38 results on '"Di Primio C"'
Search Results
2. Gene Expression of Disease-related Genes in Alzheimer’s Disease is Impaired by Tau Aggregation
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Siano, G., primary, Varisco, M., additional, Scarlatti, A., additional, Caiazza, M.C., additional, Dunville, K., additional, Cremisi, F., additional, Costa, M., additional, Pancrazi, L., additional, Di Primio, C., additional, and Cattaneo, A., additional
- Published
- 2020
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3. A proteomics approach to the study of bleomycin- induced lung fibrosis
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Di Primio C, Morales Ma, Rocchiccioli S, Terreni M, Della Latta, L. Comelli, Antonella Cecchettini, Silvia Burchielli, Nadia Ucciferri, Gualtiero Pelosi, Della, Latta V, Cecchettini, A, Comelli, L, Ucciferri, N, Di Primio, C, Terreni, M, Burchielli, S, Pelosi, G, Rocchiccioli, S, and Morales, Maria Aurora
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chemistry.chemical_compound ,chemistry ,business.industry ,Lung fibrosis ,Cancer research ,Medicine ,Settore BIO/11 - Biologia Molecolare ,Bleomycin ,Proteomics ,business - Abstract
Idiopathic pulmonary fibrosis (IPF) is the most severe lung fibrotic form and very few pharmacological therapies are available at present. Key events in the onset of the disease are the activation of fibroblasts to myofibroblasts and the production and release of extracellular matrix (ECM) and molecular factors. Primary murine lung fibroblasts were isolated and their activation induced by Bleomycin (BLM) treatment. Extracellular Vesicles (EV) were isolated and protein extracted. Released soluble proteins (Secretome) and EV-derived proteins were reduced, alkylated and trypsin digested. A nano-LC-MS/MS SWATHTM approach was used for the proteomics analyses. Specific proteins with a putative role in the transition from physiological to fibrotic conditions, such as several matrix metalloproteinases (MMPs), osteopontin (OPN), chitinase-3-like protein1 (CHI3L1) and CD44 resulted differentially released from BLM-treated fibroblasts as compared with untreated lung fibroblasts. Our results provide further understanding of the pathophysiological features of lung fibrosis, and suggest specific target for pharmacological treatments.
- Published
- 2018
4. A new FRET-based biosensor to investigate the link between hearth failure and Alzheimer disease
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Quercioli, V., primary, Siano, G., additional, Rovere, M., additional, Cattaneo, A., additional, and Di Primio, C., additional
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- 2015
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5. Nestin is a key regulator of cardioprotection and nestin-640 gene therapy rescues infarcted myocardium
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Romano, S. L., Matteucci, Marco, Zentilin, L., Di Primio, C., Scebba, Francesca, Pucci, A., Balbarini, A., Giacca, M., Recchia, FABIO ANASTASIO, and Lionetti, Vincenzo
- Published
- 2012
6. Correlation of Somatostatin Receptor Scintigraphy and [18F]FDG PET/CT Findings with Expression of the Thyroid Transcription Factor in Bronchopulmonary Neuroendocrine Tumors
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Massri, K, Betti, F, Duce, V, Di Primio, C, Antonacci, L, Vasile, E, Cacciato Insilla, A, and Volterrani, Duccio
- Published
- 2012
7. Role of Phosphorylation in the Nuclear Biology of HIV-1
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C. Francis, A., primary, Di Primio, C., additional, Allouch, A., additional, and Cereseto, A., additional
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- 2011
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8. Enhancement of gene targeting in human cells by intranuclear permeation of the Saccharomyces cerevisiae Rad52 protein
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Kalvala, A., primary, Rainaldi, G., additional, Di Primio, C., additional, Liverani, V., additional, Falaschi, A., additional, and Galli, A., additional
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- 2010
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9. Differential tissue expression of a calpastatin isoform in Xenopus embryos
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Di Primio, C., primary, Marracci, S., additional, Cecchettini, A., additional, Nardi, I., additional, Giorgi, F., additional, Fausto, A.M., additional, Gambellini, G., additional, and Mazzini, M., additional
- Published
- 2007
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10. Potentiation of gene targeting in human cells by expression of Saccharomyces cerevisiae Rad52
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Di Primio, C., primary
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- 2005
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11. A second generation HIV-IN-EGFP fluorescent viral system to analyze HIV-1 in the nuclear compartment of infected cells
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Ac, Francis, Di Primio C, Valentina Quercioli, Boll A, Arosio D, Cereseto A, Frontieres in Retrovirology 2013, Francis, A, DI PRIMIO, Cristina, Quercioli, V, Boll, A, Arosio, D, and Cereseto, A.
12. Nuclear trafficking of hiv-1 pre-integration complexes in living cells
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Cianci Gianguido, Dylla Doug, Gallo Daniel, Di Primio Cristina, Arosio Daniele, Hope Thomas, and Cereseto Anna
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Immunologic diseases. Allergy ,RC581-607 - Published
- 2009
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13. GCN5-dependent acetylation of HIV-1 integrase enhances viral integration
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Giacca Mauro, Albanese Alberto, Arosio Daniele, Tozzini Valentina, Di Fenza Armida, Di Primio Cristina, Gutierrez Maria, Liverani Vania, Terreni Mariaelena, and Cereseto Anna
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Immunologic diseases. Allergy ,RC581-607 - Published
- 2009
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14. GCN5-dependent acetylation of HIV-1 integrase enhances viral integration
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Albanese Alberto, Allouch Awatef, Tozzini Valentina, Di Fenza Armida, Di Primio Cristina, Gutierrez Maria, Liverani Vania, Valentini Paola, Terreni Mariaelena, Giacca Mauro, and Cereseto Anna
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Immunologic diseases. Allergy ,RC581-607 - Abstract
Abstract Background An essential event during the replication cycle of HIV-1 is the integration of the reverse transcribed viral DNA into the host cellular genome. Our former report revealed that HIV-1 integrase (IN), the enzyme that catalyzes the integration reaction, is positively regulated by acetylation mediated by the histone acetyltransferase (HAT) p300. Results In this study we demonstrate that another cellular HAT, GCN5, acetylates IN leading to enhanced 3'-end processing and strand transfer activities. GCN5 participates in the integration step of HIV-1 replication cycle as demonstrated by the reduced infectivity, due to inefficient provirus formation, in GCN5 knockdown cells. Within the C-terminal domain of IN, four lysines (K258, K264, K266, and K273) are targeted by GCN5 acetylation, three of which (K264, K266, and K273) are also modified by p300. Replication analysis of HIV-1 clones carrying substitutions at the IN lysines acetylated by both GCN5 and p300, or exclusively by GCN5, demonstrated that these residues are required for efficient viral integration. In addition, a comparative analysis of the replication efficiencies of the IN triple- and quadruple-mutant viruses revealed that even though the lysines targeted by both GCN5 and p300 are required for efficient virus integration, the residue exclusively modified by GCN5 (K258) does not affect this process. Conclusions The results presented here further demonstrate the relevance of IN post-translational modification by acetylation, which results from the catalytic activities of multiple HATs during the viral replication cycle. Finally, this study contributes to clarifying the recent debate raised on the role of IN acetylated lysines during HIV-1 infection.
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- 2010
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15. Gene Expression of Disease-related Genes in Alzheimer's Disease is Impaired by Tau Aggregation
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K. Dunville, Maria Claudia Caiazza, Martina Varisco, Antonino Cattaneo, C. Di Primio, A. Scarlatti, Laura Pancrazi, Giacomo Siano, Mario Costa, F. Cremisi, Siano, G., Varisco, M., Scarlatti, A., Caiazza, M. C., Dunville, K., Cremisi, F., Costa, M., Pancrazi, L., Di Primio, C., and Cattaneo, A.
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Amyloid ,Amino Acid Transport System X-AG ,Active Transport, Cell Nucleus ,Prefrontal Cortex ,tau Proteins ,Biology ,Neurotransmission ,tauopathie ,Protein Aggregation, Pathological ,03 medical and health sciences ,Glutamatergic ,Settore BIO/06 - Anatomia Comparata e Citologia ,Mice ,0302 clinical medicine ,Downregulation and upregulation ,VGluT1 ,Structural Biology ,Alzheimer Disease ,Gene expression ,Animals ,Humans ,Prefrontal cortex ,Molecular Biology ,Embryonic Stem Cells ,030304 developmental biology ,Neurons ,0303 health sciences ,prefrontal cortex ,tau aggregation ,Glutamate receptor ,Brain ,Cell biology ,Gene Expression Regulation ,Tauopathies ,Synapses ,Vesicular Glutamate Transport Protein 1 ,gene expression ,Glutamatergic synapse ,030217 neurology & neurosurgery - Abstract
Neuronal hyperexcitability linked to an increase in glutamate signalling is a peculiar trait of the early stages of Alzheimer's disease (AD) and tauopathies, however, a progressive reduction in glutamate release follows in advanced stages. We recently reported that in the early phases of the neurodegenerative process, soluble, non-aggregated Tau accumulates in the nucleus and modulates the expression of disease-relevant genes directly involved in glutamatergic transmission, thus establishing a link between Tau instability and altered neurotransmission. Here we report that while the nuclear translocation of Tau in cultured cells is not impaired by its own aggregation, the nuclear amyloid inclusions of aggregated Tau abolish Tau-dependent increased expression of the glutamate transporter. Remarkably, we observed that in the prefrontal cortex (PFC) of AD patient brain, the glutamate transporter is upregulated at early stages and is downregulated at late stages. The Gene Set Enrichment Analysis indicates that the modulation of Tau-dependent gene expression along the disease progression can be extended to all protein pathways of the glutamatergic synapse. Together, this evidence links the altered glutamatergic function in the PFC during AD progression to the newly discovered function of nuclear Tau.
- Published
- 2020
16. Alpha-synuclein fret biosensors reveal early alpha-synuclein aggregation in the endoplasmic reticulum
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Laura Betti, Cristina Di Primio, Verdiana Valvano, Emanuela Colla, Valentina Quercioli, Fabiana Miraglia, Antonino Cattaneo, Gino Giannaccini, Lucia Rota, Miraglia, F., Valvano, V., Rota, L., Di Primio, C., Quercioli, V., Betti, L., Giannaccini, G., Cattaneo, A., and Colla, E.
- Subjects
0301 basic medicine ,animal diseases ,Settore BIO/11 - Biologia Molecolare ,Immunofluorescence ,Settore BIO/09 - Fisiologia ,Alpha-synucleinopathy ,General Biochemistry, Genetics and Molecular Biology ,Article ,Alpha-synuclein ,Parkinson’s Disease ,03 medical and health sciences ,chemistry.chemical_compound ,Aggregation ,0302 clinical medicine ,In vivo ,Settore BIO/10 - Biochimica ,mental disorders ,Sense (molecular biology) ,medicine ,alpha-synucleinopathy ,lcsh:Science ,Ecology, Evolution, Behavior and Systematics ,medicine.diagnostic_test ,Endoplasmic reticulum ,Paleontology ,bacterial infections and mycoses ,nervous system diseases ,Biosensors ,FRET ,Oligomers ,030104 developmental biology ,Förster resonance energy transfer ,nervous system ,chemistry ,Oligomer ,Space and Planetary Science ,Biophysics ,lcsh:Q ,Stable cell line ,Biosensor ,030217 neurology & neurosurgery - Abstract
Endoplasmic reticulum (ER) dysfunction is important for alpha-synuclein (&alpha, S) acquired toxicity. When targeted to the ER in SH-SY5Y cells, transient or stable expression of &alpha, S resulted in the formation of compact &alpha, S-positive structures in a small subpopulation of cells, resembling &alpha, S inclusions. Thus, because of the limitations of immunofluorescence, we developed a set of &alpha, S FRET biosensors (AFBs) able to track &alpha, S conformation in cells. In native conditions, expression in i36, a stable cell line for ER &alpha, S, of intermolecular AFBs, reporters in which CFP or YFP has been fused with the C-terminal of &alpha, S (&alpha, S-CFP/&alpha, S-YFP), resulted in no Fö, rster resonance energy transfer (FRET), whereas expression of the intramolecular AFB, a probe obtained by fusing YFP and CFP with &alpha, S N- or C- termini (YFP-&alpha, S-CFP), showed a positive FRET signal. These data confirmed that &alpha, S has a predominantly globular, monomeric conformation in native conditions. Differently, under pro-aggregating conditions, the intermolecular AFB was able to sense significantly formation of &alpha, S oligomers, when AFB was expressed in the ER rather than ubiquitously, suggesting that the ER can favor changes in &alpha, S conformation when aggregation is stimulated. These results show the potential of AFBs as a new, valuable tool to track &alpha, S conformational changes in vivo.
- Published
- 2020
17. Identification of an ERK Inhibitor as a Therapeutic Drug Against Tau Aggregation in a New Cell-Based Assay
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Giacomo Siano, Maria Claudia Caiazza, Ivana Ollà, Martina Varisco, Giuseppe Madaro, Valentina Quercioli, Mariantonietta Calvello, Antonino Cattaneo, Cristina Di Primio, Siano, G., Caiazza, M. C., Olla, I., Varisco, M., Madaro, G., Quercioli, V., Calvello, M., Cattaneo, A., and Di Primio, C.
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0301 basic medicine ,Drug ,MAPK/ERK pathway ,media_common.quotation_subject ,Cell ,lcsh:RC321-571 ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,medicine ,Cytotoxicity ,lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry ,Original Research ,CST ,media_common ,Tau biosensor ,Kinase ,Chemistry ,Drug discovery ,aggregation ,Cell biology ,ERK ,030104 developmental biology ,medicine.anatomical_structure ,Förster resonance energy transfer ,FRET ,Tau ,030217 neurology & neurosurgery ,Neuroscience ,Cell based - Abstract
Formation of Tau aggregates is a common pathological feature of tauopathies and their accumulation directly correlates with cytotoxicity and neuronal degeneration. Great efforts have been made to understand Tau aggregation and to find therapeutics halting or reversing the process, however, progress has been slowed due to the lack of a suitable method for monitoring Tau aggregation. We developed a cell-based assay allowing to detect and quantify Tau aggregation in living cells. The system is based on the FRET biosensor CST able to monitor the molecular dynamic of Tau aggregation in different cellular conditions. We probed candidate compounds that could block Tau hyperphosphorylation. In particular, to foster the drug discovery process, we tested kinase inhibitors approved for the treatment of other diseases. We identified the ERK inhibitor PD-901 as a promising therapeutic molecule since it reduces and prevents Tau aggregation. This evidence establishes the CST cell-based aggregation assay as a reliable tool for drug discovery and suggests that PD-901 might be a promising compound to be tested for further preclinical studies on AD.
- Published
- 2019
18. Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes
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Maria Claudia Caiazza, Valentina Quercioli, Cristina Di Primio, Giacomo Siano, Antonino Cattaneo, Mariantonietta Calvello, Martina Varisco, Siano, G., Caiazza, M. C., Varisco, M., Calvello, M., Quercioli, V., Cattaneo, A., and Di Primio, C.
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Nucleolus ,General Chemical Engineering ,Cellular differentiation ,Tau protein ,tau Proteins ,Biology ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,0302 clinical medicine ,Alzheimer Disease ,Cell Line, Tumor ,Humans ,Gene ,030304 developmental biology ,Cell Nucleus ,Neurons ,Regulation of gene expression ,0303 health sciences ,General Immunology and Microbiology ,General Neuroscience ,Binding protein ,Cell Differentiation ,Subcellular localization ,RRNA transcription ,Cell biology ,Gene Expression Regulation ,Vesicular Glutamate Transport Protein 1 ,biology.protein ,030217 neurology & neurosurgery ,Subcellular Fractions - Abstract
Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, recent evidence indicated that Tau is present also in other subcellular compartments including the nucleus where it is implicated in DNA protection, in rRNA transcription, in the mobility of retrotransposons and in the structural organization of the nucleolus. We have recently demonstrated that nuclear Tau is involved in the expression of the VGluT1 gene, suggesting a molecular mechanism that could explain the pathological increase of glutamate release in the early stages of Alzheimer's disease. Until recently, the involvement of nuclear Tau in modulating the expression of target genes has been relatively uncertain and ambiguous due to technical limitations that prevented the exclusion of the contribution of cytoplasmic Tau or the effect of other downstream factors not related to nuclear Tau. To overcome this uncertainty, we developed a method to study the expression of target genes specifically modulated by the nuclear Tau protein. We employed a protocol that couples the use of localization signals and the subcellular fractionation, allowing the exclusion of the interference from the cytoplasmic Tau molecules. Most notably, the protocol is easy and is composed of classic and reliable methods that are broadly applicable to study the nuclear function of Tau in other cell types and cellular conditions.
- Published
- 2019
19. SARS-CoV-2 Infection Alters the Phenotype and Gene Expression of Adipocytes.
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Quaranta P, Scabia G, Storti B, Dattilo A, Quintino L, Perrera P, Di Primio C, Costa M, Pistello M, Bizzarri R, and Maffei M
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- Humans, Cells, Cultured, SARS-CoV-2, Adipocytes metabolism, Phenotype, Gene Expression, Spike Glycoprotein, Coronavirus metabolism, COVID-19 metabolism, Intellectual Disability, Heart Defects, Congenital, Genetic Diseases, X-Linked, Gigantism, Arrhythmias, Cardiac
- Abstract
Epidemiological evidence emphasizes that excess fat mass is associated with an increased risk of severe COVID-19 disease. Nevertheless, the intricate interplay between SARS-CoV-2 and adipocytes remains poorly understood. It is crucial to decipher the progression of COVID-19 both in the acute phase and on long-term outcomes. In this study, an in vitro model using the human SGBS cell line (Simpson-Golabi-Behmel syndrome) was developed to investigate the infectivity of SARS-CoV-2 in adipocytes, and the effects of virus exposure on adipocyte function. Our results show that SGBS adipocytes expressing ACE2 are susceptible to SARS-CoV-2 infection, as evidenced by the release of the viral genome into the medium, detection of the nucleocapsid in cell lysates, and positive immunostaining for the spike protein. Infected adipocytes show remarkable changes compared to uninfected controls: increased surface area of lipid droplets, upregulated expression of genes of inflammation ( Haptoglobin , MCP-1 , IL-6 , PAI-1 ), increased oxidative stress (MnSOD), and a concomitant reduction of transcripts related to adipocyte function (leptin, fatty acid synthase, perilipin). Moreover, exogenous expression of spike protein in SGBS adipocytes also led to an increase in lipid droplet size. In conclusion using the human SGBS cell line, we detected SARS-CoV-2 infectivity in adipocytes, revealing substantial morphological and functional changes in infected cells.
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- 2024
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20. The purinergic receptor P2X7 and the NLRP3 inflammasome are druggable host factors required for SARS-CoV-2 infection.
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Lécuyer D, Nardacci R, Tannous D, Gutierrez-Mateyron E, Deva Nathan A, Subra F, Di Primio C, Quaranta P, Petit V, Richetta C, Mostefa-Kara A, Del Nonno F, Falasca L, Marlin R, Maisonnasse P, Delahousse J, Pascaud J, Deprez E, Naigeon M, Chaput N, Paci A, Saada V, Ghez D, Mariette X, Costa M, Pistello M, Allouch A, Delelis O, Piacentini M, Le Grand R, and Perfettini JL
- Subjects
- Humans, NLR Family, Pyrin Domain-Containing 3 Protein metabolism, NLR Proteins, COVID-19 Drug Treatment, SARS-CoV-2 metabolism, Inflammation, Receptors, Purinergic, Inflammasomes metabolism, COVID-19
- Abstract
Purinergic receptors and NOD-like receptor protein 3 (NLRP3) inflammasome regulate inflammation and viral infection, but their effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain poorly understood. Here, we report that the purinergic receptor P2X7 and NLRP3 inflammasome are cellular host factors required for SARS-CoV-2 infection. Lung autopsies from patients with severe coronavirus disease 2019 (COVID-19) reveal that NLRP3 expression is increased in host cellular targets of SARS-CoV-2 including alveolar macrophages, type II pneumocytes and syncytia arising from the fusion of infected macrophages, thus suggesting a potential role of NLRP3 and associated signaling pathways to both inflammation and viral replication. In vitro studies demonstrate that NLRP3-dependent inflammasome activation is detected upon macrophage abortive infection. More importantly, a weak activation of NLRP3 inflammasome is also detected during the early steps of SARS-CoV-2 infection of epithelial cells and promotes the viral replication in these cells. Interestingly, the purinergic receptor P2X7, which is known to control NLRP3 inflammasome activation, also favors the replication of D614G and alpha SARS-CoV-2 variants. Altogether, our results reveal an unexpected relationship between the purinergic receptor P2X7, the NLRP3 inflammasome and the permissiveness to SARS-CoV-2 infection that offers novel opportunities for COVID-19 treatment., Competing Interests: DT and AA were employed by NH TherAguix SAS. Authors DL, DT, AA, FS, OD and J-LP are listed as co-inventors on a patent application related to SARS-CoV-2 therapy. AP and J-LP are founding members of Findimmune SAS, an Immuno-Oncology Biotech company. J-LP disclosed research funding not related to this work from NH TherAguix and Wonna Therapeutics. NC disclosed research funding not related to this work from GlaxoSmithKline, Roche, Cytune pharma and Sanofi. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Lécuyer, Nardacci, Tannous, Gutierrez-Mateyron, Deva Nathan, Subra, Di Primio, Quaranta, Petit, Richetta, Mostefa-Kara, Del Nonno, Falasca, Marlin, Maisonnasse, Delahousse, Pascaud, Deprez, Naigeon, Chaput, Paci, Saada, Ghez, Mariette, Costa, Pistello, Allouch, Delelis, Piacentini, Le Grand and Perfettini.)
- Published
- 2023
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21. Severe acute respiratory syndrome coronavirus 2 infection leads to Tau pathological signature in neurons.
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Di Primio C, Quaranta P, Mignanelli M, Siano G, Bimbati M, Scarlatti A, Piazza CR, Spezia PG, Perrera P, Basolo F, Poma AM, Costa M, Pistello M, and Cattaneo A
- Abstract
COVID-19 has represented an issue for global health since its outbreak in March 2020. It is now evident that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in a wide range of long-term neurological symptoms and is worryingly associated with the aggravation of Alzheimer's disease. Little is known about the molecular basis of these manifestations. Here, several strain variants were used to infect SH-SY5Y neuroblastoma cells and K18-hACE C57BL/6J mice. The Tau phosphorylation profile and aggregation propensity upon infection were investigated on cellular extracts, subcellular fractions, and brain tissue. The viral proteins spike, nucleocapsid, and membrane were overexpressed in SH-SY5Y cells, and the direct interaction and effect on Tau phosphorylation were checked using immunoblot experiments. Upon infection, Tau is phosphorylated at several pathological epitopes associated with Alzheimer's disease and other tauopathies. Moreover, this event increases Tau's propensity to form insoluble aggregates and alters its subcellular localization. Our data support the hypothesis that SARS-CoV-2 infection in the central nervous system triggers downstream effects altering Tau function, eventually leading to the impairment of neuronal function., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)
- Published
- 2023
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22. Tau-dependent HDAC1 nuclear reduction is associated with altered VGluT1 expression.
- Author
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Siano G, Madaro G, Caiazza MC, Allouch A, Varisco M, Mignanelli M, Cattaneo A, and Di Primio C
- Abstract
During AD pathology, Tau protein levels progressively increase from early pathological stages. Tau altered expression causes an unbalance of Tau subcellular localization in the cytosol and in the nuclear compartment leading to synaptic dysfunction, neuronal cell death and neurodegeneration as a consequence. Due to the relevant role of epigenetic remodellers in synaptic activity in physiology and in neurodegeneration, in particular of TRIM28 and HDAC1, we investigated the relationship between Tau and these epigenetic factors. By molecular, imaging and biochemical approaches, here we demonstrate that Tau altered expression in the neuronal cell line SH-SY5y does not alter TRIM28 and HDAC1 expression but it induces a subcellular reduction of HDAC1 in the nuclear compartment. Remarkably, HDAC1 reduced activity modulates the expression of synaptic genes in a way comparable to that observed by Tau increased levels. These results support a competitive relationship between Tau levels and HDAC1 subcellular localization and nuclear activity, indicating a possible mechanism mediating the alternative role of Tau in the pathological alteration of synaptic genes expression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Siano, Madaro, Caiazza, Allouch, Varisco, Mignanelli, Cattaneo and Di Primio.)
- Published
- 2023
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23. Non-Canonical Roles of Tau and Their Contribution to Synaptic Dysfunction.
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Siano G, Falcicchia C, Origlia N, Cattaneo A, and Di Primio C
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- Active Transport, Cell Nucleus, Animals, Humans, Synapses physiology, tau Proteins chemistry, Cell Nucleus metabolism, Synapses metabolism, Tauopathies metabolism, tau Proteins metabolism
- Abstract
Tau plays a central role in a group of neurodegenerative disorders collectively named tauopathies. Despite the wide range of diverse symptoms at the onset and during the progression of the pathology, all tauopathies share two common hallmarks, namely the misfolding and aggregation of Tau protein and progressive synaptic dysfunctions. Tau aggregation correlates with cognitive decline and behavioural impairment. The mechanistic link between Tau misfolding and the synaptic dysfunction is still unknown, but this correlation is well established in the human brain and also in tauopathy mouse models. At the onset of the pathology, Tau undergoes post-translational modifications (PTMs) inducing the detachment from the cytoskeleton and its release in the cytoplasm as a soluble monomer. In this condition, the physiological enrichment in the axon is definitely disrupted, resulting in Tau relocalization in the cell soma and in dendrites. Subsequently, Tau aggregates into toxic oligomers and amyloidogenic forms that disrupt synaptic homeostasis and function, resulting in neuronal degeneration. The involvement of Tau in synaptic transmission alteration in tauopathies has been extensively reviewed. Here, we will focus on non-canonical Tau functions mediating synapse dysfunction.
- Published
- 2021
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24. A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in Vero E6 cells.
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Storti B, Quaranta P, Di Primio C, Clementi N, Mancini N, Criscuolo E, Spezia PG, Carnicelli V, Lottini G, Paolini E, Freer G, Lai M, Costa M, Beltram F, Diaspro A, Pistello M, Zucchi R, Bianchini P, Signore G, and Bizzarri R
- Abstract
We exploited a multi-scale microscopy imaging toolbox to address some major issues related to SARS-CoV-2 interactions with host cells. Our approach harnesses both conventional and super-resolution fluorescence microscopy and easily matches the spatial scale of single-virus/cell checkpoints. After its validation through the characterization of infected cells and virus morphology, we leveraged this toolbox to reveal subtle issues related to the entry phase of SARS-CoV-2 variants in Vero E6 cells. Our results show that in Vero E6 cells the B.1.1.7 strain (aka Alpha Variant of Concern) is associated with much faster kinetics of endocytic uptake compared to its ancestor B.1.177. Given the cell-entry scenario dominated by the endosomal "late pathway", the faster internalization of B.1.1.7 could be directly related to the N501Y mutation in the S protein, which is known to strengthen the binding of Spike receptor binding domain with ACE2. Remarkably, we also directly observed the central role of clathrin as a mediator of endocytosis in the late pathway of entry. In keeping with the clathrin-mediated endocytosis, we highlighted the non-raft membrane localization of ACE2. Overall, we believe that our fluorescence microscopy-based approach represents a fertile strategy to investigate the molecular features of SARS-CoV-2 interactions with cells., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)
- Published
- 2021
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25. The Q336H MAPT Mutation Linked to Pick's Disease Leads to Increased Binding of Tau to the Microtubule Network via Altered Conformational and Phosphorylation Effects.
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Siano G, Micaelli M, Scarlatti A, Quercioli V, Di Primio C, and Cattaneo A
- Abstract
Tauopathies are neurodegenerative disorders characterized by Tau aggregation. Genetic studies on familial cases allowed for the discovery of mutations in the MAPT gene that increase Tau propensity to detach from microtubules and to form insoluble cytoplasmic Tau aggregates. Recently, the rare mutation Q336H has been identified to be associated with Pick's disease (PiD) and biochemical analyses demonstrated its ability to increase the microtubules (MTs) polymerization, thus revealing an opposite character compared to other Tau mutations studied so far. Here we investigated the biophysical and molecular properties of Tau
Q336H in living cells by the employment of the conformational Tau biosensor CST. We found that this mutation alters Tau conformation on microtubules, stabilizes its binding to tubulin, and is associated with a paradoxical lower level of Tau phosphorylation. Moreover, we found that this mutation impacts the cytoskeletal complexity by increasing the tubulin filament length and the number of branches. However, despite these apparently non-pathological traits, we observed the formation of intracellular inclusions confirming that Q336H leads to aggregation. Our results suggest that the Tau aggregation process might be triggered by molecular mechanisms other than Tau destabilization or post-translational modifications which are likely to be detrimental to neuronal function in vivo ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Siano, Micaelli, Scarlatti, Quercioli, Di Primio and Cattaneo.)- Published
- 2020
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26. SUGT1 controls susceptibility to HIV-1 infection by stabilizing microtubule plus-ends.
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Allouch A, Di Primio C, Paoletti A, Lê-Bury G, Subra F, Quercioli V, Nardacci R, David A, Saïdi H, Cereseto A, Ojcius DM, Montagnac G, Niedergang F, Pancino G, Saez-Cirion A, Piacentini M, Gougeon ML, Kroemer G, and Perfettini JL
- Subjects
- Acetylation, Active Transport, Cell Nucleus genetics, Anti-HIV Agents therapeutic use, Cell Cycle Proteins genetics, Drug Resistance, Viral genetics, HIV Infections drug therapy, HIV-1 genetics, Humans, Microtubule-Associated Proteins genetics, Microtubules genetics, Microtubules pathology, Raltegravir Potassium therapeutic use, Virus Replication, Cell Cycle Proteins metabolism, HIV-1 metabolism, Microtubule-Associated Proteins metabolism, Microtubules metabolism
- Abstract
Understanding the viral-host cell interface during HIV-1 infection is a prerequisite for the development of innovative antiviral therapies. Here we show that the suppressor of G2 allele of skp1 (SUGT1) is a permissive factor for human immunodeficiency virus (HIV)-1 infection. Expression of SUGT1 increases in infected cells on human brain sections and in permissive host cells. We found that SUGT1 determines the permissiveness to infection of lymphocytes and macrophages by modulating the nuclear import of the viral genome. More importantly, SUGT1 stabilizes the microtubule plus-ends (+MTs) of host cells (through the modulation of microtubule acetylation and the formation of end-binding protein 1 (EB1) comets). This effect on microtubules favors HIV-1 retrograde trafficking and replication. SUGT1 depletion impairs the replication of HIV-1 patient primary isolates and mutant virus that is resistant to raltegravir antiretroviral agent. Altogether our results identify SUGT1 as a cellular factor involved in the post-entry steps of HIV-1 infection that may be targeted for new therapeutic approaches.
- Published
- 2020
- Full Text
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27. Alpha-Synuclein FRET Biosensors Reveal Early Alpha-Synuclein Aggregation in the Endoplasmic Reticulum.
- Author
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Miraglia F, Valvano V, Rota L, Di Primio C, Quercioli V, Betti L, Giannaccini G, Cattaneo A, and Colla E
- Abstract
Endoplasmic reticulum (ER) dysfunction is important for alpha-synuclein (αS) acquired toxicity. When targeted to the ER in SH-SY5Y cells, transient or stable expression of αS resulted in the formation of compact αS-positive structures in a small subpopulation of cells, resembling αS inclusions. Thus, because of the limitations of immunofluorescence, we developed a set of αS FRET biosensors (AFBs) able to track αS conformation in cells. In native conditions, expression in i36, a stable cell line for ER αS, of intermolecular AFBs, reporters in which CFP or YFP has been fused with the C-terminal of αS (αS-CFP/αS-YFP), resulted in no Förster resonance energy transfer (FRET), whereas expression of the intramolecular AFB, a probe obtained by fusing YFP and CFP with αS N- or C- termini (YFP-αS-CFP), showed a positive FRET signal. These data confirmed that αS has a predominantly globular, monomeric conformation in native conditions. Differently, under pro-aggregating conditions, the intermolecular AFB was able to sense significantly formation of αS oligomers, when AFB was expressed in the ER rather than ubiquitously, suggesting that the ER can favor changes in αS conformation when aggregation is stimulated. These results show the potential of AFBs as a new, valuable tool to track αS conformational changes in vivo.
- Published
- 2020
- Full Text
- View/download PDF
28. Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes.
- Author
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Siano G, Caiazza MC, Varisco M, Calvello M, Quercioli V, Cattaneo A, and Di Primio C
- Subjects
- Alzheimer Disease pathology, Cell Differentiation, Cell Line, Tumor, Cell Nucleus metabolism, Humans, Neurons metabolism, Subcellular Fractions, Vesicular Glutamate Transport Protein 1 metabolism, Alzheimer Disease genetics, Gene Expression Regulation, tau Proteins metabolism
- Abstract
Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, recent evidence indicated that Tau is present also in other subcellular compartments including the nucleus where it is implicated in DNA protection, in rRNA transcription, in the mobility of retrotransposons and in the structural organization of the nucleolus. We have recently demonstrated that nuclear Tau is involved in the expression of the VGluT1 gene, suggesting a molecular mechanism that could explain the pathological increase of glutamate release in the early stages of Alzheimer's disease. Until recently, the involvement of nuclear Tau in modulating the expression of target genes has been relatively uncertain and ambiguous due to technical limitations that prevented the exclusion of the contribution of cytoplasmic Tau or the effect of other downstream factors not related to nuclear Tau. To overcome this uncertainty, we developed a method to study the expression of target genes specifically modulated by the nuclear Tau protein. We employed a protocol that couples the use of localization signals and the subcellular fractionation, allowing the exclusion of the interference from the cytoplasmic Tau molecules. Most notably, the protocol is easy and is composed of classic and reliable methods that are broadly applicable to study the nuclear function of Tau in other cell types and cellular conditions.
- Published
- 2019
- Full Text
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29. Identification of an ERK Inhibitor as a Therapeutic Drug Against Tau Aggregation in a New Cell-Based Assay.
- Author
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Siano G, Caiazza MC, Ollà I, Varisco M, Madaro G, Quercioli V, Calvello M, Cattaneo A, and Di Primio C
- Abstract
Formation of Tau aggregates is a common pathological feature of tauopathies and their accumulation directly correlates with cytotoxicity and neuronal degeneration. Great efforts have been made to understand Tau aggregation and to find therapeutics halting or reversing the process, however, progress has been slowed due to the lack of a suitable method for monitoring Tau aggregation. We developed a cell-based assay allowing to detect and quantify Tau aggregation in living cells. The system is based on the FRET biosensor CST able to monitor the molecular dynamic of Tau aggregation in different cellular conditions. We probed candidate compounds that could block Tau hyperphosphorylation. In particular, to foster the drug discovery process, we tested kinase inhibitors approved for the treatment of other diseases. We identified the ERK inhibitor PD-901 as a promising therapeutic molecule since it reduces and prevents Tau aggregation. This evidence establishes the CST cell-based aggregation assay as a reliable tool for drug discovery and suggests that PD-901 might be a promising compound to be tested for further preclinical studies on AD.
- Published
- 2019
- Full Text
- View/download PDF
30. Tau Modulates VGluT1 Expression.
- Author
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Siano G, Varisco M, Caiazza MC, Quercioli V, Mainardi M, Ippolito C, Cattaneo A, and Di Primio C
- Subjects
- Cell Line, Cell Line, Tumor, Chromatin genetics, Gene Expression genetics, HeLa Cells, Humans, Microtubules genetics, Mutation genetics, Tauopathies genetics, Vesicular Glutamate Transport Protein 1 genetics, tau Proteins genetics
- Abstract
Tau displacement from microtubules is the first step in the onset of tauopathies and is followed by toxic protein aggregation. However, other non-canonical functions of Tau might have a role in these pathologies. Here, we demonstrate that a small amount of Tau localizes in the nuclear compartment and accumulates in both the soluble and chromatin-bound fractions. We show that favoring Tau nuclear translocation and accumulation, by Tau overexpression or detachment from MTs, increases the expression of VGluT1, a disease-relevant gene directly involved in glutamatergic synaptic transmission. Remarkably, the P301L mutation, related to frontotemporal dementia FTDP-17, impairs this mechanism leading to a loss of function. Altogether, our results provide the demonstration of a direct physiological role of Tau on gene expression. Alterations of this mechanism may be at the basis of the onset of neurodegeneration., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
31. The Distance between N and C Termini of Tau and of FTDP-17 Mutants Is Modulated by Microtubule Interactions in Living Cells.
- Author
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Di Primio C, Quercioli V, Siano G, Rovere M, Kovacech B, Novak M, and Cattaneo A
- Abstract
The microtubule (MT)-associated protein Tau is a natively unfolded protein, involved in a number of neurodegenerative disorders, collectively called tauopathies, aggregating in neurofibrillary tangles (NFT). It is an open question how the conversion from a MT bound molecule to an aggregation-prone Tau species occurs and, also, if and how tauopathy-related mutations affect its behavior in the cell. To address these points, we exploited a genetically encoded FRET sensor based on the full length Tau protein, to monitor in real time Tau conformational changes in different conditions in live cells. By studying the FRET signal we found that soluble Tau molecules, detached from MTs, display an unfolded structure. On the contrary, we observed an increased FRET signal generated by Tau monomers bound to MT, indicating that the association with MTs induced a folding of Tau protein, decreasing the distance between its N and C termini. We exploited the FRET sensor to investigate the impact of FTDP-17 mutations and of phosphorylation-site mutations on Tau folding and mobility in live cells. We demonstrated that the FTDP-17 Tau mutations weaken the interaction of Tau with cellular MTs, shifting the equilibrium towards the soluble pool while, conversely, phosphorylation site mutations shift the equilibrium of Tau towards the MT-bound state and a more closed conformation.
- Published
- 2017
- Full Text
- View/download PDF
32. Post-translational selective intracellular silencing of acetylated proteins with de novo selected intrabodies.
- Author
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Chirichella M, Lisi S, Fantini M, Goracci M, Calvello M, Brandi R, Arisi I, D'Onofrio M, Di Primio C, and Cattaneo A
- Subjects
- Acetylation, Humans, Antibodies immunology, HIV Integrase immunology, HIV Integrase metabolism, Histones immunology, Histones metabolism, Protein Processing, Post-Translational immunology
- Abstract
The ability to selectively interfere with post-translationally modified proteins would have many biological and therapeutic applications. However, post-translational modifications cannot be selectively targeted by nucleic-acid-based interference approaches. Here we describe post-translational intracellular silencing antibody technology (PISA), a method for selecting intrabodies against post-translationally modified proteins. We demonstrate our method by generating intrabodies against native acetylated proteins and showing functional interference in living cells.
- Published
- 2017
- Full Text
- View/download PDF
33. Comparative Analysis of HIV-1 and Murine Leukemia Virus Three-Dimensional Nuclear Distributions.
- Author
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Quercioli V, Di Primio C, Casini A, Mulder LCF, Vranckx LS, Borrenberghs D, Gijsbers R, Debyser Z, and Cereseto A
- Subjects
- Animals, Green Fluorescent Proteins genetics, HIV-1 genetics, HIV-1 ultrastructure, HeLa Cells, Humans, Integrases genetics, Mice, Microscopy, Fluorescence, Moloney murine leukemia virus ultrastructure, Virus Integration, Cell Nucleus ultrastructure, Cell Nucleus virology, HIV-1 physiology, Moloney murine leukemia virus physiology
- Abstract
Recent advances in fluorescence microscopy allow three-dimensional analysis of HIV-1 preintegration complexes in the nuclei of infected cells. To extend this investigation to gammaretroviruses, we engineered a fluorescent Moloney murine leukemia virus (MLV) system consisting of MLV-integrase fused to enhanced green fluorescent protein (MLV-IN-EGFP). A comparative analysis of lentiviral (HIV-1) and gammaretroviral (MLV) fluorescent complexes in the nuclei of infected cells revealed their different spatial distributions. This research tool has the potential to achieve new insight into the nuclear biology of these retroviruses., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)
- Published
- 2016
- Full Text
- View/download PDF
34. Second generation imaging of nuclear/cytoplasmic HIV-1 complexes.
- Author
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Francis AC, Di Primio C, Quercioli V, Valentini P, Boll A, Girelli G, Demichelis F, Arosio D, and Cereseto A
- Subjects
- Cell Line, Tumor, Fluorescent Dyes analysis, Green Fluorescent Proteins analysis, Green Fluorescent Proteins genetics, HEK293 Cells, HIV Infections virology, HIV-1 genetics, HeLa Cells, Humans, Microscopy, Fluorescence methods, Virus Integration genetics, Virus Replication genetics, Cell Nucleus virology, HIV Infections genetics, HIV-1 physiology, Virus Internalization
- Abstract
The ability to visualize fluorescent HIV-1 particles within the nuclei of infected cells represents an attractive tool to study the nuclear biology of the virus. To this aim we recently developed a microscopy-based fluorescent system (HIV-IN-EGFP) that has proven valid to efficiently visualize HIV-1 complexes in the nuclear compartment and to examine the nuclear import efficiency of the virus. The power of this method to investigate viral events occurring between the cytoplasmic and the nuclear compartment is further shown in this study through the analysis of HIV-IN-EGFP in cells expressing the TRIMCyp restriction factor. In these cells the HIV-IN-EGFP complexes are not detected in the nuclear compartment, while treatment with MG132 reveals an accumulation of HIV-1 complexes in the cytoplasm. However, the Vpr-mediated transincorporation strategy used to incorporate IN fused to EGFP (IN-EGFP) impaired viral infectivity. To optimize the infectivity of the HIV-IN-EGFP, we used mutated forms of IN (E11K and K186E) known to stabilize the IN complexes and to partially restore viral infectivity in transcomplementation experiments. The fluorescent particles produced with the modified IN [HIV-IN(K)EGFP_IN(E)] show almost 30% infectivity as compared to wild-type NL4.3. Detailed confocal microscopy analysis revealed that the newly generated viral particles resulted in HIV-1 complexes significantly smaller in size, thus requiring the use of brighter fluorophores for nuclear visualization [HIV-IN(K)sfGFP_IN(E)]. The second-generation visualization system HIV-IN(K)sfGFP_IN(E), in addition to allowing direct visualization of HIV-1 nuclear entry and other viral events related to nuclear import, preserves intact viral properties in terms of nuclear entry and improved infectivity.
- Published
- 2014
- Full Text
- View/download PDF
35. Single-cell imaging of HIV-1 provirus (SCIP).
- Author
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Di Primio C, Quercioli V, Allouch A, Gijsbers R, Christ F, Debyser Z, Arosio D, and Cereseto A
- Subjects
- Cell Line, Tumor, Chromatin Immunoprecipitation, DNA Breaks, Double-Stranded, DNA Primers genetics, DNA Repair genetics, Deoxyribonucleases, Type II Site-Specific, Humans, RNA Interference, Real-Time Polymerase Chain Reaction, Saccharomyces cerevisiae Proteins, Single-Cell Analysis methods, HIV-1 ultrastructure, Microscopy, Fluorescence methods, Proviruses ultrastructure
- Abstract
Recent advances in fluorescence microscopy provided tools for the investigation and the analysis of the viral replication steps in the cellular context. In the HIV field, the current visualization systems successfully achieve the fluorescent labeling of the viral envelope and proteins, but not the genome. Here, we developed a system able to visualize the proviral DNA of HIV-1 through immunofluorescence detection of repair foci for DNA double-strand breaks specifically induced in the viral genome by the heterologous expression of the I-SceI endonuclease. The system for Single-Cell Imaging of HIV-1 Provirus, named SCIP, provides the possibility to individually track integrated-viral DNA within the nuclei of infected cells. In particular, SCIP allowed us to perform a topological analysis of integrated viral DNA revealing that HIV-1 preferentially integrates in the chromatin localized at the periphery of the nuclei.
- Published
- 2013
- Full Text
- View/download PDF
36. The TRIM family protein KAP1 inhibits HIV-1 integration.
- Author
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Allouch A, Di Primio C, Alpi E, Lusic M, Arosio D, Giacca M, and Cereseto A
- Subjects
- Acetylation, Cell Line, HIV Infections genetics, HIV Infections virology, HIV Integrase genetics, HIV Integrase metabolism, HIV-1 enzymology, HIV-1 genetics, Histone Deacetylase 1 genetics, Histone Deacetylase 1 metabolism, Humans, Protein Binding, Repressor Proteins genetics, Tripartite Motif-Containing Protein 28, HIV Infections metabolism, HIV-1 physiology, Repressor Proteins metabolism, Virus Integration
- Abstract
The integration of viral cDNA into the host genome is a critical step in the life cycle of HIV-1. This step is catalyzed by integrase (IN), a viral enzyme that is positively regulated by acetylation via the cellular histone acetyl transferase (HAT) p300. To investigate the relevance of IN acetylation, we searched for cellular proteins that selectively bind acetylated IN and identified KAP1, a protein belonging to the TRIM family of antiviral proteins. KAP1 binds acetylated IN and induces its deacetylation through the formation of a protein complex which includes the deacetylase HDAC1. Modulation of intracellular KAP1 levels in different cell types including T cells, the primary HIV-1 target, revealed that KAP1 curtails viral infectivity by selectively affecting HIV-1 integration. This study identifies KAP1 as a cellular factor restricting HIV-1 infection and underscores the relevance of IN acetylation as a crucial step in the viral infectious cycle., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
37. Role of phosphorylation in the nuclear biology of HIV-1.
- Author
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Francis AC, Di Primio C, Allouch A, and Cereseto A
- Subjects
- HIV Infections virology, HIV-1 drug effects, Humans, Phosphorylation drug effects, Virus Integration drug effects, Anti-HIV Agents pharmacology, HIV Infections drug therapy, HIV-1 physiology, Host-Pathogen Interactions drug effects, Protein Kinase Inhibitors pharmacology, Protein Kinases metabolism, Virus Replication drug effects
- Abstract
The central events of HIV-1 life cycle occur at the nuclear level where the viral genome is integrated into the host cellular DNA in order to be expressed and replicated. The viral pre-integration complexes (PICs) are actively transported in the nuclear compartment where integration occurs in specific regions of the cellular chromatin. Similar to all viruses, HIV-1 encodes for a limited number of proteins that are insufficient to produce new viral progenies. Several cellular pathways are thus hijacked by HIV-1 to efficiently complete the replication cycle. The majority of viral proteins are substrates for cellular kinases indicating a pivotal role of these cellular enzymes at multiple steps of the HIV-1 life cycle. The nuclear biology of the cell is highly controlled by kinases (nuclear transport, DNA replication, repair and transcription) and many of these kinases also sustain the viral nuclear events. This review summarizes our current knowledge on kinases that are involved in HIV-1 replication cycle at the nuclear level, both directly through their catalytic activity on viral proteins and indirectly being activated by the virus. Among viral proteins directly modified by kinases is integrase (IN) the factor that catalyzes the integration of HIV-1 in the cellular genome. Notably, this recent discovery may shed light onto mechanisms underlying the different susceptibility of the main cell types targeted by HIV-1 (CD-4+ T-cell) depending on their activation status. Alternatively, kinases may act indirectly such as in the case of DNA repair factors activated following HIV-1 infection and demonstrated to regulate the viral life cycle. Finally, inhibition of cellular kinases interacting with HIV-1 at the nuclear level has been shown to severely affect the viral replication cycle, thus suggesting potential new therapeutic approaches.
- Published
- 2011
- Full Text
- View/download PDF
38. GCN5-dependent acetylation of HIV-1 integrase enhances viral integration.
- Author
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Terreni M, Valentini P, Liverani V, Gutierrez MI, Di Primio C, Di Fenza A, Tozzini V, Allouch A, Albanese A, Giacca M, and Cereseto A
- Subjects
- Acetylation, Amino Acid Substitution, Cell Line, Gene Knockdown Techniques, HIV Integrase genetics, Humans, Lysine genetics, Lysine metabolism, Mutagenesis, Site-Directed, p300-CBP Transcription Factors genetics, HIV Integrase metabolism, HIV-1 pathogenicity, Host-Pathogen Interactions, Virus Integration, p300-CBP Transcription Factors metabolism
- Abstract
Background: An essential event during the replication cycle of HIV-1 is the integration of the reverse transcribed viral DNA into the host cellular genome. Our former report revealed that HIV-1 integrase (IN), the enzyme that catalyzes the integration reaction, is positively regulated by acetylation mediated by the histone acetyltransferase (HAT) p300., Results: In this study we demonstrate that another cellular HAT, GCN5, acetylates IN leading to enhanced 3'-end processing and strand transfer activities. GCN5 participates in the integration step of HIV-1 replication cycle as demonstrated by the reduced infectivity, due to inefficient provirus formation, in GCN5 knockdown cells. Within the C-terminal domain of IN, four lysines (K258, K264, K266, and K273) are targeted by GCN5 acetylation, three of which (K264, K266, and K273) are also modified by p300. Replication analysis of HIV-1 clones carrying substitutions at the IN lysines acetylated by both GCN5 and p300, or exclusively by GCN5, demonstrated that these residues are required for efficient viral integration. In addition, a comparative analysis of the replication efficiencies of the IN triple- and quadruple-mutant viruses revealed that even though the lysines targeted by both GCN5 and p300 are required for efficient virus integration, the residue exclusively modified by GCN5 (K258) does not affect this process., Conclusions: The results presented here further demonstrate the relevance of IN post-translational modification by acetylation, which results from the catalytic activities of multiple HATs during the viral replication cycle. Finally, this study contributes to clarifying the recent debate raised on the role of IN acetylated lysines during HIV-1 infection.
- Published
- 2010
- Full Text
- View/download PDF
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