Characterization of the interaction between the HIV-1 Gag structural polyprotein and the cellular ribosomal protein L7 and its implication in viral nucleic acid remodeling

Background: In HIV-1 infected cells, the integrated viral DNA is transcribed by the host cell machinery to generate the full length HIV-1 RNA (FL RNA) that serves as mRNA encoding for the Gag and GagPol precursors. Virion formation is orchestrated by Gag, and the current view is that a specific interaction between newly made Gag molecules and FL RNA initiates the process. This in turn would cause FL RNA dimerization by the NC domain of Gag (GagNC). However the RNA chaperoning activity of unprocessed Gag is low as compared to the mature NC protein. This prompted us to search for GagNC co-factors. Results: Here we report that RPL7, a major ribosomal protein involved in translation regulation, is a partner of Gag via its interaction with the NC domain. This interaction is mediated by the NC zinc fingers and the N- and C-termini of RPL7, respectively, but seems independent of RNA binding, Gag oligomerization and its interaction with the plasma membrane. Interestingly, RPL7 is shown for the first time to exhibit a potent DNA/RNA chaperone activity higher than that of Gag. In addition, Gag and RPL7 can function in concert to drive rapid nucleic acid hybridization. Conclusions: Our results show that GagNC interacts with the ribosomal protein RPL7 endowed with nucleic acid chaperone activity, favoring the notion that RPL7 could be a Gag helper chaperoning factor possibly contributing to the start of Gag assembly.Instituto de Estudios Inmunológicos y Fisiopatológico

Interaction de Gag(NC) du VIH-1 avec la protéine ribosomique RPL7 : caractérisation du complexe et rôle dans le cycle réplicatif

During the late stage of the viral cycle, the HIV-1 Gag polyprotein polymerize to form the viral particle and recruits the viral and cellular partners allowing the budding of infectious viruses. Moreover, by its nucleic acid chaperone activity Gag induce the dimerization and the encapsidation of the viral RNA. Several in vitro studies have shown that Gag has a low chaperone activity. We identified a cellular partner which, by being recruited by Gag, would be able to improve its chaperone activity. Among the identified partners, we were interested in the ribosomal protein RPL7. We characterized Gag-RPL7 complex and based on an in vitro test that follow the annealing of cTAR with dTAR, we showed that RPL7 has better nucleic acid chaperone activity than Gag and that the activity of the Gag-RPL7 complex was higher than each protein alone. Additionally, we characterized the annealing mechanism of cTAR / dTAR and showed that RPL7, Gag or Gag-RPL7 formed the cTAR / dTAR extended duplex in different ways. All these results allowed us to propose a model in which, once expressed, Gag would be able to recruit the RPL7 to boost its chaperone activity.A la fin du cycle viral, la polyprotéine Gag du VIH-1 polymérise afin de former la particule virale et recrute les partenaires viraux et cellulaires permettant le bourgeonnement de particules infectieuses. De plus, par son activité de chaperonne des acides nucléiques, elle induit la dimérisation et l’encapsidation de l’ARN viral. Plusieurs études in vitro ont montré que Gag possédait une faible activité de chaperonne. Nous avons donc cherché à identifier un partenaire cellulaire qui, en étant recruté par Gag, serait capable d’en améliorer l’activité. Parmi les partenaires identifiés, nous nous sommes intéressés à la protéine ribosomale RPL7. Nous avons caractérisé le complexe Gag-RPL7 et montré que le domaine NCp7 de Gag interagissait avec les parties N et C terminales de RPL7. En se basant sur un test in vitro consistant à suivre l’hybridation de cTAR avec dTAR, nous avons montré que la RPL7 possédait une activité chaperonne des acides nucléiques supérieure à celle de Gag et que l’activité du complexe Gag-RPL7 était supérieur à celle de chaque protéine. Enfin, nous avons déchiffré le mécanisme d’hybridation de cTAR/dTAR et montré que la RPL7, Gag ou Gag-RPL7, formaient l’hybride cTAR/dTAR suivant différentes voies. Tous ces résultats nous ont permis de proposer un modèle dans lequel, une fois exprimée, Gag serait capable de recruter la protéine RPL7 pour améliorer son activité de chaperonne

Interaction of HIV-1 Gag(NC) with ribosomal protein RPL7 : complex characterization and role in the replicative cycle

A la fin du cycle viral, la polyprotéine Gag du VIH-1 polymérise afin de former la particule virale et recrute les partenaires viraux et cellulaires permettant le bourgeonnement de particules infectieuses. De plus, par son activité de chaperonne des acides nucléiques, elle induit la dimérisation et l’encapsidation de l’ARN viral. Plusieurs études in vitro ont montré que Gag possédait une faible activité de chaperonne. Nous avons donc cherché à identifier un partenaire cellulaire qui, en étant recruté par Gag, serait capable d’en améliorer l’activité. Parmi les partenaires identifiés, nous nous sommes intéressés à la protéine ribosomale RPL7. Nous avons caractérisé le complexe Gag-RPL7 et montré que le domaine NCp7 de Gag interagissait avec les parties N et C terminales de RPL7. En se basant sur un test in vitro consistant à suivre l’hybridation de cTAR avec dTAR, nous avons montré que la RPL7 possédait une activité chaperonne des acides nucléiques supérieure à celle de Gag et que l’activité du complexe Gag-RPL7 était supérieur à celle de chaque protéine. Enfin, nous avons déchiffré le mécanisme d’hybridation de cTAR/dTAR et montré que la RPL7, Gag ou Gag-RPL7, formaient l’hybride cTAR/dTAR suivant différentes voies. Tous ces résultats nous ont permis de proposer un modèle dans lequel, une fois exprimée, Gag serait capable de recruter la protéine RPL7 pour améliorer son activité de chaperonne.During the late stage of the viral cycle, the HIV-1 Gag polyprotein polymerize to form the viral particle and recruits the viral and cellular partners allowing the budding of infectious viruses. Moreover, by its nucleic acid chaperone activity Gag induce the dimerization and the encapsidation of the viral RNA. Several in vitro studies have shown that Gag has a low chaperone activity. We identified a cellular partner which, by being recruited by Gag, would be able to improve its chaperone activity. Among the identified partners, we were interested in the ribosomal protein RPL7. We characterized Gag-RPL7 complex and based on an in vitro test that follow the annealing of cTAR with dTAR, we showed that RPL7 has better nucleic acid chaperone activity than Gag and that the activity of the Gag-RPL7 complex was higher than each protein alone. Additionally, we characterized the annealing mechanism of cTAR / dTAR and showed that RPL7, Gag or Gag-RPL7 formed the cTAR / dTAR extended duplex in different ways. All these results allowed us to propose a model in which, once expressed, Gag would be able to recruit the RPL7 to boost its chaperone activity

Interaction of HIV-1 Gag(NC) with ribosomal protein RPL7 : complex characterization and role in the replicative cycle

A la fin du cycle viral, la polyprotéine Gag du VIH-1 polymérise afin de former la particule virale et recrute les partenaires viraux et cellulaires permettant le bourgeonnement de particules infectieuses. De plus, par son activité de chaperonne des acides nucléiques, elle induit la dimérisation et l’encapsidation de l’ARN viral. Plusieurs études in vitro ont montré que Gag possédait une faible activité de chaperonne. Nous avons donc cherché à identifier un partenaire cellulaire qui, en étant recruté par Gag, serait capable d’en améliorer l’activité. Parmi les partenaires identifiés, nous nous sommes intéressés à la protéine ribosomale RPL7. Nous avons caractérisé le complexe Gag-RPL7 et montré que le domaine NCp7 de Gag interagissait avec les parties N et C terminales de RPL7. En se basant sur un test in vitro consistant à suivre l’hybridation de cTAR avec dTAR, nous avons montré que la RPL7 possédait une activité chaperonne des acides nucléiques supérieure à celle de Gag et que l’activité du complexe Gag-RPL7 était supérieur à celle de chaque protéine. Enfin, nous avons déchiffré le mécanisme d’hybridation de cTAR/dTAR et montré que la RPL7, Gag ou Gag-RPL7, formaient l’hybride cTAR/dTAR suivant différentes voies. Tous ces résultats nous ont permis de proposer un modèle dans lequel, une fois exprimée, Gag serait capable de recruter la protéine RPL7 pour améliorer son activité de chaperonne.During the late stage of the viral cycle, the HIV-1 Gag polyprotein polymerize to form the viral particle and recruits the viral and cellular partners allowing the budding of infectious viruses. Moreover, by its nucleic acid chaperone activity Gag induce the dimerization and the encapsidation of the viral RNA. Several in vitro studies have shown that Gag has a low chaperone activity. We identified a cellular partner which, by being recruited by Gag, would be able to improve its chaperone activity. Among the identified partners, we were interested in the ribosomal protein RPL7. We characterized Gag-RPL7 complex and based on an in vitro test that follow the annealing of cTAR with dTAR, we showed that RPL7 has better nucleic acid chaperone activity than Gag and that the activity of the Gag-RPL7 complex was higher than each protein alone. Additionally, we characterized the annealing mechanism of cTAR / dTAR and showed that RPL7, Gag or Gag-RPL7 formed the cTAR / dTAR extended duplex in different ways. All these results allowed us to propose a model in which, once expressed, Gag would be able to recruit the RPL7 to boost its chaperone activity

Linezolid Toxicity and Mitochondrial Susceptibility: A Novel Neurological Complication in a Lebanese Patient

The recent rise in the use of linezolid to treat a variety of resistant pathogens has uncovered many side effects. Some patients develop lactic acidosis, myelosuppression, optic or peripheral neuropathies and myopathies. We evaluated an elderly patient who presented to the Emergency Room with linezolid toxicity and a novel neurologic complication characterized by bilateral globi pallidi necrosis. Mitochondrial ribosome inhibition was described to be the predisposing factor. The patient belongs to the mitochondrial J1 haplotype known to be associated with side effects of the drug. We recommend based on the molecular profile of the illness pretreatment considerations and complication management

The nucleic acid chaperone activity of the HIV-1 Gag polyprotein is boosted by its cellular partner RPL7: a kinetic study

10.1093/nar/gkaa659Nucleic Acids Researc

Characterization of the interaction between the HIV-1 Gag structural polyprotein and the cellular ribosomal protein L7 and its implication in viral nucleic acid remodeling

Background: In HIV-1 infected cells, the integrated viral DNA is transcribed by the host cell machinery to generate the full length HIV-1 RNA (FL RNA) that serves as mRNA encoding for the Gag and GagPol precursors. Virion formation is orchestrated by Gag, and the current view is that a specific interaction between newly made Gag molecules and FL RNA initiates the process. This in turn would cause FL RNA dimerization by the NC domain of Gag (GagNC). However the RNA chaperoning activity of unprocessed Gag is low as compared to the mature NC protein. This prompted us to search for GagNC co-factors. Results: Here we report that RPL7, a major ribosomal protein involved in translation regulation, is a partner of Gag via its interaction with the NC domain. This interaction is mediated by the NC zinc fingers and the N- and C-termini of RPL7, respectively, but seems independent of RNA binding, Gag oligomerization and its interaction with the plasma membrane. Interestingly, RPL7 is shown for the first time to exhibit a potent DNA/RNA chaperone activity higher than that of Gag. In addition, Gag and RPL7 can function in concert to drive rapid nucleic acid hybridization. Conclusions: Our results show that GagNC interacts with the ribosomal protein RPL7 endowed with nucleic acid chaperone activity, favoring the notion that RPL7 could be a Gag helper chaperoning factor possibly contributing to the start of Gag assembly.Fil: El Mekdad, Hala. Centre National de la Recherche Scientifique; Francia. Université de Strasbourg; FranciaFil: Boutant, Emmanuel. Centre National de la Recherche Scientifique; Francia. Université de Strasbourg; FranciaFil: Karnib, Hassan. Université de Strasbourg; Francia. Centre National de la Recherche Scientifique; FranciaFil: Biedma, Marina Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; Argentina. Université de Strasbourg; Francia. Inserm; FranciaFil: Sharma, Kamal Kant. Centre National de la Recherche Scientifique; Francia. Université de Strasbourg; FranciaFil: Malytska, Iuliia. Université de Strasbourg; Francia. Centre National de la Recherche Scientifique; FranciaFil: Laumond, Géraldine. Université de Strasbourg; Francia. Inserm; FranciaFil: Roy, Marion. Université de Strasbourg; Francia. Centre National de la Recherche Scientifique; FranciaFil: Réal, Eléonore. Centre National de la Recherche Scientifique; Francia. Université de Strasbourg; FranciaFil: Paillart, Jean Christophe. Centre National de la Recherche Scientifique; Francia. Université de Strasbourg; FranciaFil: Moog, Christiane. Université de Strasbourg; Francia. Inserm; FranciaFil: Darlix, Jean Luc. Université de Strasbourg; Francia. Centre National de la Recherche Scientifique; FranciaFil: Mély, Yves. Université de Strasbourg; Francia. Centre National de la Recherche Scientifique; FranciaFil: de Rocquigny, Hugues. Université de Strasbourg; Francia. Centre National de la Recherche Scientifique; Franci