Publications by authors named "Alessia Zamborlini"

29 Publications

  • Page 1 of 1

The RanBP2/RanGAP1-SUMO complex gates β-arrestin2 nuclear entry to regulate the Mdm2-p53 signaling axis.

Oncogene 2021 Mar 1;40(12):2243-2257. Epub 2021 Mar 1.

Inserm, U1016, Institut Cochin, Paris, France.

Mdm2 antagonizes the tumor suppressor p53. Targeting the Mdm2-p53 interaction represents an attractive approach for the treatment of cancers with functional p53. Investigating mechanisms underlying Mdm2-p53 regulation is therefore important. The scaffold protein β-arrestin2 (β-arr2) regulates tumor suppressor p53 by counteracting Mdm2. β-arr2 nucleocytoplasmic shuttling displaces Mdm2 from the nucleus to the cytoplasm resulting in enhanced p53 signaling. β-arr2 is constitutively exported from the nucleus, via a nuclear export signal, but mechanisms regulating its nuclear entry are not completely elucidated. β-arr2 can be SUMOylated, but no information is available on how SUMO may regulate β-arr2 nucleocytoplasmic shuttling. While we found β-arr2 SUMOylation to be dispensable for nuclear import, we identified a non-covalent interaction between SUMO and β-arr2, via a SUMO interaction motif (SIM), that is required for β-arr2 cytonuclear trafficking. This SIM promotes association of β-arr2 with the multimolecular RanBP2/RanGAP1-SUMO nucleocytoplasmic transport hub that resides on the cytoplasmic filaments of the nuclear pore complex. Depletion of RanBP2/RanGAP1-SUMO levels result in defective β-arr2 nuclear entry. Mutation of the SIM inhibits β-arr2 nuclear import, its ability to delocalize Mdm2 from the nucleus to the cytoplasm and enhanced p53 signaling in lung and breast tumor cell lines. Thus, a β-arr2 SIM nuclear entry checkpoint, coupled with active β-arr2 nuclear export, regulates its cytonuclear trafficking function to control the Mdm2-p53 signaling axis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41388-021-01704-wDOI Listing
March 2021

A Genome-Wide CRISPR-Cas9 Screen Identifies the Dolichol-Phosphate Mannose Synthase Complex as a Host Dependency Factor for Dengue Virus Infection.

J Virol 2020 03 17;94(7). Epub 2020 Mar 17.

INSERM U944, CNRS UMR 7212, Genomes & Cell Biology of Disease Unit, Institut de Recherche Saint-Louis, Université de Paris, Hôpital Saint-Louis, Paris, France

Dengue virus (DENV) is a mosquito-borne flavivirus responsible for dengue disease, a major human health concern for which no specific therapies are available. Like other viruses, DENV relies heavily on the host cellular machinery for productive infection. In this study, we performed a genome-wide CRISPR-Cas9 screen using haploid HAP1 cells to identify host genes important for DENV infection. We identified DPM1 and -3, two subunits of the endoplasmic reticulum (ER) resident dolichol-phosphate mannose synthase (DPMS) complex, as host dependency factors for DENV and other related flaviviruses, such as Zika virus (ZIKV). The DPMS complex catalyzes the synthesis of dolichol-phosphate mannose (DPM), which serves as mannosyl donor in pathways leading to N-glycosylation, glycosylphosphatidylinositol (GPI) anchor biosynthesis, and C- or O-mannosylation of proteins in the ER lumen. Mutation in the DXD motif of DPM1, which is essential for its catalytic activity, abolished DPMS-mediated DENV infection. Similarly, genetic ablation of ALG3, a mannosyltransferase that transfers mannose to lipid-linked oligosaccharide (LLO), rendered cells poorly susceptible to DENV. We also established that in cells deficient for DPMS activity, viral RNA amplification is hampered and truncated oligosaccharides are transferred to the viral prM and E glycoproteins, affecting their proper folding. Overall, our study provides new insights into the host-dependent mechanisms of DENV infection and supports current therapeutic approaches using glycosylation inhibitors to treat DENV infection. Dengue disease, which is caused by dengue virus (DENV), has emerged as the most important mosquito-borne viral disease in humans and is a major global health concern. DENV encodes only few proteins and relies on the host cell machinery to accomplish its life cycle. The identification of the host factors important for DENV infection is needed to propose new targets for antiviral intervention. Using a genome-wide CRISPR-Cas9 screen, we identified DPM1 and -3, two subunits of the DPMS complex, as important host factors for the replication of DENV as well as other related viruses such as Zika virus. We established that DPMS complex plays dual roles during viral infection, both regulating viral RNA replication and promoting viral structural glycoprotein folding/stability. These results provide insights into the host molecules exploited by DENV and other flaviviruses to facilitate their life cycle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01751-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081898PMC
March 2020

The invariant arginine within the chromatin-binding motif regulates both nucleolar localization and chromatin binding of Foamy virus Gag.

Retrovirology 2018 07 11;15(1):48. Epub 2018 Jul 11.

CNRS UMR7212, Hôpital St Louis, Inserm U944, Institut Universitaire d'Hématologie, Université Paris Diderot, Sorbonne Paris Cité, Paris, France.

Background: Nuclear localization of Gag is a property shared by many retroviruses and retrotransposons. The importance of this stage for retroviral replication is still unknown, but studies on the Rous Sarcoma virus indicate that Gag might select the viral RNA genome for packaging in the nucleus. In the case of Foamy viruses, genome encapsidation is mediated by Gag C-terminal domain (CTD), which harbors three clusters of glycine and arginine residues named GR boxes (GRI-III). In this study we investigated how PFV Gag subnuclear distribution might be regulated.

Results: We show that the isolated GRI and GRIII boxes act as nucleolar localization signals. In contrast, both the entire Gag CTD and the isolated GRII box, which contains the chromatin-binding motif, target the nucleolus exclusively upon mutation of the evolutionary conserved arginine residue at position 540 (R540), which is a key determinant of FV Gag chromatin tethering. We also provide evidence that Gag localizes in the nucleolus during FV replication and uncovered that the viral protein interacts with and is methylated by Protein Arginine Methyltransferase 1 (PRMT1) in a manner that depends on the R540 residue. Finally, we show that PRMT1 depletion by RNA interference induces the concentration of Gag C-terminus in nucleoli.

Conclusion: Altogether, our findings suggest that methylation by PRMT1 might finely tune the subnuclear distribution of Gag depending on the stage of the FV replication cycle. The role of this step for viral replication remains an open question.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12977-018-0428-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6042332PMC
July 2018

Structure-function analyses unravel distinct effects of allosteric inhibitors of HIV-1 integrase on viral maturation and integration.

J Biol Chem 2018 04 5;293(16):6172-6186. Epub 2018 Mar 5.

From Biodim Mutabilis, 93230 Romainville,

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M117.816793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912470PMC
April 2018

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.

Retrovirology 2017 11 9;14(1):50. Epub 2017 Nov 9.

Biodim Mutabilis, 93230, Romainville, France.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12977-017-0373-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680779PMC
November 2017

Integration site selection by retroviruses and transposable elements in eukaryotes.

Nat Rev Genet 2017 05 13;18(5):292-308. Epub 2017 Mar 13.

Sorbonne Paris Cité, Université Paris Diderot, Institut National de la Santé et de la Recherche Médicale U944, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7212, Institut Universitaire d'Hématologie, Hôpital St. Louis, 1 Ave Claude Vellefaux, 75010 Paris, France.

Transposable elements and retroviruses are found in most genomes, can be pathogenic and are widely used as gene-delivery and functional genomics tools. Exploring whether these genetic elements target specific genomic sites for integration and how this preference is achieved is crucial to our understanding of genome evolution, somatic genome plasticity in cancer and ageing, host-parasite interactions and genome engineering applications. High-throughput profiling of integration sites by next-generation sequencing, combined with large-scale genomic data mining and cellular or biochemical approaches, has revealed that the insertions are usually non-random. The DNA sequence, chromatin and nuclear context, and cellular proteins cooperate in guiding integration in eukaryotic genomes, leading to a remarkable diversity of insertion site distribution and evolutionary strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nrg.2017.7DOI Listing
May 2017

Axl Mediates ZIKA Virus Entry in Human Glial Cells and Modulates Innate Immune Responses.

Cell Rep 2017 01;18(2):324-333

INSERM U944, CNRS 7212 Laboratoire de Pathologie et Virologie Moléculaire, Hôpital Saint-Louis, 1 avenue Claude Vellefaux, 75010 Paris, France; Institut Universitaire d'Hématologie, Hôpital Saint-Louis, 1 avenue Claude Vellefaux, 75010 Paris, France; University Paris Diderot, Sorbonne Paris Cité, Hôpital St. Louis, 1 avenue Claude Vellefaux, 75475 Paris Cedex 10, France. Electronic address:

ZIKA virus (ZIKV) is an emerging pathogen responsible for neurological disorders and congenital microcephaly. However, the molecular basis for ZIKV neurotropism remains poorly understood. Here, we show that Axl is expressed in human microglia and astrocytes in the developing brain and that it mediates ZIKV infection of glial cells. Axl-mediated ZIKV entry requires the Axl ligand Gas6, which bridges ZIKV particles to glial cells. Following binding, ZIKV is internalized through clathrin-mediated endocytosis and traffics to Rab5+ endosomes to establish productive infection. During entry, the ZIKV/Gas6 complex activates Axl kinase activity, which downmodulates interferon signaling and facilitates infection. ZIKV infection of human glial cells is inhibited by MYD1, an engineered Axl decoy receptor, and by the Axl kinase inhibitor R428. Our results highlight the dual role of Axl during ZIKV infection of glial cells: promoting viral entry and modulating innate immune responses. Therefore, inhibiting Axl function may represent a potential target for future antiviral therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2016.12.045DOI Listing
January 2017

Eleventh International Foamy Virus Conference-Meeting Report.

Viruses 2016 11 23;8(11). Epub 2016 Nov 23.

Laboratory of Retroviruses, Division of Viral Products, OVRR, CBER, U.S. Food and Drug Administration, Silver Spring, MD 20993, USA.

The Eleventh International Foamy Virus Conference took place on 9-10 June 2016 at the Institut Pasteur, Paris, France. The meeting reviewed progress on foamy virus (FV) research, as well as related current topics in retrovirology. FVs are complex retroviruses that are widespread in several animal species. Several research topics on these viruses are relevant to human health: cross-species transmission and viral emergence, vectors for gene therapy, development of antiretroviral drugs, retroviral evolution and its influence on the human genome. In this article, we review the conference presentations on these viruses and highlight the major questions to be answered.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/v8110318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127032PMC
November 2016

Vaccine and Wild-Type Strains of Yellow Fever Virus Engage Distinct Entry Mechanisms and Differentially Stimulate Antiviral Immune Responses.

mBio 2016 Feb 9;7(1):e01956-15. Epub 2016 Feb 9.

INSERM U944-CNRS 7212, Laboratoire de Pathologie et Virologie Moleculaire, Paris, France Institut Universitaire d'Hématologie, Paris, France Université Paris Diderot, Sorbonne Paris Cité, Paris, France

Unlabelled: The live attenuated yellow fever virus (YFV) vaccine 17D stands as a "gold standard" for a successful vaccine. 17D was developed empirically by passaging the wild-type Asibi strain in mouse and chicken embryo tissues. Despite its immense success, the molecular determinants for virulence attenuation and immunogenicity of the 17D vaccine are poorly understood. 17D evolved several mutations in its genome, most of which lie within the envelope (E) protein. Given the major role played by the YFV E protein during virus entry, it has been hypothesized that the residues that diverge between the Asibi and 17D E proteins may be key determinants of attenuation. In this study, we define the process of YFV entry into target cells and investigate its implication in the activation of the antiviral cytokine response. We found that Asibi infects host cells exclusively via the classical clathrin-mediated endocytosis, while 17D exploits a clathrin-independent pathway for infectious entry. We demonstrate that the mutations in the 17D E protein acquired during the attenuation process are sufficient to explain the differential entry of Asibi versus 17D. Interestingly, we show that 17D binds to and infects host cells more efficiently than Asibi, which culminates in increased delivery of viral RNA into the cytosol and robust activation of the cytokine-mediated antiviral response. Overall, our study reveals that 17D vaccine and Asibi enter target cells through distinct mechanisms and highlights a link between 17D attenuation, virus entry, and immune activation.

Importance: The yellow fever virus (YFV) vaccine 17D is one of the safest and most effective live virus vaccines ever developed. The molecular determinants for virulence attenuation and immunogenicity of 17D are poorly understood. 17D was generated by serially passaging the virulent Asibi strain in vertebrate tissues. Here we examined the entry mechanisms engaged by YFV Asibi and the 17D vaccine. We found the two viruses use different entry pathways. We show that the mutations differentiating the Asibi envelope (E) protein from the 17D E protein, which arose during attenuation, are key determinants for the use of these distinct entry routes. Finally, we demonstrate that 17D binds and enters host cells more efficiently than Asibi. This results in a higher uptake of viral RNA into the cytoplasm and consequently a greater cytokine-mediated antiviral response. Overall, our data provide new insights into the biology of YFV infection and the mechanisms of viral attenuation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.01956-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4752603PMC
February 2016

JASSA: a comprehensive tool for prediction of SUMOylation sites and SIMs.

Bioinformatics 2015 Nov 2;31(21):3483-91. Epub 2015 Jul 2.

CNRS UMR7212, Hôpital St Louis, Inserm U944, Institut Universitaire d'Hématologie, Hôpital St Louis, Université Paris Diderot, Sorbonne Paris Cité, Hôpital St Louis, Laboratoire PVM, Conservatoire national des arts et métiers (Cnam) and.

Motivation: Post-translational modification by the Small Ubiquitin-like Modifier (SUMO) proteins, a process termed SUMOylation, is involved in many fundamental cellular processes. SUMO proteins are conjugated to a protein substrate, creating an interface for the recruitment of cofactors harboring SUMO-interacting motifs (SIMs). Mapping both SUMO-conjugation sites and SIMs is required to study the functional consequence of SUMOylation. To define the best candidate sites for experimental validation we designed JASSA, a Joint Analyzer of SUMOylation site and SIMs.

Results: JASSA is a predictor that uses a scoring system based on a Position Frequency Matrix derived from the alignment of experimental SUMOylation sites or SIMs. Compared with existing web-tools, JASSA displays on par or better performances. Novel features were implemented towards a better evaluation of the prediction, including identification of database hits matching the query sequence and representation of candidate sites within the secondary structural elements and/or the 3D fold of the protein of interest, retrievable from deposited PDB files.

Availability And Implementation: JASSA is freely accessible at http://www.jassa.fr/. Website is implemented in PHP and MySQL, with all major browsers supported.

Contact: guillaume.beauclair@inserm.fr

Supplementary Information: Supplementary data are available at Bioinformatics online.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bioinformatics/btv403DOI Listing
November 2015

Integrase inhibitor reversal dynamics indicate unintegrated HIV-1 dna initiate de novo integration.

Retrovirology 2015 Mar 12;12:24. Epub 2015 Mar 12.

Background: Genomic integration, an obligate step in the HIV-1 replication cycle, is blocked by the integrase inhibitor raltegravir. A consequence is an excess of unintegrated viral DNA genomes, which undergo intramolecular ligation and accumulate as 2-LTR circles. These circularized genomes are also reliably observed in vivo in the absence of antiviral therapy and they persist in non-dividing cells. However, they have long been considered as dead-end products that are not precursors to integration and further viral propagation.

Results: Here, we show that raltegravir action is reversible and that unintegrated viral DNA is integrated in the host cell genome after raltegravir removal leading to HIV-1 replication. Using quantitative PCR approach, we analyzed the consequences of reversing prolonged raltegravir-induced integration blocks. We observed, after RAL removal, a decrease of 2-LTR circles and a transient increase of linear DNA that is subsequently integrated in the host cell genome and fuel new cycles of viral replication.

Conclusions: Our data highly suggest that 2-LTR circles can be used as a reserve supply of genomes for proviral integration highlighting their potential role in the overall HIV-1 replication cycle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12977-015-0153-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4372172PMC
March 2015

Interferon controls SUMO availability via the Lin28 and let-7 axis to impede virus replication.

Nat Commun 2014 Jun 19;5:4187. Epub 2014 Jun 19.

1] Université Paris Diderot, Sorbonne Paris Cité, Hôpital St Louis, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France [2] INSERM U944, Equipe labellisée par la Ligue Nationale contre le Cancer, Institut Universitaire d'Hématologie, Hôpital St Louis, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France [3] CNRS UMR 7212, Hôpital St Louis, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France.

Small ubiquitin-related modifier (SUMO) protein conjugation onto target proteins regulates multiple cellular functions, including defence against pathogens, stemness and senescence. SUMO1 peptides are limiting in quantity and are thus mainly conjugated to high-affinity targets. Conjugation of SUMO2/3 paralogues is primarily stress inducible and may initiate target degradation. Here we demonstrate that the expression of SUMO1/2/3 is dramatically enhanced by interferons through an miRNA-based mechanism involving the Lin28/let-7 axis, a master regulator of stemness. Normal haematopoietic progenitors indeed display much higher SUMO contents than their differentiated progeny. Critically, SUMOs contribute to the antiviral effects of interferons against HSV1 or HIV. Promyelocytic leukemia (PML) nuclear bodies are interferon-induced domains, which facilitate sumoylation of a subset of targets. Our findings thus identify an integrated interferon-responsive PML/SUMO pathway that impedes viral replication by enhancing SUMO conjugation and possibly also modifying the repertoire of targets. Interferon-enhanced post-translational modifications may be essential for senescence or stem cell self-renewal, and initiate SUMO-dependent proteolysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms5187DOI Listing
June 2014

Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage.

Retrovirology 2013 Nov 21;10:144. Epub 2013 Nov 21.

Biodim Mutabilis, Romainville 93230, France.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1742-4690-10-144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4222603PMC
November 2013

Comparative proteomic analysis of HIV-1 particles reveals a role for Ezrin and EHD4 in the Nef-dependent increase of virus infectivity.

J Virol 2013 Apr 16;87(7):3729-40. Epub 2013 Jan 16.

Hôpital Necker-Enfants Malades, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.

Nef is a human immunodeficiency virus type 1 (HIV-1) auxiliary protein that plays an important role in virus replication and the onset of acquired immunodeficiency. Although known functions of Nef might explain its contribution to HIV-1-associated pathogenesis, how Nef increases virus infectivity is still an open question. In vitro, Nef-deleted viruses have a defect that prevents efficient completion of early steps of replication. We have previously shown that this restriction is not due to the absence of Nef in viral particles. Rather, a loss of function in virus-producing cells accounts for the lower infectivity of nef-deleted viruses compared to wild-type (WT) viruses. Here we used DiGE and iTRAQ to identify differences between the proteomes of WT and nef-deleted viruses. We observe that glucosidase II is enriched in WT virions, whereas Ezrin, ALG-2, CD81, and EHD4 are enriched in nef-deleted virions. Functional analysis shows that glucosidase II, ALG-2, and CD81 have no or only Nef-independent effect on infectivity. In contrast, Ezrin and EHD4 are involved in the ability of Nef to increase virus infectivity (referred to thereafter as Nef potency). Indeed, simultaneous Ezrin and EHD4 depletion in SupT1 and 293T virus-producing cells result in an ∼30 and ∼70% decrease of Nef potency, respectively. Finally, while Ezrin behaves as an inhibitory factor counteracted by Nef, EHD4 should be considered as a cofactors required by Nef to increase virus infectivity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.02477-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624205PMC
April 2013

La fixation des cellules, un écueil expérimental à éviter.

Virologie (Montrouge) 2012 Oct;16(5):332-333

Université Paris-VII - Paris-Diderot, Institut universitaire d'hématologie, CNRS UMR 7212, Inserm U944, Hôpital Saint-Louis, 75010 Paris, France, Conservatoire national des arts et métiers, 75003 Paris, France.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1684/vir.2012.0464DOI Listing
October 2012

Ancrage chromatinien et intégration rétrovirale : implication des protéines IN et Gag.

Virologie (Montrouge) 2012 Apr;16(2):73-84

Hôpital Saint-Louis, CNRS UMR7212, 75010 Paris, France, Institut universitaire d'hématologie, hôpital Saint-Louis, Inserm U944, 75010 Paris, France, Université Paris-Diderot, Sorbonne-Paris-Cité, hôpital Saint-Louis, 1, avenue Claude-Vellefaux, 75010 Paris, France, Conservatoire national des arts et métiers, 75003 Paris, France.

Integration into the genome of the host cell is an obligatory step in the replication of retroelements. This feature accounts for the fact that these elements are both potential mutagens as well as vectors suitable for long-term gene therapy. Recently, many studies have reported that proviral insertion is not random but, rather, targets specific regions in the genome. Additionally, it has become clear that this process is highly regulated at the molecular level. Both viral proteins and cellular factors participate in the integration step, explaining why different retroelements have distinct integration profiles. This review describes recent advances about the integration of retroelements, focusing particularly on the mechanisms involved in the selectivity and specificity of integration and the chromatin-anchoring step, which precedes the insertion of the provirus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1684/vir.2012.0441DOI Listing
April 2012

Investigating the intercellular spreading properties of the foamy virus Gag protein.

PLoS One 2012 29;7(2):e31108. Epub 2012 Feb 29.

Institut Universitaire d'Hématologie, CNRS UMR7212-Inserm U944-Université Paris Diderot-Paris7, Paris, France.

Small regions called protein transduction domains (PTDs), identified in cellular and viral proteins, have been reported to efficiently cross biological membranes. Here we show that the structural Gag protein of the prototypic foamy virus (PFV) is apparently able to move from cell to cell and to transport the green fluorescent protein (GFP) from few transfected cells to the nuclei of the entire monolayer. Deletion studies showed that this property lies within the second glycine/arginine (GRII) box in the C-terminus of the protein. We also found that uptake and nuclear accumulation of Gag GRII expressed as GFP-fusion protein in recipient cells was observed only following methanol fixation, but never in living cells or when cells were fixed with glutaraldehyde or treated with trichloroacetic acid prior to methanol fixation. Absence of intercellular spreading in vivo was further confirmed using a sensitive luciferase activity assay based on transactivation of the PFV long terminal repeats. Thus, we conclude that intercellular spreading of PFV Gag represents an artificial diffusion event occurring during cell fixation, followed by nuclear retention mediated by the chromatin-binding sequence within the Gag GRII box. In light of these results, we advise caution before defining a peptide as PTD on the basis of intercellular spreading observed by fluorescence microscopy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0031108PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3290618PMC
July 2012

Impairment of human immunodeficiency virus type-1 integrase SUMOylation correlates with an early replication defect.

J Biol Chem 2011 Jun 21;286(23):21013-22. Epub 2011 Mar 21.

CNRS UMR7212, INSERM U944, Institut Universitaire d'Hématologie-Université Paris7 Diderot, 75475 Paris, France.

HIV-1 integrase (IN) orchestrates the integration of the reverse transcribed viral cDNA into the host cell genome and participates also in other steps of HIV-1 replication. Cellular and viral factors assist IN in performing its multiple functions, and post-translational modifications contribute to modulate its activities. Here, we show that HIV-1 IN is modified by SUMO proteins and that phylogenetically conserved SUMOylation consensus motifs represent major SUMO acceptor sites. Viruses harboring SUMOylation site IN mutants displayed a replication defect that was mapped during the early stages of infection, before integration but after reverse transcription. Because SUMOylation-defective IN mutants retained WT catalytic activity, we hypothesize that SUMOylation might regulate the affinity of IN for co-factors, contributing to efficient HIV-1 replication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M110.189274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3121452PMC
June 2011

Inhibition of human gamma delta [corrected] T-cell antitumoral activity through HLA-G: implications for immunotherapy of cancer.

Cell Mol Life Sci 2011 Oct 20;68(20):3385-99. Epub 2011 Feb 20.

CEAEA, I2BM, Service de Recherches en Hemato-Immunologie, 75475 Paris, France.

Vγ9Vδ2 T cells play a crucial role in the antitumoral immune response through cytokine production and cytotoxicity. Although the expression of the immunomodulatory molecule HLA-G has been found in diverse tumors, its impact on Vγ9Vδ2 T-cell functions remains unknown. Here we showed that soluble HLA-G inhibits Vγ9Vδ2 T-cell proliferation without inducing apoptosis. Moreover, soluble HLA-G inhibited the Vγ9Vδ2 T-cell production of IFN-γ induced by phosphoantigen stimulation. The reduction in Vγ9Vδ2 T-cell IFN-γ production was also induced by membrane-bound or soluble HLA-G expressed by tumor cell lines. Finally, primary tumor cells inhibited Vγ9Vδ2 T-cell proliferation and IFN-γ production through HLA-G. In this context, HLA-G impaired Vγ9Vδ2 T-cell cytotoxicity by interacting with ILT2 inhibitory receptor. These data demonstrate that HLA-G inhibits the anti-tumoral functions of Vγ9Vδ2 T cells and imply that treatments targeting HLA-G could optimize Vγ9Vδ2 T-cell-mediated immunotherapy of cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00018-011-0632-7DOI Listing
October 2011

Tax ubiquitylation and SUMOylation control the dynamic shuttling of Tax and NEMO between Ubc9 nuclear bodies and the centrosome.

Blood 2011 Jan 19;117(1):190-9. Epub 2010 Oct 19.

Department of Internal Medicine, American University of Beirut, Beirut, Lebanon.

The human T-lymphotropic virus type I oncoprotein Tax is critical for T-cell transformation, acting mainly through nuclear factor kappa B essential modulator (NEMO) binding and subsequent nuclear factor-κB activation. Tax localizes to Tax nuclear bodies and to the centrosome and is subjected to ubiquitylation and small ubiquitin-like modifier (SUMO)ylation, which are both necessary for complete transcriptional activation. Using the photoconvertible fluorophore Dendra-2 coupled with live video confocal microscopy, we show for the first time that the same Tax molecule shuttles among Tax nuclear bodies and between these nuclear bodies and the centrosome, depending on its posttranslational modifications. Ubiquitylation targets Tax to nuclear bodies to which NEMO is recruited and subsequently SUMOylated. We also demonstrate that Tax nuclear bodies contain the SUMOylation machinery including SUMO and the SUMO conjugating enzyme Ubc9, strongly suggesting that these nuclear bodies represent sites of active SUMOylation. Finally, both ubiquitylation and SUMOylation of Tax control NEMO targeting to the centrosome. Altogether, we are proposing a model where both ubiquitylation and SUMOylation of Tax control the shuttling of Tax and NEMO between the cytoplasmic and nuclear compartments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2010-05-285742DOI Listing
January 2011

Early reverse transcription is essential for productive foamy virus infection.

PLoS One 2010 Jun 11;5(6):e11023. Epub 2010 Jun 11.

CNRS UMR7212, Inserm U944, Université Paris Diderot, Institut Universitaire d'Hématologie, Paris, France.

Background: Although viral RNA constitutes the majority of nucleic acids packaged in virions, a late occurring step of reverse transcription leads to the presence of infectious viral cDNA in foamy virus particles. This peculiarity distinguishes them from the rest of the retroviral family.

Principal Findings: To evaluate the respective contribution of these viral nucleic acids in the replication of foamy viruses, their fate was studied by real-time PCR and RT-PCR early after infection, in the presence or in the absence of AZT. We found that an early reverse transcription step, which occurs during the first hours post-entry, is absolutely required for productive infection. Remarkably, sensitivity to AZT can be counteracted by increasing the multiplicity of infection (moi). We also show that 2-LTR circular viral DNA, which appears as soon as four hours post-infection, is transcriptionally competent.

Conclusion: Taken together, our data demonstrate that an early reverse transcription process, which takes place soon after viral entry, is indispensable for infectivity of FVs at low moi, when the amount of DNA-containing particles is not sufficient to lead to a productive infection. This study demonstrates a key role of the packaged viral RNA in the foamy virus infection, suggesting that the replication of this virus can be achieved by involving either viral DNA or RNA genome, depending on the condition of infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0011023PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2884000PMC
June 2010

Chromosomal tethering and proviral integration.

Biochim Biophys Acta 2010 Mar-Apr;1799(3-4):207-16. Epub 2009 Aug 13.

LBPA, CNRS UMR8113, Ecole Normale Supérieure de Cachan, 61 Avenue du Président Wilson, 94235 Cachan Cedex, France.

Since integration into the host cell genome is an obligatory step for their replication, retro-elements are both potent insertional mutagens and also suitable vectors for gene therapy. Many recent studies reported that the integration process is not random but, on the contrary, higly regulated at the molecular level. Many viral proteins and cellular factors play a key role in the integration step, explaining the reason why different retro-elements display distinct integration profiles. This review describes the recent highlights about integration of retro-elements with particular focus on the mechanisms underlying the specificity of integration target-site selection and the step of chromosomal tethering which preceeds insertion of the provirus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbagrm.2009.08.005DOI Listing
May 2010

Suppression of HIV-1 replication by microRNA effectors.

Retrovirology 2009 Mar 9;6:26. Epub 2009 Mar 9.

Institut de Génétique Humaine CNRS UPR1142, Laboratoire de Virologie Moléculaire, Montpellier, France.

The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1742-4690-6-26DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2657893PMC
March 2009

Centrosomal pre-integration latency of HIV-1 in quiescent cells.

Retrovirology 2007 Sep 10;4:63. Epub 2007 Sep 10.

CNRS UMR7151, Université Paris 7, Hôpital Saint-Louis, Paris, France.

Human immunodeficiency virus type 1 (HIV-1) efficiently replicates in dividing and non-dividing cells. However, HIV-1 infection is blocked at an early post-entry step in quiescent CD4+ T cells in vitro. The molecular basis of this restriction is still poorly understood. Here, we show that in quiescent cells, incoming HIV-1 sub-viral complexes concentrate and stably reside at the centrosome for several weeks. Upon cell activation, viral replication resumes leading to viral gene expression. Thus, HIV-1 can persist in quiescent cells as a stable, centrosome-associated, pre-integration intermediate.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1742-4690-4-63DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2014762PMC
September 2007

Characterization of reemerging chikungunya virus.

PLoS Pathog 2007 Jun;3(6):e89

Department of Virology, Unité Virus et Immunité, Institut Pasteur, Paris, France.

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.0030089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1904475PMC
June 2007

Centrosome and retroviruses: the dangerous liaisons.

Retrovirology 2007 Apr 14;4:27. Epub 2007 Apr 14.

Unité d'Epidémiologie et Physiopathologie des Virus Oncogènes, CNRS URA 3015, Département de Virologie, Institut Pasteur, Paris, France.

Centrosomes are the major microtubule organizing structures in vertebrate cells. They localize in close proximity to the nucleus for the duration of interphase and play major roles in numerous cell functions. Consequently, any deficiency in centrosome function or number may lead to genetic instability. Several viruses including retroviruses such as, Foamy Virus, HIV-1, JSRV, M-PMV and HTLV-1 have been shown to hamper centrosome functions for their own profit, but the outcomes are very different. Foamy viruses, HIV-1, JSRV, M-PMV and HTLV-1 use the cellular machinery to traffic towards the centrosome during early and/or late stages of the infection. In addition HIV-1 Vpr protein alters the cell-cycle regulation by hijacking centrosome functions. Enthrallingly, HTLV-1 Tax expression also targets the functions of the centrosome, and this event is correlated with centrosome amplification, aneuploidy and transformation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1742-4690-4-27DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1855351PMC
April 2007

Dynamin 2 is required for the enhancement of HIV-1 infectivity by Nef.

Proc Natl Acad Sci U S A 2007 Apr 5;104(16):6812-7. Epub 2007 Apr 5.

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02115, USA.

Nef is a virulence factor of HIV-1 and other primate lentiviruses that is crucial for rapid progression to AIDS. In cell culture, Nef increases the infectivity of HIV-1 progeny virions by an unknown mechanism. We now show that dynamin 2 (Dyn2), a key regulator of vesicular trafficking, is a binding partner of Nef that is required for its ability to increase viral infectivity. Dominant-negative Dyn2 or the depletion of Dyn2 by small interfering RNA potently inhibited the effect of Nef on HIV-1 infectivity. Furthermore, in Dyn2-depleted cells, this function of Nef could be rescued by ectopically expressed Dyn2 but not by Dyn1, a closely related isoform that does not bind Nef. The infectivity enhancement by Nef also depended on clathrin, because it was diminished in clathrin-depleted cells and profoundly inhibited in cells expressing the clathrin-binding domain of AP180, which blocks clathrin-coated pit formation but not clathrin-independent endocytosis. Together, these findings imply that the infectivity enhancement activity of Nef depends on Dyn2- and clathrin-mediated membrane invagination events.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0607622104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1871867PMC
April 2007

Release of autoinhibition converts ESCRT-III components into potent inhibitors of HIV-1 budding.

Proc Natl Acad Sci U S A 2006 Dec 4;103(50):19140-5. Epub 2006 Dec 4.

Program in Gene Function and Expression, Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.

The endosomal sorting complex ESCRT-III, which is formed by the structurally related CHMP proteins, is engaged by HIV-1 to promote viral budding. Here we show that progressive truncations into the C-terminal acidic domains of CHMP proteins trigger an increasingly robust anti-HIV budding activity. Together with biochemical evidence for specific intramolecular interactions between the basic and acidic halves of CHMP3 and CHMP4B, these results suggest that the acidic domains are autoinhibitory. The acidic half of CHMP3 also interacts with the endosome-associated ubiquitin isopeptidase AMSH, and the coexpression of AMSH or its CHMP3-binding domain converts wild-type CHMP3 into a potent inhibitor of HIV-1 release. Point mutations in CHMP3 that prevent binding to AMSH abrogate this effect, suggesting that binding to AMSH relieves the autoinhibition of CHMP3. Collectively, our results indicate that CHMP proteins are regulated through an autoinhibitory switch mechanism that allows tight control of ESCRT-III assembly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0603788103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1748189PMC
December 2006

Structural basis for budding by the ESCRT-III factor CHMP3.

Dev Cell 2006 Jun;10(6):821-30

European Molecular Biology Laboratory, 6 rue Jules Horowitz, 38042 Grenoble, France.

The vacuolar protein sorting machinery regulates multivesicular body biogenesis and is selectively recruited by enveloped viruses to support budding. Here we report the crystal structure of the human ESCRT-III protein CHMP3 at 2.8 A resolution. The core structure of CHMP3 folds into a flat helical arrangement that assembles into a lattice, mainly via two different dimerization modes, and unilaterally exposes a highly basic surface. The C terminus, the target for Vps4-induced ESCRT disassembly, extends from the opposite side of the membrane targeting region. Mutations within the basic and dimerization regions hinder bilayer interaction in vivo and reverse the dominant-negative effect of a truncated CHMP3 fusion protein on HIV-1 budding. Thus, the final steps in the budding process may include CHMP protein polymerization and lattice formation on membranes by employing different bilayer-recognizing surfaces, a function shared by all CHMP family members.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.devcel.2006.03.013DOI Listing
June 2006