Viral Apoptosis Evasion via the MAPK Pathway by Use of a Host Long Noncoding RNA

An emerging realization of infectious disease is that pathogens can cause a high incidence of genetic instability within the host as a result of infection-induced DNA lesions. These often lead to classical hallmarks of cancer, one of which is the ability to evade apoptosis despite the presence of numerous genetic mutations that should be otherwise lethal. The Human Immunodeficiency Virus type 1 (HIV-1) is one such pathogen as it induces apoptosis in CD4+ T cells but is largely non-cytopathic in macrophages. As a consequence there is long-term dissemination of the pathogen specifically by these infected yet surviving host cells. Apoptosis is triggered by double-strand breaks (DSBs), such as those induced by integrating retroviruses like HIV-1, and is coordinated by the p53-regulated long noncoding RNA lincRNA-p21. As is typical for a long noncoding RNA, lincRNA-p21 mediates its activities in a complex with one of its two protein binding partners, namely HuR and hnRNP-K. In this work, we monitor the cellular response to infection to determine how HIV-1 induces DSBs in macrophages yet evades apoptosis in these cells. We show that the virus does so by securing the pro-survival MAP2K1/ERK2 cascade early upon entry, in a gp120-dependent manner, to orchestrate a complex dysregulation of lincRNA-p21. By sequestering the lincRNA-p21 partner HuR in the nucleus, HIV-1 enables lincRNA-p21 degradation. Simultaneously, the virus permits transcription of pro-survival genes by sequestering lincRNA-p21's other protein partner hnRNP-K in the cytoplasm via the MAP2K1/ERK2 pathway. Of particular note, this MAP2K1/ERK2 pro-survival cascade is switched off during T cell maturation and is thus unavailable for similar viral manipulation in mature CD4+ T cells. We show that the introduction of MAP2K1, ERK2, or HDM2 inhibitors in HIV-infected macrophages results in apoptosis, providing strong evidence that the viral-mediated apoptotic block can be released, specifically by restoring the nuclear interaction of lincRNA-p21 and its apoptosis protein partner hnRNP-K. Together, these results reveal a unique example of pathogenic control over mammalian apoptosis and DNA damage via a host long noncoding RNA, and present MAP2K1/ERK2 inhibitors as a novel therapeutic intervention strategy for HIV-1 infection in macrophages

Two NEMO-like Ubiquitin-Binding Domains in CEP55 Differently Regulate Cytokinesis

International audience(F.A.) HIGHLIGHTS CEP55 contains two NEMO-like NOA and UBZ domains CEP55 NOA and UBZ are crucial for the CEP55 function in cytokinetic coordination UBZ CEP55 functions as cargo receptor to the midbody in a ubiquitin-dependent manner UBZ CEP55 preferentially binds non-degradative linear and K63 polyubiquitin chains Said Halidi et al., iScience 20, SUMMARY CEP55 regulates the final critical step of cell division termed cytokinetic abscission. We report herein that CEP55 contains two NEMO-like ubiquitin-binding domains (UBDs), NOA and ZF, which regulate its function in a different manner. In vitro studies of isolated domains showed that NOA adopts a dimeric coiled-coil structure, whereas ZF is based on a UBZ scaffold. Strikingly, CEP55 knocked-down HeLa cells reconstituted with the full-length CEP55 ubiquitin-binding defective mutants, containing structure-guided mutations either in NOA CEP55 or ZF CEP55 domains, display severe abscission defects. In addition, the ZF CEP55 can be functionally replaced by some ZF-based UBDs belonging to the UBZ family, indicating that the essential function of ZF CEP55 is to act as ubiquitin receptor. Our work reveals an unexpected role of CEP55 in non-degradative ubiquitin signaling during cytokinetic abscis-sion and provides a molecular basis as to how CEP55 mutations can lead to neurological disorders such as the MARCH syndrome

HIV Cell-to-Cell Spread Results in Earlier Onset of Viral Gene Expression by Multiple Infections per Cell

Cell-to-cell spread of HIV, a directed mode of viral transmission, has been observed to be more rapid than cell-free infection. However, a mechanism for earlier onset of viral gene expression in cell-to-cell spread was previously uncharacterized. Here we used time-lapse microscopy combined with automated image analysis to quantify the timing of the onset of HIV gene expression in a fluorescent reporter cell line, as well as single cell staining for infection over time in primary cells. We compared cell-to-cell spread of HIV to cell-free infection, and limited both types of transmission to a two-hour window to minimize differences due to virus transit time to the cell. The mean time to detectable onset of viral gene expression in cell-to-cell spread was accelerated by 19% in the reporter cell line and by 35% in peripheral blood mononuclear cells relative to cell-free HIV infection. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed onset. We recapitulated the earlier onset by infecting with multiple cell-free viruses per cell. Surprisingly, the acceleration in onset of viral gene expression was not explained by cooperativity between infecting virions. Instead, more rapid onset was consistent with a model where the fastest expressing virus out of the infecting virus pool sets the time for infection independently of the other co-infecting viruses

Die Zell-zu-Zell-Verbreitung von HIV ergibt vorzeitigen Anfang der viralen Genexpression durch vielfache Infektion pro Zelle

Knowledge about the HIV cycle in targeted human cells derives from studies of cell-free infection, whereby distant transmission occurs after viral budding and environmental dispersion. Recently, it has been proven that some viruses, including retroviruses, can be concentrated into multiply infected targets by cell-to-cell spread through virological synapses. Therefore, we investigated whether, how much and how the virus cycle can be modified by the transmission mode. Methodology We followed the onset of HIV-1 gene expression in a fluorescent reporter T-lymphocyte cell line by time-lapse microscopy together with quantitative image analysis. Cell-free was compared to coculture infection, whereby transmission can occur by cell-to-cell and cell-free spread modes. In both conditions, transmission was synchronized to a two-hour window. Target cells were separated from donors by fluorescent markers. The influence of the presence of infected donors was analysed by transwell experiment as well as co-incubation with donors unable to transmit their viruses. The effect of the multiplicity of infection (MOI) was observed in cell-free infection. Viral cooperativity was studied by co-infection with fluorescently labelled viruses. Experiments were also performed under similar conditions in primary human PBMCs and CD4+ T cells using flow cytometry. Results were summarized by mathematical modeling and the average number of viruses per target cell was confirmed by assessing the infection robustness in the face of raltegravir. Result The mean time to detectable onset of viral gene expression in coculture was accelerated by 19% in the reporter cell line and by 35% in PBMCs relative to cell-free infection. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed the dynamics. High MOI cell-free infection recapitulated the accelerated viral gene expression observed in cell-to-cell infection. Co-infection with differently labelled viruses did not reveal viral cooperativity or inhibition. Cell-free infection time courses were fitted by Gamma distributions using the different measured MOI values as parameters. The acceleration of viral gene expression with coculture was predicted by an effective MOI of 4.6 per cell, consistent with the number estimated by fitting the drug sensitivity data. Conclusion The more rapid onset of viral gene expression by cell-to-cell spread was consistent with a stochastic model where the fastest expressing virus out of the infecting virus pool sets the time for infection independently of the other co-infecting viruses. This reduction of the eclipse phase could facilitate the establishment of HIV reservoirs.Die Kentnisse über den HIV-Zyklus in menschlichen Zielzellen leitet sich aus zellfreien Infektionen ab, bei welchen nach viraler Knospung und anschließender Dispersion in das umliegenden Milieu die Übertragung erfolgt. Kürzlich wurde bewiesen, dass einige Viren, einschließlich Retroviren, durch eine virologische Synapse, beziehungsweise durch Zell-zu-Zell-Übertragung, in mehrfach infizierten Zellen konzentriert werden können. Daher untersuchten wir, ob, wie viel und wie der Viruszyklus von den Verbreitungsmodi modifiziert werden kann. Methode Wir folgten dem Beginn der HIV-1-Genexpression in einer fluoreszierenden Reporter-T- Lymphozyten-Zelllinie mit Hilfe von Zeitraffer- Mikroskopie und quantitativer Bildanalyse. Zellfreie Infektion wurde mit Kokultur verglichen, wobei Zell-zu-Zell sowie zellfreie Übertragungen erfolgen. In beiden Fällen wurde die Übertragung auf eine zweistündige Zeitspanne synchronisiert. Die Zielzellen wurden durch Fluoreszenzmarker von den Spenderzellen unterschieden. Der Einfluss des Vorhandenseins von infizierten Spendern wurde durch Transwell-Experimente sowie Koinkubation mit Spendern analysiert, welche ihre Viren nicht übertragen können. Die Wirkung der Multiplizität der Infektion (MOI) wurde in zellfreier Infektion beobachtet. Die Viruskooperativität wurde durch Koinfektion mit verschiedenen fluoreszent markierten Viren untersucht. Experimente wurden auch unter ähnlichen Bedingungen in primären humanen PBMCs und CD4+ T-Zellen anhand von Durchflusszytometrie ausgeführt. Die Ergebnisse wurden durch mathematische Modellierung zusammengefasst und die durchschnittliche Anzahl von Viren pro Zielzelle durch die Abschätzung der Robustheit der Infektion während der Behandlung mit Raltegravir bestätigt. Ergebnisse Die mittlere Zeit bis zum nachweisbaren Anfang der viralen Genexpression in Kokultur wurde im Vergleich zur zellfreien Infektion in der Reporterzelllinie um 19% und in PBMCs um 35% beschleunigt. Weder Faktoren, welche von infizierten Zellen sezerniert wurden, noch Kontakt mit infizierten Zellen ohne Übertragung veränderten die Dynamik. Zellfreie Infektion mit hoher MOI zeigte ebenfalls die beschleunigte Virusgenexpression, welche bei einer Zell- zu-Zell-Übertragung beobachtet wurde. Koinfektion mit unterschiedlich markierten Viren zeigte weder virale Kooperation noch Hemmung. Zellfreie Infektionszeitverläufe wurden durch Gammaverteilungen unter Verwendung der verschiedenen gemessenen MOI-Werte als Parameter beschrieben. Die Beschleunigung der viralen Genexpression mit Kokultur wurde durch eine effektive MOI von 4,6 pro Zelle berechnet, welche mit der durch die Modellierung der Wirkstoffempfindlichkeitsdaten geschätzten Zahl übereinstimmt. Schlussfolgerung Der vorzeitge Beginn der viralen Genexpression durch Zell-zu-Zell-Verbreitung stimmt mit einem stochastischen Modell überein, bei welchem das schnellste Virus aus dem infizierenden Viruspool die Zeit der Infektion unabhängig von den anderen koinfizierenden Viren festlegt. Diese Verringerung der Eklipsephase könnte die Bildung von HIV-Reservoirs erleichtern

High-Content RNAi Phenotypic Screening Unveils the Involvement of Human Ubiquitin-Related Enzymes in Late Cytokinesis

International audienceCEP55 is a central regulator of late cytokinesis and is overexpressed in numerous cancers. Its post-translationally controlled recruitment to the midbody is crucial to the structural coordination of the abscission sequence. Our recent evidence that CEP55 contains two ubiquitin-binding domains was the first structural and functional link between ubiquitin signaling and ESCRT-mediated severing of the intercellular bridge. So far, high-content screens focusing on cytokinesis have used multinucleation as the endpoint readout. Here, we report an automated image-based detection method of intercellular bridges, which we applied to further our understanding of late cytokinetic signaling by performing an RNAi screen of ubiquitin ligases and deubiquitinases. A secondary validation confirmed four candidate genes, i.e., LNX2, NEURL, UCHL1 and RNF157, whose downregulation variably affects interconnected phenotypes related to CEP55 and its UBDs, as follows: decreased recruitment of CEP55 to the midbody, increased number of midbody remnants per cell, and increased frequency of intercellular bridges or multinucleation events. This brings into question the Notch-dependent or independent contributions of LNX2 and NEURL proteins to late cytokinesis. Similarly, the role of UCHL1 in autophagy could link its function with the fate of midbody remnants. Beyond the biological interest, this high-content screening approach could also be used to isolate anticancer drugs that act by impairing cytokinesis and CEP55 functions

Generation of IPi001-A/B/C human induced pluripotent stem cell lines from healthy amniotic fluid cells

Human induced Pluripotent Stem Cells (hiPSCs) represent an invaluable source of primary cells to investigate development, establish cell and disease models, provide material for regenerative medicine and allow more physiological high-content screenings. Here, we generated three healthy hiPSC control lines - IPi001-A/B/C - from primary amniotic fluid cells (AFCs), an infrequently used source of cells, which can be readily obtained from amniocentesis for the prenatal diagnosis of numerous genetic disorders. These AFCs were reprogrammed by non-integrative viral transduction. The resulting hiPSCs displayed normal karyotype and expressed classic pluripotency hallmarks

A novel screening strategy to identify histone methyltransferase inhibitors reveals a crosstalk between DOT1L and CARM1

International audienceEpigenetic regulation is a dynamic and reversible process that controls gene expression. Abnormal function results in human diseases such as cancer, thus the enzymes that establish epigenetic marks, such as histone methyltransferases (HMTs), are potentially therapeutic targets. Noteworthily, HMTs form multiprotein complexes that in concert regulate gene expression. To probe epigenetic protein complexes regulation in cells, we developed a reliable chemical biology high-content imaging strategy to screen compound libraries simultaneously on multiple histone marks inside cells. By this approach, we identified that compound 4, a published CARM1 inhibitor, inhibits both histone mark H3R2me2a, regulated also by CARM1, and H3K79me2, regulated only by DOT1L, pointing out a crosstalk between CARM1 and DOT1L. Based on this interaction, we combined compound 4 and DOT1L inhibitor EPZ-5676 resulting in a stronger inhibition of cell proliferation and increase in apoptosis, indicating that our approach identifies possible effective synergistic drug combinations

Image_1_Viral Apoptosis Evasion via the MAPK Pathway by Use of a Host Long Noncoding RNA.TIFF

<p>An emerging realization of infectious disease is that pathogens can cause a high incidence of genetic instability within the host as a result of infection-induced DNA lesions. These often lead to classical hallmarks of cancer, one of which is the ability to evade apoptosis despite the presence of numerous genetic mutations that should be otherwise lethal. The Human Immunodeficiency Virus type 1 (HIV-1) is one such pathogen as it induces apoptosis in CD4+ T cells but is largely non-cytopathic in macrophages. As a consequence there is long-term dissemination of the pathogen specifically by these infected yet surviving host cells. Apoptosis is triggered by double-strand breaks (DSBs), such as those induced by integrating retroviruses like HIV-1, and is coordinated by the p53-regulated long noncoding RNA lincRNA-p21. As is typical for a long noncoding RNA, lincRNA-p21 mediates its activities in a complex with one of its two protein binding partners, namely HuR and hnRNP-K. In this work, we monitor the cellular response to infection to determine how HIV-1 induces DSBs in macrophages yet evades apoptosis in these cells. We show that the virus does so by securing the pro-survival MAP2K1/ERK2 cascade early upon entry, in a gp120-dependent manner, to orchestrate a complex dysregulation of lincRNA-p21. By sequestering the lincRNA-p21 partner HuR in the nucleus, HIV-1 enables lincRNA-p21 degradation. Simultaneously, the virus permits transcription of pro-survival genes by sequestering lincRNA-p21's other protein partner hnRNP-K in the cytoplasm via the MAP2K1/ERK2 pathway. Of particular note, this MAP2K1/ERK2 pro-survival cascade is switched off during T cell maturation and is thus unavailable for similar viral manipulation in mature CD4+ T cells. We show that the introduction of MAP2K1, ERK2, or HDM2 inhibitors in HIV-infected macrophages results in apoptosis, providing strong evidence that the viral-mediated apoptotic block can be released, specifically by restoring the nuclear interaction of lincRNA-p21 and its apoptosis protein partner hnRNP-K. Together, these results reveal a unique example of pathogenic control over mammalian apoptosis and DNA damage via a host long noncoding RNA, and present MAP2K1/ERK2 inhibitors as a novel therapeutic intervention strategy for HIV-1 infection in macrophages.</p

Modelling faster onset of HIV gene expression by multiple infections per cell.

<p>(<b>A</b>) Proposed probabilistic mechanism for the more rapid onset of HIV gene expression. (<b>B</b>) Simulation of cell-free infection times at different MOI by random draws from a Gamma distribution parametrizing the best fit cell-free infection times of single virion infections. Circles are experimental data from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005964#ppat.1005964.g003" target="_blank">Fig 3A</a>, dashed lines represent the simulation results for MOI of 0.1 (blue), 0.5 (green), 2 (red), and 4 (purple). Fitted means±std of infection times were 26.4±5.2, 25.2±5.1, 22.7±4.8, and 21.9±4.9 hours. (<b>C</b>) Fit of coculture versus cell-free infection. Circles are experimental data from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005964#ppat.1005964.g001" target="_blank">Fig 1C</a>, dashed lines represent the simulation results. Means±std for the fits were 28.0±5.0 hours for coculture and 34.5±6.2 hours for cell-free infections. Best fit MOI for the coculture infection was 4.6.</p