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Didier Trono
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office(s):
SV3805
phone(s): [+41 21 69] 31751,31634,31761
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MISSION
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Our laboratory studies molecular events that govern the replication of human immunodeficiency virus (HIV), with a particular interest for steps that lie at the interface between the pathogen and its target and thereby reveal the virus ability to exploit the host cell machinery. The goals of these studies are multiple. First, to shed light on HIV pathogenesis. Second, to suggest new avenues for the development of antiviral therapies. Third, to gain insights into evolutionary processes that shape the genetic information of all eukaryotes from yeast to mammals, in which retroelements likely play a central role. And finally, to explore the potential of HIV-derived lentiviral vectors for both experimental and therapeutic purposes. Functions and mechanisms of action of the HIV Nef protein Scientific objective : Identify the roles and dissect the mechanisms of action of a protein that alters the biology of the cell to the benefit of viral replication. The nef early gene of HIV encodes an importance virulence factor, which exerts pleiomorphic effects on the infected cell. Amongst these, Nef downregulates the cell surface expression of CD4, the primary viral receptor, thereby preserving the infectivity of HIV virions. We previously demonstrated that Nef acts by connecting CD4 first with clathrin-coated pits and then with the COP1 coatomer, thereby targeting the receptor for sequential accelerated endocytosis and lysosomal targeting. Our recent experiments indicate that the small GTPase ARF1 is the immediate downstream partner of Nef for CD4 lysosomal degradation, and further serves as a general regulator of endosomal transport. Our current efforts aim at dissecting the biochemical modalities of this event.
Early events in HIV infection
Scientific objective : unravel the steps that lead HIV from its site of entry into the cell to the chromosome in which it integrates. A key role for virion cholesterol in HIV entry The membrane of HIV-1 virions contains high levels of cholesterol and sphingomyelin, an enrichment that is explained by the preferential budding of the virus through raft microdomains of the plasma membrane. We found that the pharmacological depletion of cholesterol from HIV-1 virions abrogates their infectivity. In contrast, this treatment only has a mild effect on the infectiousness of particles pseudotyped with the G envelope of vesicular stomatitis virus. Cholesterol-depleted HIV-1virions can still bind target cells, yet are markedly defective for internalization. The cholesterol present in the HIV-1 membrane thus plays a prominent role in the fusion process that is key to viral entry. This suggests that drugs capable of disturbing the lipid composition of virions could serve as a basis for the development of microbicides.
An intracellular innate antiviral response that inteferes with the immediate early phase of HIV infection
We discovered that the nuclear proteins PML and INI-1 are recruited onto the incoming HIV nucleoprotein complex shortly after its release into the cytoplasm. We further revealed that this process represents at least in part an antiviral response that interferes with the immediate early steps of HIV infection. Our most recent analyses link this phenomenon with the proteasome-mediated degradation of HIV preintegration complexes, and further suggest that the resistance of human cell to some oncoretroviruses stems from a similar mechanism. Our prediction is that HIV not only overcomes but also ultimately subverts this response by hijacking cellular machines linked to chromatin remodeling, in order to tether the preintegration complex to transcriptionally active regions ofthe genome. The recent observation that HIV integration favors expressed genes supports such a model.
Induction of HIV permissiveness in resting T lymphocytes
Although CD4 +T lymphocytes are the main in vivo targets of HIV, their metabolic and cell cycle status limits their susceptibility to the virus. While in fully quiescent T lymphocytes the reverse transcription and nuclear import of HIV and derived vectors are impaired leading to an abortive infection, various stimuli can induce a state of virus permissiveness. We studied the modalities by which IL-7, an important controller of T cell homeostasis, exerts this effect. IL-7-exposed cord blood (CB) T lymphocytes proliferated and were efficiently transduced by HIV-derived vectors. In contrast, similarly treated adult peripheral blood (PB) T lymphocytes failed to divide and only a subset of these cells became infectable. This HIV-sensitive subset is characterized by its progresion into the G 1b phase of the cell cycle, but not by its naive, memory or activation phenotype. Our studies further indicate that the IL-7-induced HIV susceptibility of T lymphocytes is independent from NFAT and PI-3 kinase and might instead proceed through the Jak/STAT5 pathway. We are currently attempting to define the minimal changes that render a resting T cell supportive of HIV replication.
Lentiviral vectors as experimental and therapeutic tools
Scientific objective : Develop lentiviral vectors for the genetic treatment of human hereditary and acquired disorders, and exploit the resources of this system for fundamental studies. We previously developed HIV-derived lentiviral vectors, which can mediate the efficient delivery, integration, and sustained long-term expression of transgenes both in vitro and in vivo. We are currently exploiting this tool towards both experimental and therapeutic ends. Using chronic granulomatous disease (CGD) as a model, we are exploring the potential of lentivectors for the genetic treatment of lympho-hematopoietic disorders via stem cell-based gene transfer. We find that transduction of human or murine hematopoietic stem cells (HSC) with vectors expressing a transgene from proper lineage-specific promoters can restrict transgene expression to neutrophils and macrophages, the targets in which the gp91 phox protein, defective in CGD patients, is normally expressed. Using the gp91 phox mouse knockout model, we have determined that these vectors can induce physiological levels of oxidase activity in white cells of animals transplanted with lentivector-transduced hematopoietic stem cells. Infectious challenges of these mice are in progress, to assess the potential therapeutic value of this approach.
Gene therapy is attractive for the treatment of liver diseases as well.
We have now demonstrated that HIV-derived lentivectors are extremely efficient in primary human hepatocytes, but less so in rodent hepatocytes. This species-specific restriction is particularly pronounced in murine hepatocytes and reflects a block in the immediate-early phase of infection. To pave the way to clinical studies, we have developped a methodology that allows very high rates of transduction through minimal in vitro manipulation, in which hepatocytes are kept in suspension and re-implanted within a few hours of harvest with a fully preserved engraftment potential. These results have immediate implications for the treatment of liver diseases via the transplantation of genetically modified hepatocytes, an approach that could be applied to a number of hereditary and acquired hepatic disorders, including hepatitis B and C.
Finally, we have been using the formidable potential of lentiviral vectors for experimental purposes.
In particular, we have been transducing primary human cells from various organs with cocktails of genes encoding for growth promoting factors, telomerase, blockers of apoptosis, and/or proteins susceptible to affect differentiation, in order to obtain interesting immortalized cell lines. Amongst a number of such lines derived from endothelium, muscle, skin, liver and other human tissues, conditionally immortalized hepatocytes exhibit most remarkable phenotypic characteristics, which very closely resemble those of their primary counterparts. This opens interesting perspectives for the development of a bioartificial liver and for the study of hepatitis viruses.
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BIOGRAPHY
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Né en 1956, Didier Trono obtient son diplôme à la Faculté de médecine de Genève en 1981, puis son doctorat l’année suivante. Il poursuit sa carrière comme chercheur, tout d’abord au Massachusetts General Hospital, puis devient postdoctoral fellow au Whitehead Institute for Biomedical Research du MIT, à Cambridge. Professeur assistant au Salk Institute for Biological Studies de La Jolla en 1990, il devient professeur associé en 1995. En 1997, il est nommé professeur ordinaire au Département de génétique et de microbiologie, à l’Université de Genève. Il prend la tête de ce département dès 2000 et, un an plus tard, préside la Section des sciences de base de la Faculté de médecine. Depuis 2004, il est deputy director du pôle national de compétences « frontières en génétique ».
Le professeur Trono a contribué de façon notoire à différents sujets de recherche, notamment la pathogénèse du virus HIV. Il a toujours su tirer de ses expériences une information biologique fondamentale, mais l’un des aboutissements majeurs de ses travaux s’est concrétisé dans l’exploitation du potentiel des vecteurs viraux dérivés du HIV comme outil expérimental ou thérapeutique.
Tiré du Communiqué de Presse du 16 septembre 2004 du Service de Presse et d'Information de l'EPFL.
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President 