41 results on '"E. Le Rouzic"'
Search Results
2. Easy Optical Defragmentation with SDN Controlled Tunable Transmitter
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Patricia Layec, B. Haentjens, E. Le Rouzic, L. Brameriet, Arnaud Carer, Dominique Verchere, Arnaud Dupas, Q. Pham Van, Nokia Bell Labs [Nozay], Institut des Fonctions Optiques pour les Technologies de l'informatiON (Institut FOTON), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-École Nationale Supérieure des Sciences Appliquées et de Technologie (ENSSAT)-Centre National de la Recherche Scientifique (CNRS)-IMT Atlantique Bretagne-Pays de la Loire (IMT Atlantique), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT), Algorithmes et architectures adaptatifs pour les systèmes sans-fils efficaces en énergie (GRANIT), ARCHITECTURE (IRISA-D3), Institut de Recherche en Informatique et Systèmes Aléatoires (IRISA), CentraleSupélec-Télécom Bretagne-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Institut National de Recherche en Informatique et en Automatique (Inria)-École normale supérieure - Rennes (ENS Rennes)-Université de Bretagne Sud (UBS)-Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-CentraleSupélec-Télécom Bretagne-Université de Rennes 1 (UR1), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-Institut de Recherche en Informatique et Systèmes Aléatoires (IRISA), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA), Vectrawave, Orange Labs, Université de Rennes (UR)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Nationale Supérieure des Sciences Appliquées et de Technologie (ENSSAT)-Centre National de la Recherche Scientifique (CNRS), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université de Bretagne Sud (UBS)-École normale supérieure - Rennes (ENS Rennes)-Institut National de Recherche en Informatique et en Automatique (Inria)-Télécom Bretagne-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes (UR)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université de Bretagne Sud (UBS)-École normale supérieure - Rennes (ENS Rennes)-Institut National de Recherche en Informatique et en Automatique (Inria)-Télécom Bretagne-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche en Informatique et Systèmes Aléatoires (IRISA), and Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université de Bretagne Sud (UBS)-École normale supérieure - Rennes (ENS Rennes)-Institut National de Recherche en Informatique et en Automatique (Inria)-Télécom Bretagne-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)
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Service (systems architecture) ,Computer science ,business.industry ,Transmitter ,020206 networking & telecommunications ,02 engineering and technology ,01 natural sciences ,010309 optics ,[INFO.INFO-NI]Computer Science [cs]/Networking and Internet Architecture [cs.NI] ,0103 physical sciences ,0202 electrical engineering, electronic engineering, information engineering ,Defragmentation ,business ,Computer hardware - Abstract
International audience; To ease automatic optical spectrum defragmentation, we present a programmable SDN controlled 100Gbit/s transmitter prototype with a fast wavelength and bitrate switching capabilities. It can be tuned in 0.5 ms over all C-band and used for service hitless defragmentation application.
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- 2018
3. Optical Power Control to Efficiently Handle Flex-Grid Spectrum Gain over Existing Fixed-Grid Network Infrastructures
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Djamel Amar, E. Le Rouzic, Bernard Cousin, Jean-Luc Auge, Nicolas Brochier, Mohamad Kanj, Institut de Recherche Technologique b-com (IRT b-com), Orange Labs [Lannion], France Télécom, Advanced technologies for operated networks (ADOPNET), Université de Rennes (UR)-Télécom Bretagne-RÉSEAUX, TÉLÉCOMMUNICATION ET SERVICES (IRISA-D2), Institut de Recherche en Informatique et Systèmes Aléatoires (IRISA), Université de Rennes (UR)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université de Bretagne Sud (UBS)-École normale supérieure - Rennes (ENS Rennes)-Institut National de Recherche en Informatique et en Automatique (Inria)-Télécom Bretagne-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes (UR)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université de Bretagne Sud (UBS)-École normale supérieure - Rennes (ENS Rennes)-Institut National de Recherche en Informatique et en Automatique (Inria)-Télécom Bretagne-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche en Informatique et Systèmes Aléatoires (IRISA), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université de Bretagne Sud (UBS)-École normale supérieure - Rennes (ENS Rennes)-Institut National de Recherche en Informatique et en Automatique (Inria)-Télécom Bretagne-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université de Bretagne Sud (UBS)-École normale supérieure - Rennes (ENS Rennes)-Institut National de Recherche en Informatique et en Automatique (Inria)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS), Université de Rennes (UR), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Télécom Bretagne-RÉSEAUX, TÉLÉCOMMUNICATION ET SERVICES (IRISA-D2), CentraleSupélec-Télécom Bretagne-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Institut National de Recherche en Informatique et en Automatique (Inria)-École normale supérieure - Rennes (ENS Rennes)-Université de Bretagne Sud (UBS)-Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-CentraleSupélec-Télécom Bretagne-Université de Rennes 1 (UR1), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-Institut de Recherche en Informatique et Systèmes Aléatoires (IRISA), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-CentraleSupélec-Institut National de Recherche en Informatique et en Automatique (Inria)-École normale supérieure - Rennes (ENS Rennes)-Université de Bretagne Sud (UBS)-Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA), and Université de Rennes (UNIV-RENNES)
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Engineering ,Optical power budget ,Grid network ,Optical power ,02 engineering and technology ,01 natural sciences ,010309 optics ,Networking ,[INFO.INFO-NI]Computer Science [cs]/Networking and Internet Architecture [cs.NI] ,020210 optoelectronics & photonics ,Path Computation Algorithm ,Optical networks ,0103 physical sciences ,Power Control ,0202 electrical engineering, electronic engineering, information engineering ,Electronic engineering ,Optical amplifier ,business.industry ,Amplifier ,Spectral efficiency ,Optical performance monitoring ,Link Design ,Flex-Grid ,GMPLS ,business ,Power control - Abstract
International audience; The exponential traffic growth in optical networks has triggered the evolution from Fixed-Grid to Flex-Grid technology. This evolution allows better spectral efficiency and spectrum usage over current networks in order to facilitate dynamic and huge traffic demands. The integration of Flex-Grid technology increases the number of optical channels established over optical links, leading, however, to an increase in amplification power and possibly saturating optical amplifiers. In this work, we propose a power adaptation process that takes advantage of link optical signal to noise ratio (OSNR) margins to allow network operators to support this power increase while maintaining the use of legacy amplifiers. Results show that controlling channel optical power benefits from the Flex-Grid in terms of spectrum and capacity gain using in-place amplifier infrastructure.
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- 2016
4. An integrated view on monitoring and compensation for dynamic optical networks: from management to physical layer
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E. Le Rouzic, Jose A. Lazaro, Antônio Lúcio Teixeira, Lourena E. Costa, Carmen Vázquez, Ioannis Tomkos, Kyriakos Vlachos, Siamak Azodolmolky, Philippe Gravey, Szilard Zsigmond, T. Loukina, Julio Montalvo, Gerald Franzl, G.M. Tosi-Beleffi, and Tibor Cinkler
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Dynamic network analysis ,Dynamic networks ,Computer Networks and Communications ,Computer science ,Reliability (computer networking) ,Network management and routing ,02 engineering and technology ,Optical performance monitoring ,020210 optoelectronics & photonics ,0202 electrical engineering, electronic engineering, information engineering ,Network performance ,Electrical and Electronic Engineering ,Network architecture ,business.industry ,Physical layer ,020206 networking & telecommunications ,Network layer ,Impairment compensation ,Atomic and Molecular Physics, and Optics ,Reliability engineering ,Network management ,Hardware and Architecture ,Electrónica ,business ,Software ,Computer network - Abstract
A vertical perspective, ranging from management and routing to physical layer options, concerning dynamic network monitoring and compensation of impairments (M&C), is given. Feasibility, reliability, and performance improvements on reconfigurable transparent networks are expected to arise from the consolidated assessment of network management and control specifications, as a more accurate evaluation of available M&C techniques. In the network layer, physical parameters aware algorithms are foreseen to pursue reliable network performance. In the physical layer, some new M&C methods were developed and rating of the state-of-the-art reported in literature is given. Optical monitoring implementation and viability is discussed. Publicado
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- 2009
5. Alteration of C-MYB DNA binding to cognate responsive elements in HL-60 variant cells
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C Créminon, Bernard Perbal, E Le Rouzic, and C Gaillard
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animal structures ,Genes, myb ,Cellular differentiation ,Blotting, Western ,Electrophoretic Mobility Shift Assay ,HL-60 Cells ,Biology ,DNA-binding protein ,Monocytes ,Pathology and Forensic Medicine ,Proto-Oncogene Proteins c-myb ,Transcriptional regulation ,Humans ,Electrophoretic mobility shift assay ,MYB ,Cloning, Molecular ,Gene ,fungi ,Cell Differentiation ,DNA, Neoplasm ,Original Articles ,Molecular biology ,Neoplasm Proteins ,DNA-Binding Proteins ,Gene Expression Regulation, Neoplastic ,Cell culture ,Tetradecanoylphorbol Acetate ,Cell Division - Abstract
Aims: To establish whether the MYB protein expressed in HL-60 variant cells, which are cells resistant to 12-O-tetradecanoylphorbol-13-acetate (TPA) induced differentiation, is able to bind MYB recognition elements (MREs) involved in the transcriptional regulation of myb target genes. In addition, to determine whether alterations in the binding of the MYB protein to MREs affects HL-60 cell proliferation and differentiation. Methods: Nuclear extracts of HL-60 variant cells exhibiting different degrees of resistance to TPA induced monocytic differentiation were used in electrophoretic mobility shift experiments (EMSAs), bandshift experiments performed with labelled oliogonucleotides containing the MYB consensus binding sequences. Results: The MYB protein contained in nuclear extracts from HL-60 variant cells did not bind efficiently to the MYB recognition elements identified in the mim-1 and PR264 promoters. Molecular cloning of the myb gene and analysis of the MYB protein expressed in the HL-60 variant cells established that the lack of binding did not result from a structural alteration of MYB in these cells. The lack of MRE binding did not abrogate the ability of variant HL-60s to proliferate and to undergo differentiation. Furthermore, the expression of the PR264/SC35 splicing factor was not affected as a result of the altered MYB DNA binding activity. Conclusions: Because the MYB protein expressed in HL-60 variant cells did not appear to be structurally different from the MYB protein expressed in parental HL-60 cells, it is possible that the HL-60 variant cells contain a MYB binding inhibitory factor (MBIF) that interferes with MYB binding on MREs. The increased proliferation rate of HL-60 variant cells and their reduced serum requirement argues against the need for direct MYB binding in the regulation of cell growth.
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- 2002
6. Physical layer aware network architecture for the future internet
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Antonio Serrador, T. Frantti, Luis M. Correia, E. Le Rouzic, P. Mannersalo, G. Nunzi, Filipe D. Cardoso, and N. Genay
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Computer Networks and Communications ,Network security ,Computer science ,050801 communication & media studies ,02 engineering and technology ,computer.software_genre ,0508 media and communications ,0202 electrical engineering, electronic engineering, information engineering ,Wireless ,Electrical and Electronic Engineering ,Internet ,Network architecture ,Access network ,business.industry ,Physical layer aware ,05 social sciences ,Physical layer ,020206 networking & telecommunications ,Virtualization ,Computer Science Applications ,Information and Communications Technology ,The Internet ,business ,Telecommunications ,computer ,Computer network - Abstract
In this article, physical layer awareness inaccess, core, and metro networks is addressed,and a Physical Layer Aware Network Architec-ture Framework for the Future Internet is pre-sented and discussed, as proposed within theframework of the European ICT Project4WARD. Current limitations and shortcomingsof the Internet architecture are driving researchtrends at a global scale toward a novel, secure,and flexible architecture. This Future Internetarchitecture must allow for the co-existence andcooperation of multiple networks on commonplatforms, through the virtualization of networkresources. Possible solutions embrace a fullrange of technologies, from fiber backbones towireless access networks. The virtualization ofphysical networking resources will enhance thepossibility of handling different profiles, whileproviding the impression of mutual isolation.This abstraction strategy implies the use of wellelaborated mechanisms in order to deal withchannel impairments and requirements, in bothwireless (access) and optical (core) environ-ments. info:eu-repo/semantics/publishedVersion
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- 2012
7. Enhancing GMPLS Signaling Protocol for Encompassing Quality of Transmission (QoT) in All-Optical Networks
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Nicola Sambo, Alessio Giorgetti, E. Le Rouzic, Filippo Cugini, Piero Castoldi, J. Poirrier, Nicola Andriolli, and Luca Valcarenghi
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Routing protocol ,Computer science ,Resource Reservation Protocol ,computer.internet_protocol ,business.industry ,Distributed computing ,Quality of service ,ComputerSystemsOrganization_COMPUTER-COMMUNICATIONNETWORKS ,020206 networking & telecommunications ,Multiprotocol Label Switching ,02 engineering and technology ,Network topology ,Blocking (statistics) ,Atomic and Molecular Physics, and Optics ,Signaling protocol ,020210 optoelectronics & photonics ,Transmission (telecommunications) ,0202 electrical engineering, electronic engineering, information engineering ,business ,computer ,Computer network - Abstract
In this paper, quality of transmission (QoT)-aware lightpath provisioning schemes for transparent optical networks are proposed and assessed. The main idea is to overcome lightpath blocking due to excessive physical impairments (i.e., unacceptable QoT) by means of successive lightpath set up attempts performed by generalized multiprotocol label switching (GMPLS) signaling protocol along alternate routes. The schemes are enabled by the introduction into current GMPLS signaling protocol [i.e., resource reservation protocol with traffic engineering (RSVP-TE)] of extensions which encompass the QoT parameters that characterize the optical layer. Differently from previous approaches, the proposed GMPLS-based schemes are still distributed but they do not imply the introduction of additional extensions into the routing protocol (e.g., OSPF-TE). The QoT-aware provisioning schemes are first validated by simulations performed on a WDM mesh network. Results show that only few successive set up attempts are required to complete the lightpath establishment. In addition, an experimental demonstration where the proposed RSVP-TE extensions are implemented in the control plane of a transparent metro network is reported showing that impairment-aware lightpath provisioning is achieved on a time scale of few milliseconds.
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- 2008
8. Probe-based schemes to guarantee lightpath quality of transmission (QoT) in transparent optical networks
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E. Le Rouzic, Piero Castoldi, J. Poirrier, Isabella Cerutti, Nicola Sambo, Filippo Cugini, C. Pinart, and Luca Valcarenghi
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Routing protocol ,Engineering ,business.industry ,ComputerSystemsOrganization_COMPUTER-COMMUNICATIONNETWORKS ,020206 networking & telecommunications ,Data_CODINGANDINFORMATIONTHEORY ,02 engineering and technology ,Blocking (statistics) ,020210 optoelectronics & photonics ,Atmospheric measurements ,Quality (physics) ,Transmission (telecommunications) ,0202 electrical engineering, electronic engineering, information engineering ,Bit error rate ,business ,Computer network - Abstract
Two probe-based schemes are proposed to dynamically guarantee lightpath QoT in transparent optical networks. Low blocking and fast set up times are achieved within few lightpath set up attempts.
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- 2008
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9. Alterations of the MDV oncogenic regions in an MDV transformed lymphoblastoid cell line
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P Thoraval, E Le Rouzic, Y Cherel, M Afanassieff, G Dambrine, Bernard Perbal, Station de Pathologie aviaire et parasitologie [Nouzilly] (PAP), Institut National de la Recherche Agronomique (INRA), Immuno-Endocrinologie Cellulaire et Moléculaire (IECM), and Ecole Nationale Vétérinaire de Nantes-Université de Nantes (UN)-Institut National de la Recherche Agronomique (INRA)
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animal structures ,Genes, myb ,T cell ,animal diseases ,viruses ,[SDV]Life Sciences [q-bio] ,Genome, Viral ,medicine.disease_cause ,Lymphoma, T-Cell ,Polymerase Chain Reaction ,Virus ,Pathology and Forensic Medicine ,Transforming Growth Factor beta1 ,03 medical and health sciences ,Viral Proteins ,immune system diseases ,hemic and lymphatic diseases ,medicine ,Marek Disease ,Tumor Cells, Cultured ,Gammaherpesvirinae ,Animals ,Protein Precursors ,Gene ,Herpesvirus 2, Gallid ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,0303 health sciences ,biology ,Virulence ,030306 microbiology ,Lymphoblast ,Nuclear Proteins ,Oncogenes ,Original Articles ,biology.organism_classification ,Cell Transformation, Viral ,Virology ,Peptide Fragments ,medicine.anatomical_structure ,Cell culture ,Trans-Activators ,Carcinogenesis ,Chickens ,Oncovirus - Abstract
Aims: Lymphoblastoid cell lines derived from Marek’s disease virus (MDV) induced tumours have served as models of MDV latency and transformation. They are stable and can be cultured with no detectable MDV genomic alterations upon repeated passaging. An MDV transformed lymphoblastoid T cell line (T9 cell line) has been reported to contain a disrupted MDV BamHI-H fragment and a Rous associated virus insertional activation of the c-myb protooncogene. In an attempt to define the respective participation of c-myb and MDV in the transformed phenotype of T9 cells, an analysis of MDV oncogenic sequences (BamHI-H, BamHI-A, and EcoQ fragments) was performed in these cells. Methods: Using two different passages of the T9 cell line (late and early passages), the organisation of the MDV oncogenic regions and their expression in these cells were analysed. In vivo assessment of the oncogenicity of the virus contained within these cells was assessed by injecting them into 1 day old chickens. Results: In T9 cells maintained in culture for up to six months (late T9), the MDV ICP4 gene was disrupted, whereas the meq gene was actively transcribed. The alterations of the MDV genome in these cells correlated with the inability of the virus to induce the classic signs of Marek’s disease in 1 day old chickens. However, early T9 cells submitted to a limited number of passages induced classic MDV pathogenicity, as efficiently as the MDV control cell line (T5), and did not show gross structural changes in the oncogenic MDV sequences. Conclusions: Although the expression pattern of the MDV oncogenes in early T9 cells was identical to the one reported for other MDV transformed cells, longterm culture of an MDV transformed cell line containing a RAV insertional activation of the c-myb protooncogene led to the disruption of the MDV BamHI-H and BamHI-A oncogenic regions. In the late T9 cells MEQ was the only detected MDV oncoprotein. These results suggest that in the late T9 cells the truncated MYB protein compensates for the loss of MDV oncoproteins and reinforce the possibility that MEQ and MYB cooperate in the maintenance of the transformed state and the tumorigenic potential of these cells.
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- 2002
10. HIV auxiliary proteins: an interface between the virus and the host
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K, Janvier, C, Petit, E, Le Rouzic, O, Schwartz, S, Benichou, Institut Cochin (UMR_S567 / UMR 8104), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Rétrovirus et Transfert Génétique, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Laboratoire Rétrovirus et Transfert Génétique, Institut Pasteur, Institut Pasteur [Paris], Université Paris Descartes - Paris 5 (UPD5) - Institut National de la Santé et de la Recherche Médicale (INSERM) - Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] - Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)
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CD4-Positive T-Lymphocytes ,[SDV] Life Sciences [q-bio] ,Viral Proteins ,Virulence ,[SDV]Life Sciences [q-bio] ,HIV-1 ,Humans ,HIV Infections ,Virus Replication ,ComputingMilieux_MISCELLANEOUS - Abstract
International audience
- Published
- 1999
11. Algorithms for the Multi-Period Power-Aware Logical Topology Design With Reconfiguration Costs
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Luca Chiaraviglio, Edoardo Bonetto, E. Le Rouzic, and Filip Idzikowski
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Engineering ,Optimization problem ,Computer Networks and Communications ,business.industry ,Distributed computing ,Logical topology ,Control reconfiguration ,020206 networking & telecommunications ,02 engineering and technology ,Network topology ,Passive optical network ,Power (physics) ,Set (abstract data type) ,020210 optoelectronics & photonics ,0202 electrical engineering, electronic engineering, information engineering ,Heuristics ,business ,Algorithm - Abstract
We tackle the problem of reducing power consumption in IP-over-WDM networks, targeting the power-aware logical topology design (LTD). Unlike the previous work in the literature, our solution reduces the power consumption with consideration of the cost (in terms of reconfigured traffic) incurred when the network is reconfigured. We first formulate the LTD with reconfiguration costs as an optimization problem. Then, we present three heuristics to effectively solve it. We compare our algorithms over an extensive set of networks and scenarios. Results indicate that our algorithms are effective in reducing power consumption while limiting the amount of traffic that is reconfigured. Moreover, we show that the input parameters are intuitive and easy to set, which makes our algorithms more practical.
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- 2013
12. Retroviral insertional activation of the c-myb proto-oncogene in a Marek's disease T-lymphoma cell line
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E Le Rouzic and Bernard Perbal
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animal structures ,viruses ,Virus Integration ,Immunology ,Gene Expression ,Biology ,Lymphoma, T-Cell ,Transfection ,Microbiology ,Virus ,Malignant transformation ,Exon ,Proto-Oncogene Proteins c-myb ,immune system diseases ,Virology ,hemic and lymphatic diseases ,Proto-Oncogene Proteins ,medicine ,Tumor Cells, Cultured ,MYB ,RNA, Messenger ,Herpesvirus 2, Gallid ,Alleles ,Gene Rearrangement ,Marek's disease ,Avian Leukosis Virus ,Deoxyribonuclease BamHI ,medicine.disease ,biology.organism_classification ,Phenotype ,Molecular biology ,Long terminal repeat ,Lymphoma ,Insect Science ,embryonic structures ,DNA, Viral ,Trans-Activators ,Research Article - Abstract
Marek's disease virus (MDV) is an avian herpesvirus that causes, in chickens, a lymphoproliferative disease characterized by malignant transformation of T lymphocytes. The rapid onset of polyclonal tumors indicates the existence of MDV-encoded oncogenic products. However, the molecular basis of MDV-induced lymphoproliferative disease and latency remains largely unclear. Several lines of evidence suggest that MDV and Rous-associated virus (RAV) might cooperate in the development of B-cell lymphomas induced by RAV. Our present results indicate for the first time that MDV and RAV might also act synergistically in the development of T-cell lymphomas. We report an example of an MDV-transformed T-lymphoblastoid cell line (T9) expressing high levels of a truncated C-MYB protein as a result of RAV integration within one c-myb allele. The chimeric RAV-c-myb mRNA species initiated in the 5' long terminal repeat of RAV are deprived of sequences corresponding to c-myb exons 1 to 3. The attenuation of MDV oncogenicity has been strongly related to structural changes in the MDV BamHI-D and BamHI-H DNA fragments. We have established that both DNA restriction fragments are rearranged in the T9 MDV-transformed cells. Our results suggest that retroviral insertional activation of the c-myb proto-oncogene is a critical factor involved in the maintenance of the transformed phenotype and the tumorigenic potential of this T-lymphoma cell line.
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- 1996
13. Centralized vs. distributed approaches for encompassing physical impairments in transparent optical networks
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Alessio Giorgetti, Piero Castoldi, Luca Valcarenghi, Filippo Cugini, E. Le Rouzic, M. J. Poirrier, Francesco Paolucci, Nicola Andriolli, and Nicola Sambo
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Routing protocol ,Signaling protocol ,Computer science ,business.industry ,Path computation element ,Distributed computing ,Convergence (routing) ,Scalability ,Transparency (human–computer interaction) ,Routing (electronic design automation) ,business ,Optical mesh network ,Computer network - Abstract
Transparent optical mesh networks are an appealing solution to provide cost-effective high bandwidth connections eliminating the need of expensive intermediate electronic regenerators. However, the implementation of transparent optical networks requires to take into account physical impairment information for effective lightpath set-up. In this paper, we present two distributed solutions to encompass physical impairments based on enhancements of the GMPLS protocol suite. Specifically, both GMPLS routing protocol and signaling protocol extensions are presented and discussed. An alternative centralized approach based on an impairment-aware Path Computation Element (PCE) is also proposed. The distributed routing approach exhibits convergence limitations, while the distributed signaling approach is scalable and effective. The latter is then compared against the centralized PCE approach through simulations considering both a metro network and a more complex WDM network scenario. In addition, experimental implementations of the two approaches are presented. Results show the trade-off of the two approaches, demonstrating the general good performance in terms of lightpath set up time for both approaches.
14. Biological and Structural Analyses of New Potent Allosteric Inhibitors of HIV-1 Integrase.
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Bonnard D, Le Rouzic E, Singer MR, Yu Z, Le Strat F, Batisse C, Batisse J, Amadori C, Chasset S, Pye VE, Emiliani S, Ledoussal B, Ruff M, Moreau F, Cherepanov P, and Benarous R
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- Humans, Virus Replication, Antiviral Agents pharmacology, Allosteric Regulation, HIV Integrase Inhibitors pharmacology, HIV Integrase Inhibitors therapeutic use, HIV Integrase metabolism, HIV Infections drug therapy
- Abstract
HIV-1 integrase-LEDGF allosteric inhibitors (INLAIs) share the binding site on the viral protein with the host factor LEDGF/p75. These small molecules act as molecular glues promoting hyper-multimerization of HIV-1 IN protein to severely perturb maturation of viral particles. Herein, we describe a new series of INLAIs based on a benzene scaffold that display antiviral activity in the single digit nanomolar range. Akin to other compounds of this class, the INLAIs predominantly inhibit the late stages of HIV-1 replication. A series of high-resolution crystal structures revealed how these small molecules engage the catalytic core and the C-terminal domains of HIV-1 IN. No antagonism was observed between our lead INLAI compound BDM-2 and a panel of 16 clinical antiretrovirals. Moreover, we show that compounds retained high antiviral activity against HIV-1 variants resistant to IN strand transfer inhibitors and other classes of antiretroviral drugs. The virologic profile of BDM-2 and the recently completed single ascending dose phase I trial (ClinicalTrials.gov identifier: NCT03634085) warrant further clinical investigation for use in combination with other antiretroviral drugs. Moreover, our results suggest routes for further improvement of this emerging drug class.
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- 2023
- Full Text
- View/download PDF
15. Structure-function analyses unravel distinct effects of allosteric inhibitors of HIV-1 integrase on viral maturation and integration.
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Bonnard D, Le Rouzic E, Eiler S, Amadori C, Orlov I, Bruneau JM, Brias J, Barbion J, Chevreuil F, Spehner D, Chasset S, Ledoussal B, Moreau F, Saïb A, Klaholz BP, Emiliani S, Ruff M, Zamborlini A, and Benarous R
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- Allosteric Regulation, Binding Sites, Cell Line, HIV Integrase Inhibitors chemistry, Humans, Molecular Structure, Pyridines chemistry, Pyridines pharmacology, Structure-Activity Relationship, Thiophenes chemistry, Thiophenes pharmacology, HIV Integrase drug effects, HIV Integrase Inhibitors pharmacology, HIV-1 physiology, Virus Assembly drug effects, Virus Integration drug effects
- Abstract
Recently, a new class of HIV-1 integrase (IN) inhibitors with a dual mode of action, called IN-LEDGF/p75 allosteric inhibitors (INLAIs), was described. Designed to interfere with the IN-LEDGF/p75 interaction during viral integration, unexpectedly, their major impact was on virus maturation. This activity has been linked to induction of aberrant IN multimerization, whereas inhibition of the IN-LEDGF/p75 interaction accounts for weaker antiretroviral effect at integration. Because these dual activities result from INLAI binding to IN at a single binding site, we expected that these activities co-evolved together, driven by the affinity for IN. Using an original INLAI, MUT-A, and its activity on an Ala-125 (A125) IN variant, we found that these two activities on A125-IN can be fully dissociated: MUT-A-induced IN multimerization and the formation of eccentric condensates in viral particles, which are responsible for inhibition of virus maturation, were lost, whereas inhibition of the IN-LEDGF/p75 interaction and consequently integration was fully retained. Hence, the mere binding of INLAI to A125 IN is insufficient to promote the conformational changes of IN required for aberrant multimerization. By analyzing the X-ray structures of MUT-A bound to the IN catalytic core domain (CCD) with or without the Ala-125 polymorphism, we discovered that the loss of IN multimerization is due to stabilization of the A125-IN variant CCD dimer, highlighting the importance of the CCD dimerization energy for IN multimerization. Our study reveals that affinity for the LEDGF/p75-binding pocket is not sufficient to induce INLAI-dependent IN multimerization and the associated inhibition of viral maturation., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2018
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16. The HIV-1 integrase-LEDGF allosteric inhibitor MUT-A: resistance profile, impairment of virus maturation and infectivity but without influence on RNA packaging or virus immunoreactivity.
- Author
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Amadori C, van der Velden YU, Bonnard D, Orlov I, van Bel N, Le Rouzic E, Miralles L, Brias J, Chevreuil F, Spehner D, Chasset S, Ledoussal B, Mayr L, Moreau F, García F, Gatell J, Zamborlini A, Emiliani S, Ruff M, Klaholz BP, Moog C, Berkhout B, Plana M, and Benarous R
- Subjects
- Cell Line, HIV Antibodies immunology, HIV Integrase Inhibitors chemistry, HIV-1 immunology, Humans, Pyridines chemistry, Thiophenes chemistry, Virus Assembly drug effects, Virus Integration drug effects, Virus Replication drug effects, HIV Integrase metabolism, HIV Integrase Inhibitors pharmacology, HIV-1 drug effects, HIV-1 enzymology, Intercellular Signaling Peptides and Proteins metabolism, Pyridines pharmacology, Thiophenes pharmacology
- Abstract
Background: HIV-1 Integrase (IN) interacts with the cellular co-factor LEDGF/p75 and tethers the HIV preintegration complex to the host genome enabling integration. Recently a new class of IN inhibitors was described, the IN-LEDGF allosteric inhibitors (INLAIs). Designed to interfere with the IN-LEDGF interaction during integration, the major impact of these inhibitors was surprisingly found on virus maturation, causing a reverse transcription defect in target cells., Results: Here we describe the MUT-A compound as a genuine INLAI with an original chemical structure based on a new type of scaffold, a thiophene ring. MUT-A has all characteristics of INLAI compounds such as inhibition of IN-LEDGF/p75 interaction, IN multimerization, dual antiretroviral (ARV) activities, normal packaging of genomic viral RNA and complete Gag protein maturation. MUT-A has more potent ARV activity compared to other INLAIs previously reported, but similar profile of resistance mutations and absence of ARV activity on SIV. HIV-1 virions produced in the presence of MUT-A were non-infectious with the formation of eccentric condensates outside of the core. In studying the immunoreactivity of these non-infectious virions, we found that inactivated HIV-1 particles were captured by anti-HIV-specific neutralizing and non-neutralizing antibodies (b12, 2G12, PGT121, 4D4, 10-1074, 10E8, VRC01) with efficiencies comparable to non-treated virus. Autologous CD4
+ T lymphocyte proliferation and cytokine induction by monocyte-derived dendritic cells (MDDC) pulsed either with MUT-A-inactivated HIV or non-treated HIV were also comparable., Conclusions: Although strongly defective in infectivity, HIV-1 virions produced in the presence of the MUT-A INLAI have a normal protein and genomic RNA content as well as B and T cell immunoreactivities comparable to non-treated HIV-1. These inactivated viruses might form an attractive new approach in vaccine research in an attempt to study if this new type of immunogen could elicit an immune response against HIV-1 in animal models.- Published
- 2017
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17. The allosteric HIV-1 integrase inhibitor BI-D affects virion maturation but does not influence packaging of a functional RNA genome.
- Author
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van Bel N, van der Velden Y, Bonnard D, Le Rouzic E, Das AT, Benarous R, and Berkhout B
- Subjects
- Acetates chemistry, Allosteric Regulation, Cell Line, Dimerization, Genome, Viral, HEK293 Cells, HIV Integrase metabolism, HIV Integrase Inhibitors chemistry, HIV Reverse Transcriptase chemistry, HIV Reverse Transcriptase metabolism, HIV-1 enzymology, HIV-1 genetics, Humans, Quinolines chemistry, RNA, Transfer metabolism, RNA, Viral isolation & purification, Virus Assembly drug effects, Acetates pharmacology, HIV Integrase chemistry, HIV Integrase Inhibitors pharmacology, HIV-1 physiology, Quinolines pharmacology, RNA, Viral metabolism, Virus Replication drug effects
- Abstract
The viral integrase (IN) is an essential protein for HIV-1 replication. IN inserts the viral dsDNA into the host chromosome, thereby aided by the cellular co-factor LEDGF/p75. Recently a new class of integrase inhibitors was described: allosteric IN inhibitors (ALLINIs). Although designed to interfere with the IN-LEDGF/p75 interaction to block HIV DNA integration during the early phase of HIV-1 replication, the major impact was surprisingly found on the process of virus maturation during the late phase, causing a reverse transcription defect upon infection of target cells. Virus particles produced in the presence of an ALLINI are misformed with the ribonucleoprotein located outside the virus core. Virus assembly and maturation are highly orchestrated and regulated processes in which several viral proteins and RNA molecules closely interact. It is therefore of interest to study whether ALLINIs have unpredicted pleiotropic effects on these RNA-related processes. We confirm that the ALLINI BI-D inhibits virus replication and that the produced virus is non-infectious. Furthermore, we show that the wild-type level of HIV-1 genomic RNA is packaged in virions and these genomes are in a dimeric state. The tRNAlys3 primer for reverse transcription was properly placed on this genomic RNA and could be extended ex vivo. In addition, the packaged reverse transcriptase enzyme was fully active when extracted from virions. As the RNA and enzyme components for reverse transcription are properly present in virions produced in the presence of BI-D, the inhibition of reverse transcription is likely to reflect the mislocalization of the components in the aberrant virus particle.
- Published
- 2014
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18. Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage.
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Le Rouzic E, Bonnard D, Chasset S, Bruneau JM, Chevreuil F, Le Strat F, Nguyen J, Beauvoir R, Amadori C, Brias J, Vomscheid S, Eiler S, Lévy N, Delelis O, Deprez E, Saïb A, Zamborlini A, Emiliani S, Ruff M, Ledoussal B, Moreau F, and Benarous R
- Subjects
- Cell Line, Crystallography, X-Ray, HIV Integrase chemistry, HIV Integrase Inhibitors chemistry, Humans, Intercellular Signaling Peptides and Proteins chemistry, Protein Binding, Protein Conformation, HIV Integrase metabolism, HIV Integrase Inhibitors pharmacology, HIV-1 drug effects, HIV-1 physiology, Intercellular Signaling Peptides and Proteins metabolism, Virus Integration drug effects, Virus Replication drug effects
- Abstract
Background: LEDGF/p75 (LEDGF) is the main cellular cofactor of HIV-1 integrase (IN). It acts as a tethering factor for IN, and targets the integration of HIV in actively transcribed gene regions of chromatin. A recently developed class of IN allosteric inhibitors can inhibit the LEDGF-IN interaction., Results: We describe a new series of IN-LEDGF allosteric inhibitors, the most active of which is Mut101. We determined the crystal structure of Mut101 in complex with IN and showed that the compound binds to the LEDGF-binding pocket, promoting conformational changes of IN which explain at the atomic level the allosteric effect of the IN/LEDGF interaction inhibitor on IN functions. In vitro, Mut101 inhibited both IN-LEDGF interaction and IN strand transfer activity while enhancing IN-IN interaction. Time of addition experiments indicated that Mut101 behaved as an integration inhibitor. Mut101 was fully active on HIV-1 mutants resistant to INSTIs and other classes of anti-HIV drugs, indicative that this compound has a new mode of action. However, we found that Mut101 also displayed a more potent antiretroviral activity at a post-integration step. Infectivity of viral particles produced in presence of Mut101 was severely decreased. This latter effect also required the binding of the compound to the LEDGF-binding pocket., Conclusion: Mut101 has dual anti-HIV-1 activity, at integration and post-integration steps of the viral replication cycle, by binding to a unique target on IN (the LEDGF-binding pocket). The post-integration block of HIV-1 replication in virus-producer cells is the mechanism by which Mut101 is most active as an antiretroviral. To explain this difference between Mut101 antiretroviral activity at integration and post-integration stages, we propose the following model: LEDGF is a nuclear, chromatin-bound protein that is absent in the cytoplasm. Therefore, LEDGF can outcompete compound binding to IN in the nucleus of target cells lowering its antiretroviral activity at integration, but not in the cytoplasm where post-integration production of infectious viral particles takes place.
- Published
- 2013
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19. AT2 Receptor-Interacting Proteins ATIPs in the Brain.
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Rodrigues-Ferreira S, le Rouzic E, Pawlowski T, Srivastava A, Margottin-Goguet F, and Nahmias C
- Abstract
A complete renin-angiotensin system (RAS) is locally expressed in the brain and fulfills important functions. Angiotensin II, the major biologically active peptide of the RAS, acts via binding to two main receptor subtypes designated AT1 and AT2. The present paper focuses on AT2 receptors, which have been reported to have neuroprotective effects on stroke, degenerative diseases, and cognitive functions. Our group has identified a family of AT2 receptor interacting proteins (ATIPs) comprising three major members (ATIP1, ATIP3, and ATIP4) with different intracellular localization. Of interest, all ATIP members are expressed in brain tissues and carry a conserved domain able to interact with the AT2 receptor intracellular tail, suggesting a role in AT2-mediated brain functions. We summarize here current knowledge on the ATIP family of proteins, and we present new experimental evidence showing interaction defects between ATIP1 and two mutant forms of the AT2 receptor identified in cases of mental retardation. These studies point to a functional role of the AT2/ATIP1 axis in cognition.
- Published
- 2013
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20. Recruitment of the nuclear form of uracil DNA glycosylase into virus particles participates in the full infectivity of HIV-1.
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Guenzel CA, Hérate C, Le Rouzic E, Maidou-Peindara P, Sadler HA, Rouyez MC, Mansky LM, and Benichou S
- Subjects
- Cell Line, DNA Glycosylases genetics, HIV Infections virology, HIV-1 genetics, Humans, Protein Binding, Virion genetics, Virus Replication, vpr Gene Products, Human Immunodeficiency Virus genetics, vpr Gene Products, Human Immunodeficiency Virus metabolism, DNA Glycosylases metabolism, HIV Infections enzymology, HIV-1 physiology, Virion physiology
- Abstract
The HIV-1 Vpr protein participates in the early steps of the virus life cycle by influencing the accuracy of reverse transcription. This role of Vpr was related to the recruitment of the nuclear form of the uracil DNA glycosylase (UNG2) enzyme into virus particles, but several conflicting findings have been reported regarding the role of UNG2 encapsidation on viral infectivity. Here, we report that the catalytic activity of UNG2 was not required for influencing HIV-1 mutation, and this function of UNG2 was mapped within a 60-amino-acid domain located in the N-terminal region of the protein required for direct interaction with the p32 subunit of the replication protein A (RPA) complex. Importantly, enforced recruitment of overexpressed UNG2 into virions resulted in a net increase of virus infectivity, and this positive effect on infectivity was also independent of the UNG2 enzymatic activity. In contrast, virus infectivity and replication, as well as the efficiency of the viral DNA synthesis, were significantly reduced when viruses were produced from cells depleted of either endogenous UNG2 or RPA p32. Taken together, these results demonstrate that incorporation of UNG2 into virions has a positive impact on HIV-1 infectivity and replication and positively influences the reverse transcription process through a nonenzymatic mechanism involving the p32 subunit of the RPA complex.
- Published
- 2012
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21. Molecular insight into how HIV-1 Vpr protein impairs cell growth through two genetically distinct pathways.
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Maudet C, Bertrand M, Le Rouzic E, Lahouassa H, Ayinde D, Nisole S, Goujon C, Cimarelli A, Margottin-Goguet F, and Transy C
- Subjects
- Amino Acid Motifs, Carrier Proteins genetics, Cell Death genetics, Cullin Proteins genetics, Cullin Proteins metabolism, HEK293 Cells, HIV-1 genetics, HeLa Cells, Humans, Mutation, Protein Serine-Threonine Kinases, Ubiquitin-Protein Ligases, vpr Gene Products, Human Immunodeficiency Virus genetics, Carrier Proteins metabolism, Cell Cycle, HIV-1 metabolism, Models, Biological, vpr Gene Products, Human Immunodeficiency Virus metabolism
- Abstract
Vpr, a small HIV auxiliary protein, hijacks the CUL4 ubiquitin ligase through DCAF1 to inactivate an unknown cellular target, leading to cell cycle arrest at the G(2) phase and cell death. Here we first sought to delineate the Vpr determinants involved in the binding to DCAF1 and to the target. On the one hand, the three α-helices of Vpr are necessary and sufficient for binding to DCAF1; on the other hand, nonlinear determinants in Vpr are required for binding to the target, as shown by using protein chimeras. We also underscore that a SRIG motif conserved in the C-terminal tail of Vpr proteins from HIV-1/SIVcpz and HIV-2/SIVsmm lineages is critical for G(2) arrest. Our results suggest that this motif may be predictive of the ability of Vpr proteins from other SIV lineages to mediate G(2) arrest. We took advantage of the characterization of a subset of G(2) arrest-defective, but DCAF1 binding-proficient mutants, to investigate whether Vpr interferes with cell viability independently of its ability to induce G(2) arrest. These mutants inhibited cell colony formation in HeLa cells and are cytotoxic in lymphocytes, unmasking a G(2) arrest-independent cytopathic effect of Vpr. Furthermore these mutants do not block cell cycle progression at the G(1) or S phases but trigger apoptosis through caspase 3. Disruption of DCAF1 binding restored efficiency of colony formation. However, DCAF1 binding per se is not sufficient to confer cytopathicity. These data support a model in which Vpr recruits DCAF1 to induce the degradation of two host proteins independently required for proper cell growth.
- Published
- 2011
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22. The CDK inhibitor p21Cip1/WAF1 is induced by FcgammaR activation and restricts the replication of human immunodeficiency virus type 1 and related primate lentiviruses in human macrophages.
- Author
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Bergamaschi A, David A, Le Rouzic E, Nisole S, Barré-Sinoussi F, and Pancino G
- Subjects
- Animals, Cells, Cultured, Cyclin-Dependent Kinase Inhibitor p21 antagonists & inhibitors, Cyclin-Dependent Kinase Inhibitor p21 genetics, Gene Knockdown Techniques, HIV-1 immunology, HIV-2 growth & development, HIV-2 immunology, Humans, Lentiviruses, Primate immunology, Primates, Simian Immunodeficiency Virus growth & development, Simian Immunodeficiency Virus immunology, Virus Integration immunology, Virus Replication immunology, Cyclin-Dependent Kinase Inhibitor p21 biosynthesis, HIV-1 growth & development, Lentiviruses, Primate growth & development, Macrophages immunology, Macrophages virology, Receptors, IgG immunology
- Abstract
Macrophages are major targets of human immunodeficiency virus type 1 (HIV-1). We have previously shown that aggregation of activating immunoglobulin G Fc receptors (FcgammaR) by immune complexes inhibits reverse transcript accumulation and integration of HIV-1 and related lentiviruses in monocyte-derived macrophages. Here, we show that FcgammaR-mediated restriction of HIV-1 is not due to enhanced degradation of incoming viral proteins or cDNA and is associated to the induction of the cyclin-dependent kinase inhibitor p21(Cip1/WAF1) (p21). Small interfering RNA-mediated p21 knockdown rescued viral replication in FcgammaR-activated macrophages and enhanced HIV-1 infection in unstimulated macrophages by increasing reverse transcript and integrated DNA levels. p21 induction by other stimuli, such as phorbol myristate acetate and the histone deacetylase inhibitor MS-275, was also associated with preintegrative blocks of HIV-1 replication in macrophages. Binding of p21 to reverse transcription/preintegration complex-associated HIV-1 proteins was not detected in yeast two-hybrid, pulldown, or coimmunoprecipitation assays, suggesting that p21 may affect viral replication independently of a specific interaction with an HIV-1 component. Consistently, p21 silencing rescued viral replication from the FcgammaR-mediated restriction also in simian immunodeficiency virus SIV(mac)- and HIV-2-infected macrophages. Our results point to a role of p21 as an inhibitory factor of lentiviral infection in macrophages and to its implication in FcgammaR-mediated restriction.
- Published
- 2009
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23. Characterization of the molecular determinants of primary HIV-1 Vpr proteins: impact of the Q65R and R77Q substitutions on Vpr functions.
- Author
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Jacquot G, Le Rouzic E, Maidou-Peindara P, Maizy M, Lefrère JJ, Daneluzzi V, Monteiro-Filho CM, Hong D, Planelles V, Morand-Joubert L, and Benichou S
- Subjects
- Alleles, Amino Acid Sequence, Apoptosis, Genetic Variation, Humans, Leukocytes, Mononuclear cytology, Models, Genetic, Molecular Sequence Data, Mutation, Protein Binding, Ubiquitin-Protein Ligases metabolism, Gene Products, vpr genetics, Genes, vpr, Terminal Repeat Sequences, vpr Gene Products, Human Immunodeficiency Virus genetics
- Abstract
Although HIV-1 Vpr displays several functions in vitro, limited information exists concerning their relevance during infection. Here, we characterized Vpr variants isolated from a rapid and a long-term non-progressor (LTNP). Interestingly, vpr alleles isolated from longitudinal samples of the LTNP revealed a dominant sequence that subsequently led to diversity similar to that observed in the progressor patient. Most of primary Vpr proteins accumulated at the nuclear envelope and interacted with host-cell partners of Vpr. They displayed cytostatic and proapoptotic activities, although a LTNP allele, harboring the Q65R substitution, failed to bind the DCAF1 subunit of the Cul4a/DDB1 E3 ligase and was inactive. This Q65R substitution correlated with impairment of Vpr docking at the nuclear envelope, raising the possibility of a functional link between this property and the Vpr cytostatic activity. In contradiction with published results, the R77Q substitution, found in LTNP alleles, did not influence Vpr proapoptotic activity.
- Published
- 2009
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24. The human immunodeficiency virus type 2 Vpx protein usurps the CUL4A-DDB1 DCAF1 ubiquitin ligase to overcome a postentry block in macrophage infection.
- Author
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Bergamaschi A, Ayinde D, David A, Le Rouzic E, Morel M, Collin G, Descamps D, Damond F, Brun-Vezinet F, Nisole S, Margottin-Goguet F, Pancino G, and Transy C
- Subjects
- CD4-Positive T-Lymphocytes virology, Cullin Proteins metabolism, DNA-Binding Proteins metabolism, Gene Knockdown Techniques, Gene Silencing, HIV-2 genetics, HIV-2 physiology, HeLa Cells, Humans, Simian Immunodeficiency Virus metabolism, Simian Immunodeficiency Virus physiology, Virus Replication, HIV Infections virology, HIV-2 metabolism, Macrophages virology, Ubiquitin-Protein Ligases metabolism, Viral Regulatory and Accessory Proteins metabolism
- Abstract
The human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) genomes encode several auxiliary proteins that have increasingly shown their importance in the virus-host relationship. One of these proteins, Vpx, is unique to the HIV-2/SIVsm lineage and is critical for viral replication in macrophages. The functional basis for this requirement, as well as the Vpx mode of action, has remained unexplained, and it is all the more enigmatic that HIV type 1 (HIV-1), which has no Vpx counterpart, can infect macrophages. Here, we underscore DCAF1 as a critical host effector of Vpx in its ability to mediate infection and long-term replication of HIV-2 in human macrophages. Vpx assembles with the CUL4A-DDB1 ubiquitin ligase through DCAF1 recruitment. Precluding Vpx present in the incoming virions from recruiting DCAF1 in target macrophages leads to a postentry block characterized by defective accumulation of HIV-2 reverse transcripts. In addition, Vpx from SIVsm functionally complements Vpx-defective HIV-2 in a DCAF1-binding-dependent manner. Altogether, our data point to a mechanism in which Vpx diverts the Cul4A-DDB1(DCAF1) ligase to inactivate an evolutionarily conserved factor, which restricts macrophage infection by HIV-2 and closely related simian viruses.
- Published
- 2009
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25. Assembly with the Cul4A-DDB1DCAF1 ubiquitin ligase protects HIV-1 Vpr from proteasomal degradation.
- Author
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Le Rouzic E, Morel M, Ayinde D, Belaïdouni N, Letienne J, Transy C, and Margottin-Goguet F
- Subjects
- Cell Cycle, Cell Line, DNA-Binding Proteins metabolism, G2 Phase, Gene Silencing, HeLa Cells, Humans, Models, Biological, Mutation, RNA, Small Interfering metabolism, Virus Replication, Cullin Proteins metabolism, HIV-1 metabolism, Proteasome Endopeptidase Complex metabolism, vpr Gene Products, Human Immunodeficiency Virus metabolism
- Abstract
Many viruses subvert the host ubiquitin-proteasome system to optimize their life cycle. We recently documented such a mechanism for the human immunodeficiency virus type 1 Vpr protein, which promotes cell cycle arrest by recruiting the DCAF1 adaptor of the Cul4A-DDB1 ubiquitin ligase, a finding now confirmed by several groups. Here we examined the impact of Cul4A-DDB1(DCAF1) on Vpr stability. We show that the Vpr(Q65R) mutant, which is defective in DCAF1 binding, undergoes proteasome-mediated degradation at a higher rate than wild-type Vpr. DCAF1 overexpression stabilizes wild-type Vpr and leads to its cytoplasmic accumulation, whereas it has no effect on the Vpr(Q65R) mutant. Conversely, small interfering RNA-mediated silencing of DCAF1 decreases the steady state amount of the viral protein. Stabilization by DCAF1, which is conserved by Vpr species from human immunodeficiency virus type 2 and the SIVmac strain, results in increased G(2) arrest and requires the presence of DDB1, indicating that it occurs through assembly of Vpr with a functional Cul4A-DDB1(DCAF1) complex. Furthermore, in human immunodeficiency virus type 1-infected cells, the Vpr protein, issued from the incoming viral particle, is destabilized under DCAF1 or DDB1 silencing. Together with our previous findings, our data suggest that Cul4A-DDB1(DCAF1) acts at a dual level by providing Vpr with the equipment for the degradation of specific host proteins and by counter-acting its proteasome targeting by another cellular E3 ubiquitin ligase. This protection mechanism may represent an efficient way to optimize the activity of Vpr molecules that are delivered by the incoming virus before neosynthesis takes place. Targeting the Vpr-DCAF1 interaction might therefore present therapeutic interest.
- Published
- 2008
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26. Localization of HIV-1 Vpr to the nuclear envelope: impact on Vpr functions and virus replication in macrophages.
- Author
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Jacquot G, Le Rouzic E, David A, Mazzolini J, Bouchet J, Bouaziz S, Niedergang F, Pancino G, and Benichou S
- Subjects
- Active Transport, Cell Nucleus physiology, Cell Cycle, HIV-1 chemistry, HIV-1 metabolism, HeLa Cells, Humans, Two-Hybrid System Techniques, vpr Gene Products, Human Immunodeficiency Virus biosynthesis, vpr Gene Products, Human Immunodeficiency Virus genetics, vpr Gene Products, Human Immunodeficiency Virus metabolism, Cell Nucleus virology, HIV-1 physiology, Macrophages virology, Nuclear Envelope metabolism, Virus Replication, vpr Gene Products, Human Immunodeficiency Virus physiology
- Abstract
Background: HIV-1 Vpr is a dynamic protein that primarily localizes in the nucleus, but a significant fraction is concentrated at the nuclear envelope (NE), supporting an interaction between Vpr and components of the nuclear pore complex, including the nucleoporin hCG1. In the present study, we have explored the contribution of Vpr accumulation at the NE to the Vpr functions, including G2-arrest and pro-apoptotic activities, and virus replication in primary macrophages., Results: In order to define the functional role of Vpr localization at the NE, we have characterized a set of single-point Vpr mutants, and selected two new mutants with substitutions within the first alpha-helix of the protein, Vpr-L23F and Vpr-K27M, that failed to associate with hCG1, but were still able to interact with other known relevant host partners of Vpr. In mammalian cells, these mutants failed to localize at the NE resulting in a diffuse nucleocytoplasmic distribution both in HeLa cells and in primary human monocyte-derived macrophages. Other mutants with substitutions in the first alpha-helix (Vpr-A30L and Vpr-F34I) were similarly distributed between the nucleus and cytoplasm, demonstrating that this helix contains the determinants required for localization of Vpr at the NE. All these mutations also impaired the Vpr-mediated G2-arrest of the cell cycle and the subsequent cell death induction, indicating a functional link between these activities and the Vpr accumulation at the NE. However, this localization is not sufficient, since mutations within the C-terminal basic region of Vpr (Vpr-R80A and Vpr-R90K), disrupted the G2-arrest and apoptotic activities without altering NE localization. Finally, the replication of the Vpr-L23F and Vpr-K27M hCG1-binding deficient mutant viruses was also affected in primary macrophages from some but not all donors., Conclusion: These results indicate that the targeting of Vpr to the nuclear pore complex may constitute an early step toward Vpr-induced G2-arrest and subsequent apoptosis; they also suggest that Vpr targeting to the nuclear pore complex is not absolutely required, but can improve HIV-1 replication in macrophages.
- Published
- 2007
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27. Regulated degradation of the HIV-1 Vpu protein through a betaTrCP-independent pathway limits the release of viral particles.
- Author
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Estrabaud E, Le Rouzic E, Lopez-Vergès S, Morel M, Belaïdouni N, Benarous R, Transy C, Berlioz-Torrent C, and Margottin-Goguet F
- Subjects
- Animals, Cell Cycle Proteins metabolism, Cell Line, Chlorocebus aethiops, F-Box Proteins metabolism, HIV-1 pathogenicity, Human Immunodeficiency Virus Proteins, Humans, Molecular Sequence Data, Phosphorylation, Ubiquitin metabolism, cdc25 Phosphatases metabolism, Gene Expression Regulation, Viral, HIV-1 genetics, Viral Regulatory and Accessory Proteins metabolism, Virus Replication physiology, beta-Transducin Repeat-Containing Proteins genetics, beta-Transducin Repeat-Containing Proteins metabolism
- Abstract
Viral protein U (Vpu) of HIV-1 has two known functions in replication of the virus: degradation of its cellular receptor CD4 and enhancement of viral particle release. Vpu binds CD4 and simultaneously recruits the betaTrCP subunit of the SCF(betaTrCP) ubiquitin ligase complex through its constitutively phosphorylated DS52GXXS56 motif. In this process, Vpu was found to escape degradation, while inhibiting the degradation of betaTrCP natural targets such as beta-catenin and IkappaBalpha. We further addressed this Vpu inhibitory function with respect to the degradation of Emi1 and Cdc25A, two betaTrCP substrates involved in cell-cycle progression. In the course of these experiments, we underscored the importance of a novel phosphorylation site in Vpu. We show that, especially in cells arrested in early mitosis, Vpu undergoes phosphorylation of the serine 61 residue, which lies adjacent to the betaTrCP-binding motif. This phosphorylation event triggers Vpu degradation by a betaTrCP-independent process. Mutation of Vpu S61 in the HIV-1 provirus extends the half-life of the protein and significantly increases the release of HIV-1 particles from HeLa cells. However, the S61 determinant of regulated Vpu turnover is highly conserved within HIV-1 isolates. Altogether, our results highlight a mechanism where differential phosphorylation of Vpu determines its fate as an adaptor or as a substrate of distinct ubiquitin ligases. Conservation of the Vpu degradation determinant, despite its negative effect on virion release, argues for a role in overall HIV-1 fitness.
- Published
- 2007
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28. RASSF1C, an isoform of the tumor suppressor RASSF1A, promotes the accumulation of beta-catenin by interacting with betaTrCP.
- Author
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Estrabaud E, Lassot I, Blot G, Le Rouzic E, Tanchou V, Quemeneur E, Daviet L, Margottin-Goguet F, and Benarous R
- Subjects
- Amino Acid Motifs, Cell Nucleus metabolism, Cytoplasm metabolism, Gene Silencing, HeLa Cells, Humans, Protein Binding, RNA, Small Interfering genetics, Tumor Suppressor Proteins genetics, beta Catenin antagonists & inhibitors, beta Catenin biosynthesis, beta Catenin genetics, Tumor Suppressor Proteins metabolism, beta Catenin metabolism, beta-Transducin Repeat-Containing Proteins metabolism
- Abstract
The Ras-association domain family 1 (RASSF1) gene has seven different isoforms; isoform A is a tumor-suppressor gene (RASSF1A). The promoter of RASSF1A is inactivated in many cancers, whereas the expression of another major isoform, RASSF1C, is not affected. Here, we show that RASSF1C, but not RASSF1A, interacts with betaTrCP. Binding of RASSF1C to betaTrCP involves serine 18 and serine 19 of the SS(18)GYXS(19) motif present in RASSF1C but not in RASSF1A. This motif is reminiscent of the canonical phosphorylation motif recognized by betaTrCP; however, surprisingly, the association between RASSF1C and betaTrCP does not occur via the betaTrCP substrate binding domain, the WD40 repeats. Overexpression of RASSF1C, but not of RASSF1A, resulted in accumulation and transcriptional activation of the beta-catenin oncogene, due to inhibition of its betaTrCP-mediated degradation. Silencing of RASSF1A by small interfering RNA was sufficient for beta-catenin to accumulate, whereas silencing of both RASSF1A and RASSF1C had no effect. Thus, RASSF1A and RASSF1C have opposite effects on beta-catenin degradation. Our results suggest that RASSF1C expression in the absence of RASSF1A could play a role in tumorigenesis.
- Published
- 2007
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29. Human immunodeficiency virus type 1 KK26-27 matrix mutants display impaired infectivity, circularization and integration but not nuclear import.
- Author
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Mannioui A, Nelson E, Schiffer C, Felix N, Le Rouzic E, Benichou S, Gluckman JC, and Canque B
- Subjects
- Cell Cycle, Cell Line, Chromatin metabolism, DNA, Viral metabolism, Gene Products, gag genetics, HIV Antigens genetics, HIV-1 metabolism, Humans, Mutation, T-Lymphocytes virology, Viral Proteins genetics, Virus Integration, Virus Replication, gag Gene Products, Human Immunodeficiency Virus, Gene Products, gag physiology, HIV Antigens physiology, HIV-1 physiology, Viral Proteins physiology
- Abstract
We analyzed the role of human immunodeficiency virus (HIV)-1 matrix protein (MA) during the virus replication afferent phase. Single-round infection of H9 T lymphocytes showed that the combined mutation of MA Lys residues 26-27 in MA reported nuclear localization signal (NLS)-1 impaired infectivity, abrogated 2-LTR-circle formation and significantly reduced integration. However, the mutation did not affect viral DNA docking to chromatin in either interphasic or mitotic cells, indicating that MA N-terminal basic domain should not represent a major determinant of HIV-1 nuclear import in T lymphocytes. These data point to a previously unreported role of MA in the late, post-chromatin-binding, afferent phase of HIV-1 replication cycle.
- Published
- 2005
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30. The Vpr protein from HIV-1: distinct roles along the viral life cycle.
- Author
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Le Rouzic E and Benichou S
- Subjects
- Gene Products, vpr chemistry, Gene Products, vpr genetics, Genes, vpr, HIV-1 genetics, HIV-1 pathogenicity, Humans, Models, Molecular, vpr Gene Products, Human Immunodeficiency Virus, Gene Products, vpr metabolism, HIV-1 physiology, Virus Replication
- Abstract
The genomes of human and simian immunodeficiency viruses (HIV and SIV) encode the gag, pol and env genes and contain at least six supplementary open reading frames termed tat, rev, nef, vif, vpr, vpx and vpu. While the tat and rev genes encode regulatory proteins absolutely required for virus replication, nef, vif, vpr, vpx and vpu encode for small proteins referred to "auxiliary" (or "accessory"), since their expression is usually dispensable for virus growth in many in vitro systems. However, these auxiliary proteins are essential for viral replication and pathogenesis in vivo. The two vpr- and vpx-related genes are found only in members of the HIV-2/SIVsm/SIVmac group, whereas primate lentiviruses from other lineages (HIV-1, SIVcpz, SIVagm, SIVmnd and SIVsyk) contain a single vpr gene. In this review, we will mainly focus on vpr from HIV-1 and discuss the most recent developments in our understanding of Vpr functions and its role during the virus replication cycle.
- Published
- 2005
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- View/download PDF
31. Vpr-mediated incorporation of UNG2 into HIV-1 particles is required to modulate the virus mutation rate and for replication in macrophages.
- Author
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Chen R, Le Rouzic E, Kearney JA, Mansky LM, and Benichou S
- Subjects
- Blotting, Western, Catalysis, Cell Line, DNA Glycosylases metabolism, DNA Repair, Glutathione Transferase metabolism, HeLa Cells, Humans, Leukocytes, Mononuclear metabolism, Macrophages metabolism, Monocytes virology, Plasmids metabolism, Protein Binding, Recombinant Fusion Proteins metabolism, Retroviridae genetics, Time Factors, Transfection, Two-Hybrid System Techniques, Uracil-DNA Glycosidase, DNA Glycosylases genetics, DNA Replication, Genes, vpr, HIV-1 metabolism, Macrophages virology, Mutation, Virus Replication
- Abstract
Human immunodeficiency virus type 1 is able to infect nondividing cells, such as macrophages, and the viral Vpr protein has been shown to participate in this process. Here, we investigated the impact of the recruitment into virus particles of the nuclear form of uracil DNA glycosylase (UNG2), a cellular DNA repair enzyme, on the virus mutation rate and on replication in macrophages. We demonstrate that the interaction of Vpr with UNG2 led to virion incorporation of a catalytically active enzyme that is directly involved with Vpr in modulating the virus mutation rate. The lack of UNG in virions during virus replication in primary monocyte-derived macrophages further exacerbated virus mutant frequencies to an 18-fold increase compared with the 4-fold increase measured in actively dividing cells. Because the presence of UNG is also critical for efficient infection of macrophages, these observations extend the role of Vpr to another early step of the virus life cycle, e.g. viral DNA synthesis, that is essential for replication of human immunodeficiency virus type 1 in nondividing cells.
- Published
- 2004
- Full Text
- View/download PDF
32. Influence of reverse transcriptase variants, drugs, and Vpr on human immunodeficiency virus type 1 mutant frequencies.
- Author
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Mansky LM, Le Rouzic E, Benichou S, and Gajary LC
- Subjects
- Amino Acid Substitution, HIV-1 drug effects, Thioguanine pharmacology, vpr Gene Products, Human Immunodeficiency Virus, Anti-HIV Agents pharmacology, Gene Products, vpr genetics, HIV Reverse Transcriptase genetics, HIV-1 genetics, Mutation
- Abstract
The evolution of drug resistance is a major complication of human immunodeficiency virus type 1 (HIV-1) chemotherapy. HIV-1 reverse transcriptase (RT) is a major target of antiretroviral therapy and ultimately the target of drug resistance mutations. Previous studies have indicated that drug-resistant HIV-1 RTs can alter HIV-1 mutant frequencies. In this study, we have tested a panel of HIV-1 RT variants for their ability to influence virus mutant frequencies. The RT variants tested included drug-resistant RT variants as well as other variants analyzed in enzyme fidelity studies with the lacZalpha gene as a mutation target and/or implicated as being important for enzyme fidelity by structural studies. Combinations of mutations that alone had a statistically significant influence on virus mutant frequencies resulted in different mutant frequency phenotypes. Furthermore, when virus replication occurred in the presence of drugs [e.g., 3'-azido-3'-deoxythymidine, (-)2/,3'-dideoxy-3'-thiacytidine, hydroxyurea, thymidine, or thioguanine] with selected RT variants, virus mutant frequencies increased. Similarly, Vpr variants deficient for binding to the uracil DNA glycosylase repair enzyme were observed to influence HIV-1 virus mutant frequencies when tested alone or in combination with RT variants. In summary, these observations indicate that HIV-1 mutant frequencies can significantly change by single amino acid substitutions in RT and that these effects can be altered by additional mutations in RT, by drugs, and/or by expression of Vpr variants. Such altered virus mutant frequencies could impact HIV-1 dynamics and evolution in small population sizes.
- Published
- 2003
- Full Text
- View/download PDF
33. Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1.
- Author
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Le Rouzic E, Mousnier A, Rustum C, Stutz F, Hallberg E, Dargemont C, and Benichou S
- Subjects
- Active Transport, Cell Nucleus physiology, Fluorescence Recovery After Photobleaching, Gene Products, vpr genetics, Genes, Reporter, Genes, myc, Green Fluorescent Proteins, HeLa Cells, Humans, Luminescent Proteins genetics, Luminescent Proteins metabolism, Protein Binding, Protein Structure, Secondary, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Two-Hybrid System Techniques, vpr Gene Products, Human Immunodeficiency Virus, Gene Products, vpr metabolism, HIV-1 metabolism, Nuclear Envelope metabolism, Nuclear Pore Complex Proteins metabolism, Nuclear Proteins metabolism, Nucleocytoplasmic Transport Proteins
- Abstract
The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast two-hybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.
- Published
- 2002
- Full Text
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34. Differential nucleocytoplasmic shuttling of beta-arrestins. Characterization of a leucine-rich nuclear export signal in beta-arrestin2.
- Author
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Scott MG, Le Rouzic E, Périanin A, Pierotti V, Enslen H, Benichou S, Marullo S, and Benmerah A
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Arrestins chemistry, Binding Sites, Green Fluorescent Proteins, HeLa Cells, Humans, Kinetics, Leucine, Luminescent Proteins metabolism, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3, Mitogen-Activated Protein Kinases metabolism, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Isoforms chemistry, Protein Isoforms metabolism, Recombinant Fusion Proteins metabolism, Recombinant Proteins metabolism, Signal Transduction, Transfection, beta-Arrestins, Active Transport, Cell Nucleus physiology, Arrestins metabolism, Cell Nucleus metabolism, Cytoplasm metabolism, MAP Kinase Signaling System physiology, Saccharomyces cerevisiae metabolism
- Abstract
beta-arrestins (betaarrs) are two highly homologous proteins that uncouple G protein-coupled receptors from their cognate G proteins, serve as adaptor molecules linking G protein-coupled receptors to clathrin-coat components (AP-2 complex and clathrin), and act as scaffolding proteins for ERK1/2 and JNK3 cascades. A striking difference between the two betaarrs (betaarr1 and betaarr2) is that betaarr1 is evenly distributed throughout the cell, whereas betaarr2 shows an apparent cytoplasmic localization at steady state. Here, we investigate the molecular determinants underlying this differential distribution. betaarr2 is constitutively excluded from the nucleus by a leptomycin B-sensitive pathway because of the presence of a classical leucine-rich nuclear export signal in its C terminus (L395/L397) that is absent in betaarr1. In addition, using a nuclear import assay in yeast we showed that betaarr2 is actively imported into the nucleus, suggesting that betaarr2 undergoes constitutive nucleocytoplasmic shuttling. In cells expressing betaarr2, JNK3 is mostly cytosolic. A point mutation of the nuclear export signal (L395A) in betaarr2, which was sufficient to redistribute betaarr2 from the cytosol to the nucleus, also caused the nuclear relocalization of JNK3. These data indicate that the nucleocytoplasmic shuttling of betaarr2 controls the subcellular distribution of JNK3.
- Published
- 2002
- Full Text
- View/download PDF
35. Alteration of C-MYB DNA binding to cognate responsive elements in HL-60 variant cells.
- Author
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Gaillard C, Le Rouzic E, Créminon C, and Perbal B
- Subjects
- Blotting, Western, Cell Differentiation drug effects, Cell Division, Cloning, Molecular, DNA, Neoplasm genetics, DNA-Binding Proteins metabolism, Electrophoretic Mobility Shift Assay methods, Gene Expression Regulation, Neoplastic, Genes, myb, HL-60 Cells pathology, Humans, Monocytes cytology, Monocytes drug effects, Tetradecanoylphorbol Acetate pharmacology, HL-60 Cells metabolism, Neoplasm Proteins metabolism, Proto-Oncogene Proteins c-myb metabolism
- Abstract
Aims: To establish whether the MYB protein expressed in HL-60 variant cells, which are cells resistant to 12-O-tetradecanoylphorbol-13-acetate (TPA) induced differentiation, is able to bind MYB recognition elements (MREs) involved in the transcriptional regulation of myb target genes. In addition, to determine whether alterations in the binding of the MYB protein to MREs affects HL-60 cell proliferation and differentiation., Methods: Nuclear extracts of HL-60 variant cells exhibiting different degrees of resistance to TPA induced monocytic differentiation were used in electrophoretic mobility shift experiments (EMSAs), bandshift experiments performed with labelled oliogonucleotides containing the MYB consensus binding sequences., Results: The MYB protein contained in nuclear extracts from HL-60 variant cells did not bind efficiently to the MYB recognition elements identified in the mim-1 and PR264 promoters. Molecular cloning of the myb gene and analysis of the MYB protein expressed in the HL-60 variant cells established that the lack of binding did not result from a structural alteration of MYB in these cells. The lack of MRE binding did not abrogate the ability of variant HL-60s to proliferate and to undergo differentiation. Furthermore, the expression of the PR264/SC35 splicing factor was not affected as a result of the altered MYB DNA binding activity., Conclusions: Because the MYB protein expressed in HL-60 variant cells did not appear to be structurally different from the MYB protein expressed in parental HL-60 cells, it is possible that the HL-60 variant cells contain a MYB binding inhibitory factor (MBIF) that interferes with MYB binding on MREs. The increased proliferation rate of HL-60 variant cells and their reduced serum requirement argues against the need for direct MYB binding in the regulation of cell growth.
- Published
- 2002
- Full Text
- View/download PDF
36. Alterations of the MDV oncogenic regions in an MDV transformed lymphoblastoid cell line.
- Author
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Le Rouzic E, Thoraval P, Afanassieff M, Cherel Y, Dambrine G, and Perbal B
- Subjects
- Animals, Chickens, Genes, myb, Genome, Viral, Herpesvirus 2, Gallid pathogenicity, Nuclear Proteins genetics, Oncogenes, Peptide Fragments genetics, Polymerase Chain Reaction methods, Protein Precursors genetics, Trans-Activators genetics, Transforming Growth Factor beta1, Tumor Cells, Cultured, Virulence genetics, Cell Transformation, Viral genetics, Herpesvirus 2, Gallid genetics, Lymphoma, T-Cell virology, Marek Disease virology, Viral Proteins
- Abstract
Aims: Lymphoblastoid cell lines derived from Marek's disease virus (MDV) induced tumours have served as models of MDV latency and transformation. They are stable and can be cultured with no detectable MDV genomic alterations upon repeated passaging. An MDV transformed lymphoblastoid T cell line (T9 cell line) has been reported to contain a disrupted MDV BamHI-H fragment and a Rous associated virus insertional activation of the c-myb protooncogene. In an attempt to define the respective participation of c-myb and MDV in the transformed phenotype of T9 cells, an analysis of MDV oncogenic sequences (BamHI-H, BamHI-A, and EcoQ fragments) was performed in these cells., Methods: Using two different passages of the T9 cell line (late and early passages), the organisation of the MDV oncogenic regions and their expression in these cells were analysed. In vivo assessment of the oncogenicity of the virus contained within these cells was assessed by injecting them into 1 day old chickens., Results: In T9 cells maintained in culture for up to six months (late T9), the MDV ICP4 gene was disrupted, whereas the meq gene was actively transcribed. The alterations of the MDV genome in these cells correlated with the inability of the virus to induce the classic signs of Marek's disease in 1 day old chickens. However, early T9 cells submitted to a limited number of passages induced classic MDV pathogenicity, as efficiently as the MDV control cell line (T5), and did not show gross structural changes in the oncogenic MDV sequences., Conclusions: Although the expression pattern of the MDV oncogenes in early T9 cells was identical to the one reported for other MDV transformed cells, longterm culture of an MDV transformed cell line containing a RAV insertional activation of the c-myb protooncogene led to the disruption of the MDV BamHI-H and BamHI-A oncogenic regions. In the late T9 cells MEQ was the only detected MDV oncoprotein. These results suggest that in the late T9 cells the truncated MYB protein compensates for the loss of MDV oncoproteins and reinforce the possibility that MEQ and MYB cooperate in the maintenance of the transformed state and the tumorigenic potential of these cells.
- Published
- 2002
- Full Text
- View/download PDF
37. Characterization of the nuclear import pathway for HIV-1 integrase.
- Author
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Depienne C, Mousnier A, Leh H, Le Rouzic E, Dormont D, Benichou S, and Dargemont C
- Subjects
- Biological Transport, HeLa Cells, Humans, Virus Replication, Cell Nucleus metabolism, HIV Infections virology, HIV Integrase metabolism, HIV-1 physiology
- Abstract
The karyophilic properties of the human immunodeficiency virus, type I (HIV-1) pre-integration complex (PIC) allow the virus to infect non-dividing cells. To better understand the mechanisms responsible for nuclear translocation of the PIC, we investigated nuclear import of HIV-1 integrase (IN), a PIC-associated viral enzyme involved in the integration of the viral genome in the host cell DNA. Accumulation of HIV-1 IN into nuclei of digitonin-permeabilized cells does not result from passive diffusion but rather from an active transport that occurs through the nuclear pore complexes. HIV-1 IN is imported by a saturable mechanism, implying that a limiting cellular factor is responsible for this process. Although IN has been previously proposed to contain classical basic nuclear localization signals, we found that nuclear accumulation of IN does not involve karyopherins alpha, beta1, and beta2-mediated pathways. Neither the non-hydrolyzable GTP analog, guanosine 5'-O-(thiotriphosphate), nor the GTP hydrolysis-deficient Ran mutant, RanQ69L, significantly affects nuclear import of IN, which depends instead on ATP hydrolysis. Therefore these results support the idea that IN import is not mediated by members of the karyopherin beta family. More generally, in vitro nuclear import of IN does not require addition of cytosolic factors, suggesting that cellular factor(s) involved in this active but atypical pathway process probably remain associated with the nuclear compartment or the nuclear pore complexes from permeabilized cells.
- Published
- 2001
- Full Text
- View/download PDF
38. Interaction of human immunodeficiency virus type 1 Vpr with the HHR23A DNA repair protein does not correlate with multiple biological functions of Vpr.
- Author
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Mansky LM, Preveral S, Le Rouzic E, Bernard LC, Selig L, Depienne C, Benarous R, and Benichou S
- Subjects
- Amino Acid Substitution, Animals, COS Cells, Cell Cycle, Cell Nucleus metabolism, DNA Repair Enzymes, Genetic Vectors, HeLa Cells, Humans, Transfection, Virus Replication, vpr Gene Products, Human Immunodeficiency Virus, DNA Repair, DNA-Binding Proteins metabolism, Gene Products, vpr metabolism, HIV-1 metabolism, Retroviridae Proteins metabolism
- Abstract
The virion-associated Vpr protein of human immunodeficiency virus type 1 (HIV-1) alters cell cycle progression from the G2 phase, influences the virus in vivo mutation rate, and participates in the nuclear translocation of viral DNA. While many Vpr-interacting proteins have been identified, the functional relevance of these interactions remains to be thoroughly documented. We have explored the contribution of the interaction of HIV-1 Vpr with HHR23A, a cellular protein implicated in DNA repair, to the known phenotypes of Vpr. The association of Vpr with HHR23A required the core region of Vpr, which encompasses the two alpha-helical structures of the protein. No binding of HHR23A was detected with the Vpr and Vpx proteins of other primate lentiviruses. HIV-1 Vpr variants containing single amino acid substitutions in each alpha-helix and deficient for binding to HHR23A were isolated. The functional characterization of these Vpr variants indicated that binding to HHR23A did not correlate with the ability of Vpr to induce cell cycle arrest, even though it was previously proposed that HHR23A is a mediator of the Vpr-induced G2 arrest. Also, the Vpr-HHR23A interaction did not influence the HIV-1 in vivo mutation rate. Finally, Vpr and HHR23A are both localized in the nucleus, but no correlation was observed between the nuclear targeting of Vpr and the interaction with HHR23A. Further analysis is needed to determine the functional role(s) of the Vpr-HHR23A association during the HIV-1 life cycle., (Copyright 2001 Academic Press.)
- Published
- 2001
- Full Text
- View/download PDF
39. HIV auxiliary proteins: an interface between the virus and the host.
- Author
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Janvier K, Petit C, Le Rouzic E, Schwartz O, and Benichou S
- Subjects
- CD4-Positive T-Lymphocytes immunology, CD4-Positive T-Lymphocytes virology, HIV-1 metabolism, Humans, Virulence, Virus Replication, HIV Infections virology, HIV-1 pathogenicity, Viral Proteins metabolism
- Published
- 2000
40. Characterization of SRp46, a novel human SR splicing factor encoded by a PR264/SC35 retropseudogene.
- Author
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Soret J, Gattoni R, Guyon C, Sureau A, Popielarz M, Le Rouzic E, Dumon S, Apiou F, Dutrillaux B, Voss H, Ansorge W, Stévenin J, and Perbal B
- Subjects
- 3T3 Cells, Amino Acid Sequence, Animals, Base Sequence, COS Cells, Cell Line, Chlorocebus aethiops, DNA, Complementary, Gene Expression, HL-60 Cells, HeLa Cells, Humans, Mice, Molecular Sequence Data, Sequence Homology, Amino Acid, Sequence Homology, Nucleic Acid, Serine-Arginine Splicing Factors, Alternative Splicing, Phosphoproteins genetics, Pseudogenes
- Abstract
The highly conserved SR family contains a growing number of phosphoproteins acting as both essential and alternative splicing factors. In this study, we have cloned human genomic and cDNA sequences encoding a novel SR protein designated SRp46. Nucleotide sequence analyses have revealed that the SRp46 gene corresponds to an expressed PR264/SC35 retropseudogene. As a result of mutations and amplifications, the SRp46 protein significantly differs from the PR264/SC35 factor, mainly at the level of its RS domain. Northern and Western blot analyses have established that SRp46 sequences are expressed at different levels in several human cell lines and normal tissues, as well as in simian cells. In contrast, sequences homologous to SRp46 are not present in mice. In vitro splicing studies indicate that the human SRp46 recombinant protein functions as an essential splicing factor in complementing a HeLa cell S100 extract deficient in SR proteins. In addition, complementation analyses performed with beta-globin or adenovirus E1A transcripts and different splicing-deficient extracts have revealed that SRp46 does not display the same activity as PR264/SC35. These results demonstrate, for the first time, that an SR splicing factor, which represents a novel member of the SR family, is encoded by a functional retropseudogene.
- Published
- 1998
- Full Text
- View/download PDF
41. Retroviral insertional activation of the c-myb proto-oncogene in a Marek's disease T-lymphoma cell line.
- Author
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Le Rouzic E and Perbal B
- Subjects
- Alleles, DNA, Viral analysis, Deoxyribonuclease BamHI metabolism, Gene Expression, Gene Rearrangement, Lymphoma, T-Cell pathology, Proto-Oncogene Proteins c-myb, RNA, Messenger, Transfection, Tumor Cells, Cultured, Virus Integration, Avian Leukosis Virus genetics, Herpesvirus 2, Gallid genetics, Proto-Oncogene Proteins genetics, Trans-Activators genetics
- Abstract
Marek's disease virus (MDV) is an avian herpesvirus that causes, in chickens, a lymphoproliferative disease characterized by malignant transformation of T lymphocytes. The rapid onset of polyclonal tumors indicates the existence of MDV-encoded oncogenic products. However, the molecular basis of MDV-induced lymphoproliferative disease and latency remains largely unclear. Several lines of evidence suggest that MDV and Rous-associated virus (RAV) might cooperate in the development of B-cell lymphomas induced by RAV. Our present results indicate for the first time that MDV and RAV might also act synergistically in the development of T-cell lymphomas. We report an example of an MDV-transformed T-lymphoblastoid cell line (T9) expressing high levels of a truncated C-MYB protein as a result of RAV integration within one c-myb allele. The chimeric RAV-c-myb mRNA species initiated in the 5' long terminal repeat of RAV are deprived of sequences corresponding to c-myb exons 1 to 3. The attenuation of MDV oncogenicity has been strongly related to structural changes in the MDV BamHI-D and BamHI-H DNA fragments. We have established that both DNA restriction fragments are rearranged in the T9 MDV-transformed cells. Our results suggest that retroviral insertional activation of the c-myb proto-oncogene is a critical factor involved in the maintenance of the transformed phenotype and the tumorigenic potential of this T-lymphoma cell line.
- Published
- 1996
- Full Text
- View/download PDF
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