the pools from Brazilians subtypes. The most frequently recognized pools were NEF3 (68%) and NEF2 (63%) from HXB2 isolated. The most frequently recognized pool from Brazilians subtypes were NEF F (87%), NEF C (62%), GAG F1 (62%). On average, the magnitude of responses to HXB2 isolate pools were 2174 SFCX106 PBMC and 1331 SFCX106 PBMC for Brazilians pools. The highest magnitude of response was directed to GAG p24-1 from HXB2 subtype with 3290 SFCX106 PBMC. These results indicated that peptides from NEF and GAG proteins from HXB2 isolated or Brazilians subtypes BBR, C and F sequences are recognized by HIV-1 infected patients and may have the potential for inclusion in a vaccine against HIV-1.
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15

Bräu, Tanja [Verfasser], and Thomas [Akademischer Betreuer] Winkler. "Die Funktion HIV-1 Nef-induzierter Exosomen / Tanja Bräu. Betreuer: Thomas Winkler." Erlangen : Universitätsbibliothek der Universität Erlangen-Nürnberg, 2013. http://d-nb.info/1038871328/34.

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16

Ye, Michael. "HIV-1 and SIV Nef and Env modulate p21-activated kinase function /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2004. http://uclibs.org/PID/11984.

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17

Salvador, Carlos Eduardo de Melo. "Design e síntese de possíveis inibidores da proteína auxiliar nef do vírus HIV-1." reponame:Repositório Institucional da UnB, 2011. http://repositorio.unb.br/handle/10482/9476.

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Dissertação (mestrado)—Universidade de Brasília, Instituto de Química, 2011.
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Aproximadamente após 25 anos da implantação da terapia anti-retroviral, a pesquisa em HIV/SIDA encontra-se em uma encruzilhada marcada pelo surgimento de novos paradigmas, entre os quais se destacam o aparecimento de variantes resistentes a inibidores presentes no coquetel anti-retroviral; a permanência de reservatórios virais latentes; a presença de efeitos tóxicos colaterais causados pelo tratamento e o alto custo das drogas disponíveis no mercado. Diante deste quadro, a pesquisa de mecanismos básicos de patogêneses, assim como a identificação de novos alvos terapêuticos, volta a desempenhar um papel crucial no desenvolvimento de novas drogas anti-retro virais. A diminuição da expressão do receptor CD4 na superfície das células infectadas é um dos mais importantes eventos durante a infecção pelo HIV-1. A identificação de inibidores desta função de Nef é de grande valia no tratamento da infecção pelo HIV. Para atingir este objetivo, foram sintetizados, com base em cálculos teóricos de modelagem molecular, três compostos (I, 15 e 18) que se demonstraram relevantes para futuros testes farmacológicos como possíveis inibidores da degradação de CD4 pela Nef. _______________________________________________________________________________ ABSTRACT
Approximately after 25 years of the implantation of the anti-retroviral therapy, the research in HIV, aids is at a crossroad marked for the sprouting of new paradigms: the emergence of variants resistant to protease inhibitors present in the anti-retroviral cocktail; the persistence of latent viral reservoirs, and the presence of collateral toxic effect caused by the treatment and the high cost of the available drugs in the market. Because of this situation, the research of basic mechanisms of pathogenesis and the identification of new therapeutic targets should play a crucial role in the development of new anti-retro viral drugs. Decrease in the expression of receiver CD4 on the surface of the infected cells is one of the most important events during the infection by HIV-1. The identification of inhibitors through function of Nef is very important to the treatment of HIV infection. To achieve this goal, three compounds (I, 15 and 18) were synthesized based on theoretical calculations of molecular modeling, which proved to be relevant for future pharmacological tests as potential inhibitors of the degradation of CD4 by Nef.
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18

Collette, Yves. "Rôle du gène nef du virus de l'immunodéficience humaine (HIV-1): étude et bases structurales du déficit lymphocytaire Th-1 induit par nef." Doctoral thesis, Universite Libre de Bruxelles, 1994. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/212629.

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19

Choma, Maja. "In search of cell machinery contributing to the activity of HIV-1 Nef." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648443.

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20

Thomson, Cassandra. "The HIV-1 protein Nef disrupts T cell receptor signalling and thymic function." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=119458.

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The HIV-1 Nef protein is a virulence factor and major determinant of pathogenicity of the virus. Nef impairs T cell function by uncoupling TcR signaling from antigen stimulation and by down regulating CD4 on the cell surface. In Nef infected thymocytes, the activity of intracellular T signaling molecule Lck is increased in basal conditions and attenuated with stimulation. This phenomenon contributes to partial T cell activation allowing viral replication meanwhile preventing full activation of the cell in order to prevent cell death. In addition, Nef infection results in intracellular accumulation of Lck and delocalization from its regular compartments. In the first part of this study we use biochemical techniques and confocal microscopy to study other proteins that are involved in the tightly regulated Lck localization and activity in a normal thymocytes to attempt to understand the pathway in the context of Nef infection. We find that the delocalization of Lck disrupts the regulatory feedback loop of the protein and subsequently interferes with the activation of downstream mediators. TcR signaling and Lck are also important for thymic selection and progression from CD4+CD8+ double positive to CD4+ and CD8+single positive thymocytes. In the second part of this study, we show that the loss of CD4+ thymocytes results in the loss of an incredibly important mediator of central tolerance, the transcription factor AIRE. We correlate this loss of AIRE to an impairment of maturation of thymic epithelial cells and a profound loss of expression of tissue restricted antigens necessary for inducing thymic tolerance. Such loss in the thymus is directly correlated to autoimmunity in mouse models and human studies. This study proposes a novel mechanism for autoimmunity frequently observed in patients with HIV/AIDS.
La protéine Nef du VIH-1 joue un rôle important dans la pathogenèse du virus en modulant les voies de signalisation du récepteur de la lymphocyte T et en baissant l'expression du corécepteur CD4 à la membrane. Ainsi, dans une cellule infectée par Nef, l'activation de la molécule Lck est augmentée avant la stimulation de la cellule et diminuée après stimulation par rapport une cellule normale. Cette activation partiale faitque le virus peut répliquer en évitant l'apoptose. De plus, Nef mène à une accumulation intracellulaire de la protéine Lck ce qui la délocalise de ses compartiments habituels. Dans la première partie de cette étude, nous employons des essais biochimiques ainsi que la microscopie confocale afin d'étudier les protéines que sont impliquées dans la régulation précise de Lck pour que nous puissions comprendre sa signalisation sous le contrôle de Nef. Nous avons trouvé que la délocalisation de Lck perturbe la rétroaction régulatrice de la protéine ce qui interfére avec l'activation des messagers secondaires. La signalisation du lymphocyte T et de la protéine Lck sont importantes pour la séléction des thymocytes et la progression des cellules doubles positives CD4+ CD8+ aux cellules simples positives CD4+ ou CD8+. Dans la deuxième partie de ce mémoire, nous démontrons que la perte des thymocytes CD4+ mène à une perte d'une protéine essentielle à la tolérance centrale immunitaire, AIRE. Nous avons lié cette perte d'AIRE à une déficience des cellules épithéliales médullaires matures. Une telle perte est directement corrélée à l'autoimmunité dans les modèles de souris et les études chez l'homme. Notre étude suggère un nouveau mécanisme pour l'autoimmunité que l'on voit souvent chez les patients qui sont atteints du VIH-1.
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21

Rafie, Salomeh. "La protéine Nef du VIH-1 : Contribution des complexes adaptateurs de la voie d'endocytose aux fonctions de Nef." Thesis, Paris 5, 2012. http://www.theses.fr/2012PA05T085/document.

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La protéine Nef des virus de l’immunodéficience humaine (VIH-1 et VIH-2) joue un rôle essentiel dans la physiopathologie de l’infection et induction du SIDA. La capacité de Nef à perturber le trafic intracellulaire de protéines membranaires, et notamment du récepteur CD4, circulant entre les compartiments de la voie d’endocytose pourrait rendre compte de son importance comme facteur de virulence au cours de l’infection naturelle. Les mécanismes responsables des perturbations de la voie d’endocytose induites par Nef au cours de l’infection ne sont pas totalement élucidés, mais il est admis qu’elles résultent d’interactions avec les complexes adaptateurs (AP) associés à la clathrine et participant au transport vésiculaire entre les différents compartiments de la voie d’endocytose. Notre objectif était de déterminer les mécanismes par lesquels Nef influe positivement sur le pouvoir infectieux du VIH-1 en interagissant avec la machinerie cellulaire de la voie d’endocytose. Notre programme s’est organisé autour de deux axes principaux: le premier a consisté à étudier l’implication respective des différents types de complexes AP (AP-1, -2 et -3) sur les perturbations du fonctionnement de la voie d’endocytose induites par Nef en analysant son impact sur le niveau d’expression de surface de CD4; le deuxième axe a consisté à évaluer l’impact de l’interaction de Nef avec les complexes AP sur les capacités infectieuses des particules virales. Le rôle respectif des différents complexes AP dans ces fonctions de Nef a donc été étudié après déplétion de l’expression des complexes AP-1, AP-2 et AP-3 par une approche d’ARN interférence. Les résultats obtenus montrent que contrairement à certaines données de la littérature, la déplétion des complexes AP de la voie d’endocytose ne semble pas avoir un impact majeur sur la capacité de Nef à moduler l’expression de surface de CD4, même si une légère diminution de l’activité de Nef a pu être révélée dans notre étude réalisée sur des cellules HeLa-CD4 transduites par les shRNA ciblant les complexes AP-2. Inversement, nos résultats confirment que la déplétion des complexes AP-1, AP-2 et AP-3 dans les cellules productrices des particules virales se traduit par une diminution importante des propriétés infectieuses de ces particules sur lesquelles l’impact positif de Nef n’est plus alors capable de se manifester. En conclusion, ce travail a donc permis de montrer que les complexes AP de la voie d’endocytose sont indispensables pour que Nef puisse exercer son rôle positif sur le pouvoir infectieux du VIH-1. Il est maintenant important de confirmer ces résultats en analysant le rôle fonctionnel des complexes AP sur les activités de Nef dans les cibles cellulaires naturelles du VIH-1, lymphocytes et macrophages
Nef protein of human immunodeficiency virus (HIV-1 and HIV-2) plays an essential role in the pathophysiology of infection and induction of AIDS. The ability of Nef to disrupt intracellular trafficking of membrane proteins, including the CD4 receptor, moving between the compartments of the endocytic pathway could account for its importance as a virulence factor during natural infection. The mechanisms responsible for disruption of the endocytic pathway induced by Nef during infection are not fully understood, but it is accepted that they arise from interactions with adaptor complexes (AP) associated with clathrin and participant in vesicular transport between the different compartments of the endocytic pathway. Our objective was to determine the mechanisms by which Nef positively affects the infectivity of HIV-1 by interacting with the cellular machinery of the endocytic pathway. Our program has been organized around two main axes: the first was to investigate the respective involvement of different types of complexes (AP-1, -2 and -3) on the Nef induced disruption of the endocytic pathway by analyzing its impact on the level of surface expression of CD4; the second axis was to evaluate the impact of the interaction of Nef with AP complexes on the infectious capacity of the viral particles. The respective roles of the different AP complexes in these functions of Nef has been studied after depletion of the expression of complex AP-1, AP-2 and AP-3 by RNA interference approach. The results show that, contrary to some literature data, depletion of AP complex endocytic pathway does not appear to have a major impact on the ability of Nef to modulate the surface expression of CD4, although a slight decreased activity of Nef could be revealed in our study on HeLa-CD4 cells transduced with the shRNA targeting complex AP-2. Conversely, our results confirm that the depletion of complex AP-1, AP-2 and AP-3 in the cells producing viral particles resulted in a significant decrease in infectious properties of these particles on which the positive impact of Nef is no longer able to manifest. In conclusion, this work has shown that complex AP of endocytic pathway are essential for Nef to exercise its positive role in the infectivity of HIV-1. It is now important to confirm these findings by analyzing the functional role of AP complexes on the activities of Nef in the natural cellular targets of HIV-1, lymphocytes and macrophages
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22

Fausther, Bovendo Hugues F. B. "Sensibilité des CD4 à la lyse NK : effet du peptide 3S de la gp41 et des anticorps spécifiques." Paris 6, 2009. http://www.theses.fr/2009PA066418.

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L’infection VIH est caractérisée par une détérioration du système immunitaire et notamment par une chute des lymphocytes T (LT) CD4. Les LT CD4 jouant un rôle important dans la réponse immunitaire, cette perte est cruciale dans la pathologie du VIH. Étrangement, la plupart des LT CD4 mourantes ne sont pas infectées. Cependant, bien que plusieurs hypothèses ont été émises, les causes exactes de cette déplétion sont toujours contestées. Le motif 3S de la gp41 du VIH-1 induit l’expression de NKp44L, un ligand d’un des récepteurs activateurs des cellules NK, à la surface des LT CD4. Les LT CD4 exprimant NKp44L sont plus sensibles à la lyse NK. Au cours de l’infection VIH-1 chez l’homme et chez un modèle macaque de cette infection, de nombreuses données suggèrent que la destruction des LT CD4 par les cellules NK est en partie responsable de la déplétion CD4 observée. Au cours de ma thèse, j’ai identifié le gC1qR comme étant le récepteur du motif 3S de la gp41 à la surface des LT CD4. Le peptide 3S ainsi que deux des ligands du gC1qR, le C1q et le HK, induisent la translocation de NKp44L. La liaison du peptide 3S au gC1qR active la PI3K, la NADPH oxidase, p190A RhoGAP et TC10 induisant l’expression de NKp44L à la surface des LT CD4. J’ai également découvert un mécanisme d’échappement viral ou les LT CD4 infectés n’expriment pas NKp44L à la surface. J’ai ensuite montré que Nef séquestre NKp44L à l’intérieur des LT CD4 infectés, ce qui les protège contre la lyse NK. Ces résultats identifient de nouvelles cibles pour d’éventuelles stratégies thérapeutiques visant à empêcher la chute des LT CD4 ou à détruire le réservoir du VIH-1.
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23

Ono, Takeshi. "Functional association between the nef gene product and gag-pol region of HIV-1." Kyoto University, 2000. http://hdl.handle.net/2433/180853.

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24

Florido, Camila [UNIFESP]. "Análise da diversidade genética no gene nef do vírus da imunodeficiência humana do tipo 1(HIV-1) em crianças e em adultos recém infectados." Universidade Federal de São Paulo (UNIFESP), 2007. http://repositorio.unifesp.br/handle/11600/23570.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Diversos estudos clínicos têm demonstrado que a dinâmica da AIDS na infecção pediátrica difere da observada em adultos. Em especial, diferenças na carga viral, taxa de depleção de linfócitos e rapidez no aparecimento dos sintomas são mais acentuados em crianças infectadas pelo HIV-1 (Luzuriaga et al., 1999). Contudo, os processos evolutivos que ocorrem nos genes do HIV-1 durante a infecção de crianças ainda são pouco caracterizados. Em razão do sistema imunológico das crianças não estar completamente desenvolvido sugere que a pressão seletiva nos genes do HIV é distinta da observada em adultos. Para explorar a diversificação viral nesse contexto, comparamos a pressão seletiva do gene nef do HIV-1 em crianças (transmissão materno-infantil). com idades diferentes (i.e. 0-3 e 4-6 anos ) e comparamos com a observada em adultos. Para esse propósito comparamos a razão entre o número de substituições não-sinônimas por sítio não-sinônimo (dN) e o número de substituições sinônimas por sítio sinônimo (dS) medidos códon a códon no gene nef do HIV-1 entre adultos e crianças. Os resultados mostraram que, independente do tempo de diversificação do HIV-1, não existem diferenças na entendida de seleção e nem na localização dos códons positivamente selecionadas entre crianças e adultos no nível populacional. Em adição, detectamos uma região no gene nef que concentra mais os códons sob seleção positiva. Essa região é densa em epitopos descritos para anticorpos e ausente de epitopos para CTL ou CD4-hekper (de acordo com os mapas de epitopos com os mapas de Los Alamos HIV databank). Esses resultados são importantes pois mostram que a pressão mediada por anticorpos é bastante significativa para a diversificação do HIV-1 no nível populacional.
FAPESP: 04/12044-3
BV UNIFESP: Teses e dissertações
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25

Mukerji, Joya. "A Novel Exocyst-Based Mechanism for HIV Nef-Mediated Enhancement of Intercellular Nanotube Formation." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10441.

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HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunnelling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. In this dissertation, we developed and characterized a lentiviral vector-based system to express Nef in T-cell lines and primary human peripheral blood mononuclear cells, and then used this system to perform a proteomic screen to identify Nef-associated host cell factors and better understand how Nef hijacks the T-cell machinery to maximize HIV production and dissemination. Bioinformatic and cell-based analysis of the resulting host factors revealed a mechanism by which Nef enhances nanotube formation. To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3). Wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6), an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Together, our findings identify the exocyst complex as a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Furthermore, linkages revealed between Nef and the exocyst complex suggest a new paradigm of exocyst involvement in polarized targeting for intercellular transfer of viral proteins and viruses.
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Bandawe, Gama. "Expression of HIV-1 subtype C nef in E. coli and Nicotiana benthamiana : development of plant-based vaccines for HIV." Master's thesis, University of Cape Town, 2003. http://hdl.handle.net/11427/4242.

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Bibliography: leaves 126-145.
Expression of the nefgene from HIV-1 subtype C in Nicotiana benthamiana was carried out using a TMV -based vector with the aim of developing a plant-based candidate vaccine for HIV-1. The nef gene of the DU151 isolate of mV-l subtype C taken from a recently seroconverted individual was amplified by PCR with a deletion of 59 amino acids from the cytotoxic N-terminal. The amplified gene was inserted into a bacterial expression vector pProEXHTb for rapid expression of Nef protein, which was used as a diagnostic tool in the development of an indirect ELISA assay for detection of Nef in Nicotiana benthamiana. An indirect ELISA assay was developed using a commercially available polyclonal anti Nef antiserum raised in sheep. The role of codon optimization in expression of Nef in benthamiana was investigated. A synthetic nef gene was constructed based on the codon usage of benthamiana. The plant codon optimized gene and the wild type nef genes were inserted into the TMV -based vector pBSG1057. RNA transcripts from both constructs were used to infect young benthamiana plants. Expression of nefmRNA was confirmed by RT -PCR analysis of total RNA extracted from plants inoculated with respective constructs. The Nef protein was expressed at low levels which were detectable by ELISA. Nef was detectable by Western blot after concentration of plant extract using a membrane filter device. Quantitative analysis of Nef expression in plants was done by western blot on concentrated plant extract from three separate infections. Codon optimization of the nef gene improved the expression of Nef by a factor of about two.
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Tavares, Lucas Alves. "O envolvimento da proteína adaptadora 1 (AP-1) no mecanismo de regulação negativa do receptor CD4 por Nef de HIV-1." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/17/17136/tde-06012017-113215/.

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O Vírus da Imunodeficiência Humana (HIV) é o agente etiológico da Síndrome da Imunodeficiência Adquirida (AIDS). A AIDS é uma doença de distribuição mundial, e estima-se que existam atualmente pelo menos 36,9 milhões de pessoas infectadas com o vírus. Durante o seu ciclo replicativo, o HIV promove diversas alterações na fisiologia da célula hospedeira a fim de promover sua sobrevivência e potencializar a replicação. A rápida progressão da infecção pelo HIV-1 em humanos e em modelos animais está intimamente ligada à função da proteína acessória Nef. Dentre as diversas ações de Nef está a regulação negativa de proteínas importantes na resposta imunológica, como o receptor CD4. Sabe-se que esta ação resulta da indução da degradação de CD4 em lisossomos, mas os mecanismos moleculares envolvidos ainda são totalmente elucidados. Nef forma um complexo tripartite com a cauda citosólica de CD4 e a proteína adaptadora 2 (AP-2), em vesículas revestidas por clatrina nascentes, induzindo a internalização e degradação lisossomal de CD4. Pesquisas anteriores demonstraram que o direcionamento de CD4 aos lisossomos por Nef envolve a entrada do receptor na via dos corpos multivesiculares (MVBs), por um mecanismo atípico, pois, embora não necessite da ubiquitinação de carga, depende da ação de proteínas que compõem os ESCRTs (Endosomal Sorting Complexes Required for Transport) e da ação de Alix, uma proteína acessória da maquinaria ESCRT. Já foi reportado que Nef interage com subunidades dos complexos AP-1, AP-2, AP-3 e Nef não parece interagir com subunidades de AP-4 e AP-5. Entretanto, o papel da interação de Nef com AP-1 e AP-3 na regulação negativa de CD4 ainda não está totalmente elucidado. Ademais, AP-1, AP-2 e AP-3 são potencialmente heterogêneos devido à existência de isoformas múltiplas das subunidades codificadas por diferentes genes. Todavia, existem poucos estudos para demonstrar se as diferentes combinações de isoformas dos APs são formadas e se possuem propriedades funcionais distintas. O presente trabalho procurou identificar e caracterizar fatores celulares envolvidos na regulação do tráfego intracelular de proteínas no processo de regulação negativa de CD4 induzido por Nef. Mais especificamente, este estudo buscou caracterizar a participação do complexo AP-1 na modulação negativa de CD4 por Nef de HIV-1, através do estudo funcional das duas isoformas de ?-adaptina, subunidades de AP-1. Utilizando a técnica de Pull-down demonstramos que Nef é capaz de interagir com ?2. Além disso, nossos dados de Imunoblot indicaram que a proteína ?2-adaptina, e não ?1-adaptina, é necessária no processo de degradação lisossomal de CD4 por Nef e que esta participação é conservada para degradação de CD4 por Nef de diferentes cepas virais. Ademais, por citometria de fluxo, o silenciamento de ?2, e não de ?1, compromete a diminuição dos níveis de CD4 por Nef da membrana plasmática. A análise por imunofluorêsncia indireta também revelou que a diminuição dos níveis de ?2 impede a redistribuição de CD4 por Nef para regiões perinucleares, acarretando no acúmulo de CD4, retirados por Nef da membrana plasmática, em endossomos primários. A depleção de ?1A, outra subunidade de AP-1, acarretou na diminuição dos níveis celulares de ?2 e ?1, bem como, no comprometimento da eficiente degradação de CD4 por Nef. Além disso, foi possível observar que, ao perturbar a maquinaria ESCRT via super-expressão de HRS (uma subunidade do complexo ESCRT-0), ocorreu um acumulo de ?2 em endossomos dilatados contendo HRS-GFP, nos quais também detectou-se CD4 que foi internalizado por Nef. Em conjunto, os resultados indicam que ?2-adaptina é uma importante molécula para o direcionamento de CD4 por Nef para a via ESCRT/MVB, mostrando ser uma proteína relevante no sistema endo-lisossomal. Ademais, os resultados indicaram que as isoformas ?-adaptinas não só possuem funções distintas, mas também parecem compor complexos AP-1 com diferentes funções celulares, já que apenas a variante AP-1 contendo ?2, mas não ?1, participa da regulação negativa de CD4 por Nef. Estes estudos contribuem para o melhor entendimento dos mecanismos moleculares envolvidos na atividade de Nef, que poderão também ajudar na melhor compreensão da patogênese do HIV e da síndrome relacionada. Em adição, este trabalho contribui para o entendimento de processos fundamentais da regulação do tráfego de proteínas transmembrana no sistema endo-lisossomal.
The Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). AIDS is a disease which has a global distribution, and it is estimated that there are currently at least 36.9 million people infected with the virus. During the replication cycle, HIV promotes several changes in the physiology of the host cell to promote their survival and enhance replication. The fast progression of HIV-1 in humans and animal models is closely linked to the function of an accessory protein Nef. Among several actions of Nef, one is the most important is the down-regulation of proteins from the immune response, such as the CD4 receptor. It is known that this action causes CD4 degradation in lysosome, but the molecular mechanisms are still incompletely understood. Nef forms a tripartite complex with the cytosolic tail of the CD4 and adapter protein 2 (AP-2) in clathrin-coated vesicles, inducing CD4 internalization and lysosome degradation. Previous research has demonstrated that CD4 target to lysosomes by Nef involves targeting of this receptor to multivesicular bodies (MVBs) pathway by an atypical mechanism because, although not need charging ubiquitination, depends on the proteins from ESCRTs (Endosomal Sorting Complexes Required for Transport) machinery and the action of Alix, an accessory protein ESCRT machinery. It has been reported that Nef interacts with subunits of AP- 1, AP-2, AP-3 complexes and Nef does not appear to interact with AP-4 and AP-5 subunits. However, the role of Nef interaction with AP-1 or AP-3 in CD4 down-regulation is poorly understood. Furthermore, AP-1, AP-2 and AP-3 are potentially heterogeneous due to the existence of multiple subunits isoforms encoded by different genes. However, there are few studies to demonstrate if the different combinations of APs isoforms are form and if they have distinct functional properties. This study aim to identify and characterize cellular factors involved on CD4 down-modulation induced by Nef from HIV-1. More specifically, this study aimed to characterize the involvement of AP-1 complex in the down-regulation of CD4 by Nef HIV-1 through the functional study of the two isoforms of ?-adaptins, AP-1 subunits. By pull-down technique, we showed that Nef is able to interact with ?2. In addition, our data from immunoblots indicated that ?2- adaptin, not ?1-adaptin, is required in Nef-mediated targeting of CD4 to lysosomes and the ?2 participation in this process is conserved by Nef from different viral strains. Furthermore, by flow cytometry assay, ?2 depletion, but not ?1 depletion, compromises the reduction of surface CD4 levels induced by Nef. Immunofluorescence microscopy analysis also revealed that ?2 depletion impairs the redistribution of CD4 by Nef to juxtanuclear region, resulting in CD4 accumulation in primary endosomes. Knockdown of ?1A, another subunit of AP-1, resulted in decreased cellular levels of ?1 and ?2 and, compromising the efficient CD4 degradation by Nef. Moreover, upon artificially stabilizing ESCRT-I in early endosomes, via overexpression of HRS, internalized CD4 accumulates in enlarged HRS-GFP positive endosomes, where co-localize with ?2. Together, the results indicate that ?2-adaptin is a molecule that is essential for CD4 targeting by Nef to ESCRT/MVB pathway, being an important protein in the endo-lysosomal system. Furthermore, the results indicate that ?-adaptins isoforms not only have different functions, but also seem to compose AP-1 complex with distinct cell functions, and only the AP-1 variant comprising ?2, but not ?1, acts in the CD4 down-regulation induced by Nef. These studies contribute to a better understanding on the molecular mechanisms involved in Nef activities, which may also help to improve the understanding of the HIV pathogenesis and the related syndrome. In addition, this work contributes with the understanding of primordial process regulation on intracellular trafficking of transmembrane proteins.
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28

Corrêa, Lucília Zeymer Alves. "Síntese de compostos 1,2,3-triazólicos : potenciais inibidores da proteína auxiliar Nef do vírus HIV-1." reponame:Repositório Institucional da UnB, 2011. http://repositorio.unb.br/handle/10482/10123.

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Dissertação (mestrado)—Universidade de Brasília, Instituto de Química, 2011.
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O vírus da imunodeficiência humana infecta linfócitos T, que possuem o receptor CD 4 na membrana plasmática. Inicialmente, o CD 4 é responsável pela entrada do vírus na célula hospedeira. Em um segundo momento, ocorre a modulação negativa do CD 4 pela proteína auxiliar Nef do vírus HIV-1, o que possibilita a saída do vírus para infectar outra célula. O antibiótico Ikarugamicina impede a modulação negativa do CD4 pela proteína auxiliar Nef e reestabelece o nível de CD 4 na membrana plasmática, mas é citotóxico em testes in vitro. Inibidores da proteína auxiliar Nef vêm sendo amplamente estudados como possíveis fármacos para o tratamento antirretroviral. O objetivo deste trabalho é a síntese de possíveis inibidores da proteína auxiliar Nef do vírus HIV -1. A partir de estudos de interação molecular, propusemos a síntese de seis moléculas, que contêm os grupos 3-amino-1,2,4-triazol, uracila ou 1,2,3-triazol, como possíveis inibidores da proteína auxiliar Nef do vírus HIV-1. Após análise retrossintética, diversas rotas foram propostas para a síntese das moléculas. Após várias tentativas de síntese, não se obteve sucesso na síntese da molécula com o grupo 3-amino-1,2,4-triazol, e da molécula com o grupo uracila. Um total de quatro moléculas contendo o anel 1,2,3-triazólico foram sintetizadas por rota sintética direta, obtendo-se excelentes rendimentos. A síntese envolveu a preparação do grupo azida, seguida por reação de Huisgen – reação click – e acoplamento com DCC/DMAP. ______________________________________________________________________________ ABSTRACT
The human immunodeficiency virus infects T lymphocytes that have the CD4 receptor on the plasma membrane. Initially, CD4 is responsible for virus entry into the host cell. In a second stage, the CD 4 downregulation by the auxiliary protein of HIV-1 Nef occurs, allowing the exit of the virus to infect another cell. The antibiotic Ikarugamycin prevents downregulation of CD 4 by the Nef auxiliary protein and restores the level of CD4 at the plasma membrane, but in vitro tests it is cytotoxic. Nef inhibitors have been investigated as potential drugs for antiretroviral treatment. The aim of this work is the synthesis of potential inhibitors of auxiliary protein HIV-1 Nef. Based on studies of molecular interaction we proposed the synthesis of six molecules, which contain the groups 3-amino-1,2,4-triazole, 1,2,3-triazole or uracil as potential inhibitors of the auxiliary protein HIV -1 Nef. After retrosynthetic analysis, several routes have been proposed for the synthesis of these molecules. Several attempts to synthesize molecules with the 3-amino-1,2,4-triazole and uracil groups, were performed without success. The molecules containing 1,2,3-triazole ring were synthesized by a direct synthetic route with excellent yields. The synthesis involved the preparation of the azide group, followed by the Huisgen reaction – click reaction – and DCC/DMAP coupling.
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Ornelas, Sócrates Souza. "Estudo da relevância fisiológica da tioesterase humana II (hTE) na modulação de CD4 mediada pela proteína Nef do HIV – 1." reponame:Repositório Institucional da UnB, 2007. http://repositorio.unb.br/handle/10482/2845.

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Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, 2007.
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A diminuição da expressão do receptor CD4 da superfície celular é um dos eventos mais importantes durante a infecção pelo vírus da Imunodeficiência Adquirida (HIV-1). Dentre as três proteínas virais que participam neste processo, Nef, Vpu e Env, a primeira se apresenta como a mais relevante. Estudos realizados por outros grupos e o nosso, evidenciaram claramente a relação entre a capacidade em diminuir a expressão da molécula CD4, a capacidade replicativa e a infectividade viral, sugerindo a participação deste evento na patogênese e progressão à doença. Resultados encorajadores obtidos por nosso grupo demonstraram que o bloqueio da modulação de CD4 mediada por Nef em células infectadas pode ser uma boa abordagem terapêutica. Baseados nestes resultados e com o intuito de identificar novos alvos e abordagens terapêuticas, traçamos como principal objetivo neste estudo determinar o real papel fisiológico da tioesterase humana II (hTE) na modulação do receptor CD4 mediada por Nef. Esta proteína foi descrita como um dos principais parceiros celulares de Nef neste processo fisiopatológico. Utilizando a ferramenta molecular do RNA de interferência (RNAi) para bloquear a expressão de proteínas celulares e virais envolvidas neste fenômeno, foi possível observar que hTE não desempenharia um papel relevante na modulação de CD4, contrariamente ao assinalado em trabalhos prévios. Porém, esta proteína mostrou participar na regulação dos níveis de CD4, possivelmente através da despalmitoilação do receptor viral na superfície celular, levando a internalização da molécula independentemente da presença de Nef. Outros resultados obtidos neste estudo também indicam que ao menos em variantes de Nef que se ligam a esta proteína, como a presente na cepa NL43, a ação da enzima celular poderia ser potencializada pela proteína viral. No entanto a confirmação desta como outras hipóteses relacionadas às possíveis conseqüências biológicas da interação entre hTE e Nef requerem estudos adicionais. _________________________________________________________________________________________ Abstract
The down-modulation of CD4 receptor expression is one of the most important events during the HIV-1 infection. Among the three viral proteins involved in this process Nef, Env and Vpu, the first one is the most relevant. Results obtained by our group and others showed a clear relationship between the virus mediated receptor down-modulation, the increasing of infectivity and viral replication of HIV-1, suggesting a pathogenic role in this phenomenon and disease progression. More recently, we provided proof-of-concept that specific inhibition of Nef mediated CD4 down-modulation could be a good therapeutical strategy. Based on these results and aiming to identify new targets and therapeutical strategies, we investigated the physiological role of the human tioesterase II (hTE) in the down-modulation of CD4 receptor mediated by Nef. The hTE was described as one of the main cellular partners of Nef in this process. Using the interference RNA (RNAi) mechanism, as a molecular tool to block cellular and viral proteins expression involved in this phenomenon, we observed that hTE does not play a relevant role on Nef-CD4 modulation, as assigned by previous works. We showed here that the participation in the regulation of cellular surface CD4 levels possibly by a depalmitolation process, making the internalization independent of Nef. The hTE enzymatic action on Nef-NL43 transfected cells showed a probably synergism between Nef and hTE. The confirmation of these preliminary findings as well as, a more thoroughly understanding of the general biological consequences arising from the interaction between hTE and Nef, requires additional studies.
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Gondim, Marcos Vinícius Pereira. "Caracterização da interação de proteínas celulares envolvidas na degradação de Cd4 mediada por diferentes alelos da proteína Nef do Hiv-1." reponame:Repositório Institucional da UnB, 2011. http://repositorio.unb.br/handle/10482/9469.

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Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, 2011.
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A diminuição da expressão do receptor CD4 na superfície das células infectadas é um dos mais importantes eventos durante a infecção pelo HIV-1. Foi observada uma clara relação entre a degradação do receptor viral, o aumento da infectuosidade e a replicação viral, sugerindo a participação deste fenômeno na patogênese e progressão da infecção. Para vencer os efeitos negativos exercidos pela expressão da molécula CD4, o HIV possui três proteínas: Nef, Vpu e Env, sendo Nef a proteína mais relevante neste fenômeno. Nef agiria na superfície celular como um conector da cauda citoplasmática da molécula CD4, com membros do complexo adaptador heterotetramérico de clatrina, redirecionando a molécula CD4 para vesículas endocíticas. Uma bomba de prótons vacuolar (V-ATPase) e a proteína Epsn15 foram envolvidas no aumento da força de ligação entre Nef e a subunidade μ2 de AP-2. Com a finalidade de evitar a reciclagem de CD4 para a superfície celular, uma segunda conexão entre CD4 e a proteína β-COP é estabelecida por Nef, o que permite direcionar o receptor para degradação lisossômica. Adicionalmente, duas proteínas foram também envolvidas no mecanismo a tioesterase humana II (hTE-II) e a dinamina 2 (Dyn2). Experimentos para validar o papel dessas proteínas celulares e sua relevância fisiológica não foram conclusivos. Por esse motivo nos propomos neste trabalho a caracterizar as interações entre algumas das proteínas celulares e diferentes alelos da proteína Nef. Os níveis de interação entre as proteínas celulares hTEII, CD4, V1H, β-COP, AP1σ, AP2μ e AP2σ e diferentes alelos da proteína Nef do HIV-1 e de SIV (Nef.NA7, NL4.3 e Mac239, respectivamente), assim como mudança de localização intracelular foram avaliados pelas técnicas de transferência relativa de energia de fluorescência (FRET) e microscopia confocal. As proteínas hTEII, AP2μ e β-COP, embora tenham mostrado elevados níveis de interação com os alelos de Nef NL4.3, NA7 e Mac239, demonstraram importantes diferençaspor FRET e confirmados por microscopia confocal, sendo que algumas proteínas tiveram também sua localização intracelular alterada. Resultados similares foram observados quando analisadas as interações entre as proteínas celulares e o alelo Mac239 de SIV, detectando-se elevados níveis de interação entre este alelo e a proteína celular AP2μ, assim como uma mudança na localização da proteína celular. Já as proteínas V1H, AP1σ e AP2σ AP1 não evidenciaram níveis significativos de interação com os alelos de Nef por ambas as técnicas. As proteínas AP2μ e β-COP e o receptor CD4 mostraram uma significativa interação, mas não foi observado mudanças na localização das mesmas. Adicionalmente, foram construídos vetores lentivirais codificando RNAs de interferência (RNAis) contra as diferentes proteínas celulares e virais, com o intuito de avaliar o impacto fisiológico das mesmas na degradação de CD4 no contexto da infecção viral. A eficiência dos diferentes vetores no silenciamento gênico foi confirmada, entretanto, experimentos exploratórios para avaliar o impacto da inibição da expressão das proteínas celulares na degradação de CD4 mediada por Nef não foram conclusivos, precisando–se realizar experimentos adicionais com mudança nas condições experimentais. _______________________________________________________________________________ ABSTRACT
The reduction of the receiver’s expression CD4 in the infected cells surfasse is one of the most importante events during the infectious process by HIV-1. It was observed a clear relationship among the viruses’ receiver degradation, the increase of infectious and virus’ replication, suggesting the participation of this phenomenon in infection pathogenesis and progress. To overcome the negative effects carried on by the molecular expression. CD4, HIV has three proteins: Nef, Vpu and Env, being Nef the most relevant protein in this phenomenon. Nef could act in cell surfasse as a link of the molecular cytoplasmic tail CD4 with the members of the complex adapter hetero tatrameter of clathrin, redirecting the CD4 molecule to the endocytic vesicles. A bomb of vacuole prótons (V- ATPase) and the protein Espn 15 were involved in the increase of bond strength between Nef and the subunit μ2 of AP-2. In order to avoid CD4 recycling to cell surfasse, another connection between CD4 and β-COP proteins is stabilished by Nef, which allows direct the receiver to the lysosomal degradation. Additionally, two proteins were also involved in this mechanism, human tioesterase II (hTEII) and the dynamin 2 (Dyn2). Experiments to validate the role of these cell proteins and its physiological relevance were not conclusive. For this reason we propose in this work characterize the interactions between some cell proteins and Nef’s protein diferente alleles. In both, the interactions and the location levels among some cell proteins as: hTEII, CD4, V1H,β- COP, AP1α, AP2μ and AP2α and diferente alleles of Nef protein of HIV-1 and SIV (Nef. NA7, NL4.3 and Mac 234, respectively), were evaluated by the techniques Relative transference of fluorescence energy (FRET) and Confocal microscopy. The proteins hTEII, Ap2μ and β- COP, although have shown high levels of interaction with Nef NL 4.3, NA7 and mac 239 alleles, demonstrated importante diferences among them. These levels of interaction were detected by FRET and confirmed by Confocal microscopy, of which some proteins also had their intracelular locations changed. Similar results were observed when interactions have happened between cell proteins and similar alleles Mac 239 of SIV, detecting high levels between this allele and the cell protein AP2μ, observing a change in the location of the protein, too. On the other hand, the proteins V1H, AP1α and AP2α, AP1 did not evidence significant levels of interaction with Ned alleles for both techniques. The analysis of interactions between cell proteins and CD4 receiver showed a significant interaction with AP2μ and β- COP proteins, but they did not induce changes in their location. Furthermore, there were lentiviral vectors encoding Interference RNAs (RNAis) against different cell proteins and viral. For the purposes of evaluate the physiological impact of CD4 degradation in viral infection context. The efficiency of different vectors in gene sileencing was confirmed, however, exploratory experiments to rate the impact of inibition in cell protein expression. in CD4 degradation, mediated by Nef, were not conclusive. It is necessary carry out additional experiments with change of experimental conditons.
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31

Götz, Nicola [Verfasser]. "Regain of Nef-mediated tetherin antagonism in a chimpanzee experimentally infected with HIV-1 / Nicola Götz." Ulm : Universität Ulm. Medizinische Fakultät, 2014. http://d-nb.info/1049238524/34.

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32

Noviello, Colleen M. "Downregulation of MHC-I by HIV-1 Nef evolution after sexual transmission and mechanism of action /." Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p3249651.

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Thesis (Ph. D.)--University of California, San Diego, 2007.
Title from first page of PDF file (viewed March 22, 2007). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
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33

Camaur, Diana M. "Roles of virion associated proteins in HIV-1 infectivity : Vif, Nef and the matrix tyrosine kinase /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1997. http://wwwlib.umi.com/cr/ucsd/fullcit?p9814538.

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34

Pandori, Mark W. "Producer cell modifications of HIV-1 by the nef gene : nef is a virion protein and augments viral infectivity by a CD4-independent mechanism /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1998. http://wwwlib.umi.com/cr/ucsd/fullcit?p9911850.

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35

Silva, Bosco Christiano Maciel da. "Estudo do reconhecimento de epitopos das proteínas Gag e Nef do HIV-1 por linfócitos T em indivíduos cronicamente infectados pelo HIV-1 não progressores por longo tempo." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/5/5133/tde-04082008-102804/.

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Os linfócitos T têm um papel central no controle da infecção pelo HIV-1. As respostas mediadas por esses linfócitos contra epitopos do HIV-1 restritos a moléculas HLA de classe I podem estar associadas à proteção natural em indivíduos LTNP. Relatos sugerem que determinados alelos HLA apresentamse mais representados entre os LTNP. Para avaliar esses aspectos na coorte francesa ALT, coletamos células mononucleares de sangue periférico (CMSP) de 24 indivíduos LTNP e verificamos a freqüência de respostas específicas para o HIV-1. Para isso, utilizamos pools de peptídeos sobrepostos de Gag e regiões imunodominantes da RT e Nef, e identificamos epitopos do HIV-1 restritos a moléculas HLA de classe I, associados ou não à proteção, através do ensaio de ELISPOT IFN-?. Todos os indivíduos apresentaram respostas específicas aos pools testados, com uma mediana de 5 (2-12). Todas as proteínas do HIV-1 foram reconhecidas, sendo que Gag-p24 e Nef foram as mais freqüentemente reconhecidas pelas CMSP dos indivíduos avaliados. A intensidade total de resposta de linfócitos T específicos aos pools de Gag, RT e Nef do HIV-1 em cada indivíduo variou de 160 a 12307 SFC/106 CMSP (mediana: 2025). Observamos o reconhecimento de 22 epitopos já descritos na literatura, contidos nas proteínas Gag-p17, Gag-p24 e Nef do HIV-1, restritos a moléculas HLA de classe I, a maioria descrita como protetoras da progressão para a doença. Quatro novos epitopos ainda não descritos na literatura também foram observados. Concluímos que: respostas específicas mediadas por linfócitos T, eficazes e dirigidas contra um amplo painel de epitopos do HIV-1, estão presentes nos indivíduos LTNP; a presença de moléculas HLA de classe I associadas à proteção favorece o reconhecimento preferencial de epitopos do HIV-1 restritos por elas na maioria dos indivíduos LTNP; esses aspectos devem ser levados em conta na perspectiva do desenvolvimento de uma vacina candidata contra o HIV-1.
T lymphocytes (T-L) have a paramount role in the control of HIV-1 infection. The responses mediated by these cells against HLA class I epitopes may be associated to the natural protection in long-term non-progressors (LTNP). The literature suggests that some HLA alleles relate to the protection against the immune dysfunction. The aim of this research is to study the recognition of HIV-1 Gag, Nef and RT epitopes by T-L through an ELISPOT IFN-? assay in the peripheral blood mononuclear cells (PBMC) of 24 LTNP selected from French ALT study group. We evaluated the frequency of anti-HIV-1 responses and identified HLA class I epitopes. All individuals presented specific responses to the pools of peptides tested with a median of 5 (2-12). Gag-p24 and Nef were the most frequently recognized proteins. The magnitude of the responses varied from 160 to 12307 SFC/106 PBMC (median=2025). We observed the recognition of 22 epitopes already described in HIV-1 Gag-p17, Gag-p24 and Nef, restricted to HLA class I molecules reported as protective. We have also observed four new epitopes not already described in the literature. Our results suggest that: HIV-1 responses by T-L are present in LTNP; the presence of HLA class I molecules associated with protection in the majority of LTNP are related to the recognition of MHC restricted HIV-1 epitopes; these aspects must be taken into account in the development of a candidate vaccine against HIV-1.
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36

Boulton, Victoria J. "An investigation into the effect of myristoylation on the interactions between HIV-1 NEF and cellular proteins." Thesis, University of Glasgow, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244253.

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37

Lülf, Sebastian [Verfasser], Martin [Akademischer Betreuer] Engelhard, and Roland [Gutachter] Winter. "Molekulare Strategien zur Inhibition des HIV-1 Nef Proteins / Sebastian Lülf. Betreuer: Martin Engelhard. Gutachter: Roland Winter." Dortmund : Universitätsbibliothek Dortmund, 2014. http://d-nb.info/1101595515/34.

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38

Dai, Lue. "A Novel Motif in HIV-1 Nef that Regulates MIP-1β Chemokine Release in Macrophages: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/485.

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Nef is an accessory protein encoded by human and simian immunodeficiency viruses (HIV and SIV), and is critical for viral pathogenicity in vivo.The structure of Nef has been resolved and the major cellular activities of Nef are generally described as down-regulation of cell surface molecules, enhancement of virus infectivity and regulation of cell signaling and activation. Macrophages represent a key target of HIV-1 infection and may contribute significantly to viral pathogenesis by facilitating viral propagation, maintaining a viral reservoir and regulating viral replication. During HIV-1 infection, various cytokines and chemokines are induced for viral advantages more than for host defense. We have previously demonstrated that HIV-1 Nef regulates the release of chemokines, MIP-1α and MIP-1ß, from infected macrophages and have proposed that this may enhance conditions for viral replication by promoting recruitment of substrate lymphocytes to sites of infection (1). However, the molecular basis for this Nef activity remains to be defined. The main goals of this thesis are to identify the functional motif in Nef that is responsible for chemokine induction in macrophages and to elucidate the relevance of this motif to other Nef functions. Using a mutagenesis approach, we have eventually identified a novel motif (KEK) that regulates chemokine production in infected macrophages after we excluded several previously described Nef motifs. This motif is conserved in both HIV-1 and SIV Nef proteins. Mutations in this domain abrogated MIP-1ß induction as well as the Nef-dependent release of other secretory factors by macrophages. However, disruption of this motif did not affect other Nef-ascribed activities such as CD4 and MHC-I down-regulation. In addition, we have determined the involvement of viral Env proteins in Nef-induced chemokine production. Distinct signaling pathways that regulate chemokine release in macrophage will also be described. Finally, several possible roles of the KEK motif are proposed and some preliminary results of co-immunoprecipitation experiments will be presented which aim to characterize cellular proteins involved in chemokine regulation by Nef. Collectively, our studies reveal a specific determinant within Nef that is critical for chemokine release by Nef. Identification of this motif paves the way for future studies to explore the molecular machanisms of Nef-regulated cell signaling pathways. Such knowledge may point to new therapeutic strategies that interrupt Nef function and limit the course of HIV-1 infection.
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39

Weiss, Eric R. "Investigating the Roles of NEDD4.2s and Nef in the Release and Replication of HIV-1: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/641.

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Replication of HIV-1 requires the assembly and release of mature and infectious viral particles. In order to accomplish this goal, HIV-1 has evolved multiple methods to interact with the host cell. HIV-1 recruits the host cell ESCRT machinery to facilitate the release of nascent viral particles from the host cell membrane. Recruitment of these cellular factors is dependent on the presence of short motifs in Gag referred to as Late-domains. Deletion or mutation of these domains results in substantial decrease in the release of infectious virions. However, previously published work has indicated that over-expression of the E3 ubiquitin ligase, NEDD4.2s is able to robustly rescue release of otherwise budding-defective HIV-1 particles. This rescue is specific to the NEDD4.2s isoform as related E3 ubiquitin ligases display no ability to rescue particle release. In addition, rescue of particle release is dependent on the presence of the partial C2 domain and a catalytically active HECT domain of NEDD4.2s. Here I provide evidence supporting the hypothesis that a partial C2 domain of NEDD4.2s constitutes a Gag interacting module capable of targeting the HECT domains of other E3 ubiquitin ligases to HIV-1 Gag. Also, by generating chimeras between HECT domains shown to form poly-ubiquitin chains linked through either K48 or K63 of ubiquitin, I demonstrate that the ability of NEDD4.2s to catalyze the formation of K63-polyubiquitin chains is required for its stimulation of HIV-1 L-domain mutant particle release. In addition, I present findings from on-going research into the role of the HIV-1 accessory protein Nef during viral replication using the culture T-cell line, MOLT3. My current findings indicate that downregulation of CD4 from the host cell membrane does not solely account for the dramatic dependence of HIV-1 replication on Nef expression in this system. In addition, I present evidence indicating that Nef proteins from diverse HIV-1 Groups and strains are capable of enhancing HIV-1 replication in this system. Analysis of a range of mutations in Nef known to impact interaction with cellular proteins suggest that the observed replication enhancement requires Nef targeting to the host cell membrane and may also require the ability to interact with select Src-kinases. Lastly, we find that the ability of Nef to enhance replication in this system is separate from any increase in viral particle infectivity, in agreement with current literature.
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40

Jao, Kevin. "The requirement of p56Lck tyrosine kinase in the modulation of fas-mediated apoptosis by HIV-1 Nef protein /." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=80295.

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The HIV-1 protein Nef is a critical factor in the viral pathogenesis and decline of CD4 T-cells during HIV infection. Nef has been implicated in modulating several cellular pathways, including apoptosis. This thesis herein describes our attempt to elucidate the mechanism by which Nef modulates apoptosis in T-cells. Using Jurkat cells inducibly expressing wild-type Nef, we observe that Nef renders cells more sensitive to apoptosis upon cross-linking with anti-Fas or TRAIL. Enhancement of Fas-mediated apoptosis required the presence of Lck, as apoptosis was abrogated in Nef expressing Lck-/- cells as compared to wild type Jurkat cells. Nef does not modulate expression of pro or anti-apoptotic proteins, or cell surface Fas receptor. Furthermore, Nef differentially mediates activation signaling pathways upon anti-CD3 stimulation. Enhancement of apoptosis by Nef may represent one of the mechanisms by which HIV depletes CD4 T-cells during infection.
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41

Bell, Ian. "T-cell signalling dysfunction associated with expression of NEF derived from the primate lentiviruses, HIV-1 and SIV." Thesis, Imperial College London, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299904.

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42

Kutscher, Sarah. "Immunomonitoring technologies for the evaluation of Modified Vaccinia Virus Ankara expressing HIV-1 nef as a vaccine against AIDS." Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-117303.

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43

Boeske, Alexandra [Verfasser]. "GABARAPs vermitteln den anterograden Transport und die Sekretion von HIV-1 Nef durch Autophagie-basierte unkonventionelle Sekretionsmechanismen / Alexandra Boeske." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2015. http://d-nb.info/107997122X/34.

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44

Scriba, Thomas Jens. "Accessory gene components for an HIV-1 subtype C vaccine : functional analysis of mutated Tat, Rev and Nef antigens." Thesis, Stellenbosch : Stellenbosch University, 2002. http://hdl.handle.net/10019.1/52621.

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Thesis (MSc)--University of Stellenbosch, 2002.
ENGLISH ABSTRACT: HIV has attained a global distribution and the number of infected people reached an estimated 28.1 million in sub-Saharan Africa at the end of 2001. HIV-1 subtype C is overwhelmingly prevalent in Botswana and South Africa and to date no interventions have been successful enough to curb the rapid spread of the virus. A number of HIV-1 vaccine strategies are being developed, however the breadth and efficacy of such candidate vaccines, many of which are based on the HIV-1 structural genes pol, gag and env, have mostly been found to be inadequate. The HIV-1 accessory genes are attractive components of HIV vaccines due to their role in viral pathogenesis, early expression and the high ratio of conserved CTl epitopes. Yet, because of undesirable properties questions regarding their safety as vaccine components are raised. In this study candidate tat, rev and nefmutants were assessed for efficient expression and inactivation of undesirable functionality. / Plasmid constructs that encode the South African HIV-1 subtype C consensus Tat, Rev and Nef proteins were constructed. The coding sequences of the genes were codon-optimised for optimum protein expression and these synthetic genes were constructed using overlapping 50-mer oligonucleotides. Furthermore, the proteins were mutated at previously described sites by PCR-based site-directed mutagenesis to render them inactive for their respective functions. Corresponding wild-type Tat, Rev and Nef constructs were also made from viral isolates that were least dissimilar to the respective consensus amino acid sequences. tn vitro expression of the different constructs were assessed in 293 cells by Western blotting with polyclonal mouse sera, which were generated by DNA immunisation with one of the Tat, Rev and Nef constructs. The transactivation activity of Tat variants and Rev-mediated nuclear export activity of RRE-containing transcripts were studied in cotransfection experiments using reporter-gene-based assays while Nef functionality was assessed in a cotransfection assay with subsequent flow cytometric analysis of surface CD4 and MHC-I expression on 293 cells. Sequence analysis of the South African HIV-1 subtype C consensus sequences of Tat, Rev and Nef revealed a high degree of similarity with a consensus sequence that was drawn up from a large number of viruses from southern Africa. These consensus sequences were also closer to individual viral isolate sequences than any individual sequences were, indicating that the use of a consensus sequence may serve to reduce genetic diversity between a vaccine and circulating viruses. Expression levels of the sequence-modified tat and nef gene constructs were not significantly higher than the wild-type constructs, however, the codon-optimised rev mutant exhibited markedly higher expression than the wild-type rev construct. Immunoreactivity of the protein with the mouse sera demonstrates expression and immunogenicity of the Tat, Rev and Nef immunogens in mice. In the background of the subtype C Tat, a single C22 mutation was insufficient to inactivate l TRdependent CAT expression in 293T and Hela cells. Yet, this activity was significantly impaired using the single mutation, C3?, or the double mutation, C22C3? Compared to the wild-type Rev, the function of the Rev with a double mutation, M5M10, was completely abrogated. Similarly, while the wild-type Nef and native, codon-optimised consensus Nef proteins mediated CD4 and MHC-I downregulation, CD4 downregulation was completely abrogated in one of the mutants, while both Nef mutants were entirely deficient for MHC-I downregulation. These data demonstrate the high expression levels and impaired functionality of sequence-modified HIV-1 subtype C consensus Tat, Rev and Nef DNA immunogens that may be used as single-standing vaccine components or form part of a multicomponent HIV-1 vaccine.
AFRIKAANSE OPSOMMING: Sedert die eerste gevalle van MIV in die vroeë 1980's beskryf is het die virus wêreldwyd versprei en 'n beraamde 28.1 miljoen mense in sub-Sahara Afrika was teen die einde van 2001 geïnfekteer. MIV-1 subtipe C kom verreweg die meeste voor in Botswana en Suid-Afrika en tans is daar geen suksesvolle tussenkoms wat die vinnige verspreiding van die virus kan stuit nie. 'n Aantal MIV-1 subtipe C entstofstrategieë word tans ontwikkel maar die spektrum en effektiwiteit van sulke entstowwe, waarvan baie op die MIV strukturele gene gag, pol en env gebaseer is, is tans onvoldoende. Die MIV-1 bykomstige gene is aantreklike entstofkomponente omdat hulle vroeg uitgedruk word, 'n belangrike rol in virale patogenese speel en omdat hulle 'n hoë verhouding van gekonserveerde sitotoksiese T-limfosiet (STL) epitope tot grootte besit. Vanweë hierdie gene se verskeie ongewenste eienskappe word vrae ten opsigte van hul veilige insluiting in enstofstrategieë geopper. Hierdie studie omskryf die evaluasie van kandidaat tat, reven nef mutante vir doeltreffende proteïenuitdrukking en funksionele onaktiwiteit. Plasmiedkonstrukte wat vir die Suid-Afrikaanse MIV-1 subtipe C konsensus Tat, Rev en Nef proteïene kodeer is saamgestel. Die koderingsvolgordes van die gene is geoptimiseer vir optimale uitdrukking en die sintetiese gene is van oorvleuelende 50- mer oligonukleotiede vervaardig. Deur van PKR-gebaseerde site-directed mutagenese gebruik te maak is hierdie proteïene gemuteer op posisies wat voorheen geïdentifiseer is. Ooreenstemmende wilde-tipe Tat, Reven Nef konstrukte is gemaak vanaf virale isolate waarvan die aminosuurvolgordes die meeste ooreenstem met dié van die konsensusvolgorde. In vitro uitdrukking van die konstrukte in 293 selle is met behulp van immunoklad met poliklonale muissera bepaal. Die serum is gegenereer deur DNS immunisasie van muise met een elk van die Tat, Reven Nef konstrukte. Die transaktiverings-aktiwiteit van Tat variante en Rev bemiddelde uitvoer van RREbesittende transkripte uit die nukleus is in verklikkergeen kotransfeksie-eksperimente bestudeer. Nef se funksionaliteit is deur kotransfeksie en die daaropvolgende vloeisitometriese analise van 293 selle se oppervlak-CD4 en MHC-I uitdrukking bestudeer. Nukleotiedvolgorde-analise van die Suid-Afrikaanse MIV-1 subtipe C konsensus Tat, Reven Nef proteiëne toon 'n hoë vlak van ooreenkoms met 'n konsensusvolgorde wat afgelei is vanaf 'n groot aantal suider-Afrikaanse virusse. Hierdie konsensusvolgordes is ook meer soortgelyk aan individuele virale isolate as enige individuele volgordes. Vanuit hierdie data kan afgelei word dat die gebruik van so 'n konsensusvolgorde die genetiese diversiteit tussen 'n entstof en sirkuierende virusse kan verminder. Uitdrukkingsvlakke van die volgorde-geoptimiseerde tat en nef geenkonstrukte is nie merkbaar hoër as die van die wilde-tipe konstrukte nie. In teenstelling het die volgorde-geoptimiseerde rev mutant merkbaar hoër uitdrukkingsvlakke as die wildetipe getoon. Immunoreaktiwiteit van die proteïene met die muissera demonstreer dat die Tat, Reven Nef proteïene uitgedruk word en immunogenies in muise is. 'n Enkele C22 mutasie in Tat is nie genoeg om lTR-afhanklike CAT uitdrukking in 293T en Hela selle te inaktiveer nie. In teenstelling is hierdie aktiwiteit geïnhibeer vir Tat proteïene met die enkel mutasie C37 en die dubbel mutasie C22C37. In vergelyking met die funksionele aktiwiteit van die wilde-tipe Rev is dié van die Rev mutant M5M10 heeltemal geïnhibeer. Die wilde-tipe en geoptimiseerde, konsensus Nef proteïene het seloppervlak-CD4 en -MHC-I uitdrukking verlaag, maar hierdie effek van afregulering van CD4 uitdrukking was heeltemaal opgehef in een Nef mutant en van MHC-I uitdrukking in beide Nef mutante. Hierdie data demonstreer die hoë uitdrukkingsvlakke en geïnhibeerde funksionaliteit van volgorde-gemodifiseerde MIV-1 subtipe C konsensus Tat, Reven Nef DNS immunogene wat as enkelstaande enstof kan optree of deel kan uitmaak van 'n multi-komponent MIV-1 entstof.
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45

Tarosso, Leandro Fagundes da Silva. "Resposta Vif- e Nef-específica mediada por células T CD8+ em indivíduos HIV-1-positivos que espontaneamente controlam a replicação viral." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/5/5146/tde-22122010-103356/.

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Indivíduos infectados pelo vírus da imunodeficiência humana do tipo 1 (HIV-1) que controlam a replicação viral, mesmo na ausência de tratamento com drogas antirretrovirais, representam um exemplo de contenção bemsucedida do vírus. O entendimento das respostas imunes antivirais presentes nestes indivíduos pode auxiliar no delineamento de vacinas, particularmente no caso de estratégias vacinais desenvolvidas para induzir um fenótipo de controle da replicação viral e, assim, diminuir o ritmo da progressão à AIDS e/ou a taxa de transmissão para terceiros. A resposta imune celular contra HIV-1 é geralmente mapeada em ensaios de ELISPOT-IFN-γ empregando-se peptídeos pentadecâmeros sobrepostos por 11 aminoácidos sintetizados a partir de seqüências consensuais do vírus. Contudo, este método pode subestimar a detecção da real amplitude da resposta imune celular contra epitopos contidos na seqüência autóloga do vírus infectivo. Neste trabalho, foram comparadas respostas imunes celulares contra peptídeos 15-meros baseados nas seqüências de vif e nef do consenso do subtipo B do HIV-1 e respostas imunes contra peptídeos HLA-restritos de nove ou 10 aminoácidos baseados tanto nas seqüências de vif e nef do consenso do subtipo B do HIV-1, quanto nas seqüências autólogas dos vírus seqüenciados a partir de seis pacientes controladores da replicação do HIV-1. Nossa análise revelou que três dos seis pacientes investigados mostraram maior amplitude de resposta imune celular contra epitopos em Vif e Nef quando os peptídeos HLA-restritos foram empregados, tenham sido eles preditos a partir da seqüência consensual ou a partir das seqüências do vírus autólogo. O número de respostas positivas aumentou de quatro para 16 em Vif e de oito para 22 em Nef, com o uso dos reagentes HLA-restritos. Estes resultados sugerem que emprego de peptídeos 15-meros pode sub-representar a amplitude real da resposta imune celular envolvidas no controle da replicação do HIV-1 e que o conhecimento acerca das respostas imunes de sucesso em indivíduos controladores pode ser melhorado e ampliado com a revisão dos métodos empregados.
Human immunodeficiency virus type 1 (HIV-1)-infected individuals who spontaneously control viral replication represent an example of successful containment of the AIDS virus. Understanding the anti-viral immune responses in these individuals may help in vaccine design, particularly vaccine strategies designed to induce a controller phenotype and thus, prevent disease progression and decrease risk of transmission. Immune responses against HIV-1 are normally screened using 15-mer peptides overlapped by 11 amino acids from HIV-1 consensus sequences in ELISPOT-IFN-γ assays. However, this method may underestimate the real breadth of the cellular immune responses against the autologous sequence of the infecting virus. We compared cellular immune responses against nef and vif-encoded consensus B 15-mer peptides to responses against HLA class I-predicted minimal optimal epitopes from consensus B and autologous sequences in six patients who have controlled HIV-1 replication. Interestingly, our analysis revealed that three of our patients had broader cellular immune responses against Vif- and Nef-HLA class I-predicted minimal optimal epitopes from either autologous viruses or from the consensus B sequence, when compared to responses against the 15-mer HIV-1 consensus B peptides. The number of positive responses against epitopes in these two HIV-1 proteins increased from four to 16 for Vif and from eight to 22 for Nef. These findings suggest that immune responses assessed using 15-mers peptides may underrepresent the real breadth of the immune control of the infecting virus and the knowledge about the successful responses in controller individuals could be improved after reviewing the employed methods.
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46

Pennington, Daniel John. "The analysis of the in vivo effects of the HIV-1 Nef and Tat regulatory genes using a transgenic mouse system." Thesis, University College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.309192.

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47

Meunier, Clémence. "Effects of the HIV-1 protein Nef on the stromal cells of mouse peripheral lymph nodes and on mouse keratinocytes." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=119682.

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The HIV-1 protein Nef plays an important role in HIV-1 pathogenesis, both in humans and mouse models. Indeed, transgenic (Tg) mice expressing Nef under the control of the human CD4 promoter develop many phenotypes that closely resemble those of AIDS patients. Using these Tg mice, we have studied the effects of Nef on lymph node stroma, namely on fibroblastic reticular cells (FRCs), and blood and lymphatic endothelial cells (BECs and LECs, respectively). In human patients, a severe and irreversible loss of LN structure and function is observed, and this is correlated with the depletion of FRCs. We show that, in our model, Nef does not significantly change the size of the FRC population, nor does it affect its functions in resting lymph nodes (LNs). We hypothesized that FRCs are supported by lymphoid tissue inducer cells, which prevent their depletion. No fibrosis or loss of structure could be observed in the LNs of Tg mice, contrary to what is seen in human AIDS patients. Nef did, however, cause a localized expansion of BECs and LECs in medullary blood vessels and the subcapsular sinus, respectively, sometimes to the point of completely obstructing these structures. The mechanism driving this expansion is still under investigation. We also studied an unexpected effect of Nef expression on skin keratinocytes. This expression led to the development of an atopic dermatitis-like disease. Atopic dermatitis is one of the most prevalent skin diseases associated with AIDS but, to our knowledge, no HIV-1 protein had previously been directly linked to it. We show here that Nef causes this atopic dermatitis-like disease by inhibiting the Notch1 signalling pathway in keratinocytes. Thus, our data adds to the list of known Nef effects, and provides potential new insights for therapy.
La protéine Nef du VIH-1 joue un rôle important dans la pathogenèse de ce virus, chez les humains et chez des modèles murins. En effet, des souris transgéniques (Tg) exprimant Nef sous le contrôle du promoteur CD4 humain développent des phénotypes très similaires à ceux retrouvés chez les patients atteints du SIDA. Nous avons étudié, chez ce modèle murin, les effets de Nef sur le stroma des ganglions lymphatiques (GL), c'est-à-dire sur les cellules réticulaires fibroblastiques (CRFs) et les cellules endothéliales vasculaires et lymphatiques (respectivement CEVs et CELs). Chez les humains infectés par le VIH-1, une perte sévère et irréversible de la structure et de la fonction des GLs est observée et est corrélée avec la déplétion des CRFs. Nous montrons ici que, dans notre modèle, Nef ne change pas significativement la taille de la population de CRFs et n'affecte pas ses fonctions. Nous proposons que la population de CRFs est maintenue par les cellules inductrices de tissu lymphoïde. Aucune fibrose ou perte de structure n'a été observée dans les GLs des souris Tg, contrairement à ce qui se retrouve chez les patients humains. Par contre, Nef cause une expansion localisée des CEVs et des CELs dans les vaisseaux sanguins médullaires et dans le sinus subcapsulaire, respectivement, parfois au point de complètement obstruer ces structures. Le mécanisme de cette expansion est à l'étude. Nous avons également étudié des effets inattendus de l'expression de Nef dans les kératinocytes de la peau. Cette expression a provoqué le développement d'une maladie similaire à la dermatite atopique. La dermatite atopique est l'une des maladies de peau les plus fréquentes chez les patients atteints du SIDA. Cependant, à notre connaissance, aucune protéine du VIH-1 n'y avait été directement associée à ce jour. Nous montrons ici que Nef cause cette maladie en inhibant la voie de signalisation de Notch1 dans les kératinocytes. Ainsi, nos résultats identifient de nouveaux effets de Nef et fournissent de nouvelles perspectives potentielles pour des thérapies.
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Fournier, Cécile. "Caractérisation de la fixation de la protéine NEF du virus HIV-1 à l'ARN et mise en évidence de son rôle dans la rétrotranscription." Montpellier 1, 2000. http://www.theses.fr/2000MON1T015.

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49

Santerre, Maryline. "Étude de l'action sur l'épissage de protéines nucléaires se liant à la région de l'ARN du virus VIH-1 contenant le site d'épissage A7 et role de ces protéines sur d'autres sites accepteurs d'épissage de VIH-1." Thesis, Nancy 1, 2010. http://www.theses.fr/2010NAN10115/document.

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L'épissage est une étape clef de la multiplication du VIH-1. Par utilisation de 4 sites donneurs et 8 sites accepteurs d'épissage, plus de 40 ARNm différents sont produits. Une approche protéomique nous a permis d'identifier de nouvelles protéines interagissant avec la région de l'ARN viral contenant le site A7. Nous avons démontré l'interaction directe avec l'ARN viral de 5 des protéines identifiées (nucléoline, hnRNP A1/B, hnRNP H et hnRNP K). Nous avons montré que hnRNP K a plusieurs sites de fixation dans la région du site A7 et que hnRNP A1et hnRNP K se lient de façon coopérative. Nous avons montré un effet inhibiteur de hnRNP K sur l'épissage au site A7. Comme la protéine hnRNP A1 est un régulateur négatif de plusieurs sites accepteurs d'épissage (A1, A2, A3, A7), nous avons testé si la protéine hnRNP K pouvait renforcer l'inhibition à ces sites. En fait, hnRNP K active l'épissage in vitro des introns entre le site donneur D1 et les sites accepteurs A1, A2 et A3. Nous avons montré que la protéine hnRNP K renforce fortement l'activité de ASF/SF2 au site A2, ce qui indique que selon le contexte, la protéine hnRNP K peut être activatrice ou inhibitrice de l'épissage du VIH-1. J'ai observé de plus que la surexpression de la protéine hnRNP K dans des cellules HeLa, transfectées avec le plasmide p PSP contenant le virus VIH-1 dépourvu de ses capacités d'encapsidation, produit un changement très marqué de l'épissage alternatif de l'ARN PSP, ce qui confirme la forte influence de hnRNP K sur l'épissage alternatif du VIH-1. L'augmentation de la concentration cellulaire de hnRNP K dans les cellules HeLa conduit aussi à une diminution de la protéine virale Nef. La protéine hnRNP K intervient donc non seulement dans la régulation du site A7, mais aussi dans celle de la majorité des sites d'épissage régulés de l'ARN du VIH. L'action de cette protéine sur plusieurs des sites d'épissage montre que la protéine hnRNP K est probablement un régulateur général de l'épissage de VIH-1
HIV-1 pre-mRNA splicing depends upon 4 donor and 8 acceptor sites, which are used in combination to produce more than 40 different mRNAs. To further characterize nuclear factors involved in these processes, we purified RNP complexes formed by incubation of SLS2-A7 transcripts in HeLa cell nuclear extracts by affinity chromatography to identify new associated proteins. We showed that, in addition to the well known hnRNP A1 inhibitor of site A7, nucleolin, hnRNP H and hnRNP K interact directly with SLS2-A7 RNA. We demonstrated that hnRNP K has multiple binding sites in the vicinity of site A7 and that binds cooperatively to hnRNP A1 to the A7 RNA region and limits the A7 utilization in vitro. As hnRNP A1 is a negative regulator of several HIV-1 splicing sites (A1, A2, A3), we tested whether hnRNP K may also reinforce hnRNP A1 inhibition at these sites. Surprisingly, hnRNP K activated in vitro splicing of the D1-A1, D1-A2 and D1-A3 introns. Interestingly, hnRNP K was found to reinforce strongly the ASF/SF2 activity at site A2, which indicates that depending on the splicing site hnRNP K can be a splicing activator or inhibitor. To test how hnRNP K influences the relative utilization of HIV-1 splicing sites in cellulo, we used plasmid p PSP containing all the HIV-1 splicing sites and tested the effect of over-expression in HeLa cells on alternative splicing of the PSP RNA. Doubling the amount of hnRNP K in HeLa cells led to a drastic change of the PSP RNA alternative splicing, which confirms the strong influence of hnRNP K on alternative splicing. Moreover, increase of cellular concentration of hnRNP K strongly decrease the viral Nef protein production. hnRNP K protein affects A7 splicing regulation but also regulates the majority of regulated splicing sites of HIV. By extension of the study of hnRNP K effect to other HIV-1 splicing sites, we discovered that hnRNP K is a general regulator of HIV-1 splicing
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Lee, Jung-Hyun [Verfasser], and Lars [Akademischer Betreuer] Nitschke. "HIV-1 Nef-associated Paxillin and Pak1/2 regulate activation and secretion of ADAM17/10 proteases / Jung-Hyun Lee. Gutachter: Lars Nitschke." Erlangen : Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 2014. http://d-nb.info/1054342636/34.

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