RESUMO
BACKGROUND: The Human T-cell Lymphotropic Virus Type-1 (HTLV-1) is a blood-borne pathogen and etiological agent of Adult T-cell Leukemia/Lymphoma (ATLL) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). HTLV-1 has currently infected up to 10 million globally with highly endemic areas in Japan, Africa, the Caribbean and South America. We have previously shown that Extracellular Vesicles (EVs) enhance HTLV-1 transmission by promoting cell-cell contact. RESULTS: Here, we separated EVs into subpopulations using differential ultracentrifugation (DUC) at speeds of 2 k (2000×g), 10 k (10,000×g), and 100 k (100,000×g) from infected cell supernatants. Proteomic analysis revealed that EVs contain the highest viral/host protein abundance in the 2 k subpopulation (2 k > 10 k > 100 k). The 2 k and 10 k populations contained viral proteins (i.e., p19 and Tax), and autophagy proteins (i.e., LC3 and p62) suggesting presence of autophagosomes as well as core histones. Interestingly, the use of 2 k EVs in an angiogenesis assay (mesenchymal stem cells + endothelial cells) caused deterioration of vascular-like-tubules. Cells commonly associated with the neurovascular unit (i.e., astrocytes, neurons, and macrophages) in the blood-brain barrier (BBB) showed that HTLV-1 EVs may induce expression of cytokines involved in migration (i.e., IL-8; 100 k > 2 k > 10 k) from astrocytes and monocyte-derived macrophages (i.e., IL-8; 2 k > 10 k). Finally, we found that EVs were able to promote cell-cell contact and viral transmission in monocytic cell-derived dendritic cell. The EVs from both 2 k and 10 k increased HTLV-1 spread in a humanized mouse model, as evidenced by an increase in proviral DNA and RNA in the Blood, Lymph Node, and Spleen. CONCLUSIONS: Altogether, these data suggest that various EV subpopulations induce cytokine expression, tissue damage, and viral spread.
Assuntos
Células Endoteliais/virologia , Vesículas Extracelulares/virologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Animais , Comunicação Celular , Citocinas/análise , Citocinas/genética , Citocinas/imunologia , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/fisiologia , Feminino , Infecções por HTLV-I/virologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Proteômica , Células THP-1 , Células U937RESUMO
BACKGROUND: HIV-1 patients receiving combination antiretroviral therapy (cART) survive infection but require life-long adherence at high expense. In chronic cART-treated patients with undetectable viral titers, cell-associated viral RNA is still detectable, pointing to low-level viral transcriptional leakiness. To date, there are no FDA-approved drugs against HIV-1 transcription. We have previously shown that F07#13, a third generation Tat peptide mimetic with competitive activity against Cdk9/T1-Tat binding sites, inhibits HIV-1 transcription in vitro and in vivo. RESULTS: Here, we demonstrate that increasing concentrations of F07#13 (0.01, 0.1, 1 µM) cause a decrease in Tat levels in a dose-dependent manner by inhibiting the Cdk9/T1-Tat complex formation and subsequent ubiquitin-mediated Tat sequestration and degradation. Our data indicate that complexes I and IV contain distinct patterns of ubiquitinated Tat and that transcriptional inhibition induced by F07#13 causes an overall reduction in Tat levels. This reduction may be triggered by F07#13 but ultimately is mediated by TAR-gag viral RNAs that bind suppressive transcription factors (similar to 7SK, NRON, HOTAIR, and Xist lncRNAs) to enhance transcriptional gene silencing and latency. These RNAs complex with PRC2, Sin3A, and Cul4B, resulting in epigenetic modifications. Finally, we observed an F07#13-mediated decrease of viral burden by targeting the R region of the long terminal repeat (HIV-1 promoter region, LTR), promoting both paused polymerases and increased efficiency of CRISPR/Cas9 editing in infected cells. This implies that gene editing may be best performed under a repressed transcriptional state. CONCLUSIONS: Collectively, our results indicate that F07#13, which can terminate RNA Polymerase II at distinct sites, can generate scaffold RNAs, which may assemble into specific sets of "RNA Machines" that contribute to gene regulation. It remains to be seen whether these effects can also be seen in various clades that have varying promoter strength, mutant LTRs, and in patient samples.
Assuntos
Regulação Viral da Expressão Gênica/efeitos dos fármacos , HIV-1/genética , RNA não Traduzido/genética , Transcrição Gênica , Antirretrovirais/farmacologia , Biomimética , Sistemas CRISPR-Cas , Linhagem Celular , Edição de Genes , Inativação Gênica , HIV-1/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas , RNA Viral/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/químicaRESUMO
Background: Ebola virus (EBOV) mainly targets myeloid cells; however, extensive death of T cells is often observed in lethal infections. We have previously shown that EBOV VP40 in exosomes causes recipient immune cell death. Methods: Using VP40-producing clones, we analyzed donor cell cycle, extracellular vesicle (EV) biogenesis, and recipient immune cell death. Transcription of cyclin D1 and nuclear localization of VP40 were examined via kinase and chromatin immunoprecipitation assays. Extracellular vesicle contents were characterized by mass spectrometry, cytokine array, and western blot. Biosafety level-4 facilities were used for wild-type Ebola virus infection studies. Results: VP40 EVs induced apoptosis in recipient T cells and monocytes. VP40 clones were accelerated in growth due to cyclin D1 upregulation, and nuclear VP40 was found bound to the cyclin D1 promoter. Accelerated cell cycling was related to EV biogenesis, resulting in fewer but larger EVs. VP40 EV contents were enriched in ribonucleic acid-binding proteins and cytokines (interleukin-15, transforming growth factor-ß1, and interferon-γ). Finally, EBOV-infected cell and animal EVs contained VP40, nucleoprotein, and glycoprotein. Conclusions: Nuclear VP40 upregulates cyclin D1 levels, resulting in dysregulated cell cycle and EV biogenesis. Packaging of cytokines and EBOV proteins into EVs from infected cells may be responsible for the decimation of immune cells during EBOV pathogenesis.
Assuntos
Ciclo Celular/fisiologia , Ebolavirus/metabolismo , Vesículas Extracelulares/metabolismo , Doença pelo Vírus Ebola/metabolismo , Doença pelo Vírus Ebola/virologia , Nucleoproteínas/metabolismo , Proteínas do Core Viral/metabolismo , Apoptose/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Ciclina D1/metabolismo , Exossomos/metabolismo , Vesículas Extracelulares/virologia , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Regiões Promotoras Genéticas/fisiologia , Ligação Proteica/fisiologia , Células U937 , Regulação para Cima/fisiologia , Proteínas da Matriz Viral/metabolismoRESUMO
Currently, there is no cure for human immunodeficiency virus type 1 (HIV-1) infection. However, combined antiretroviral therapy (cART) aids in viral latency and prevents the progression of HIV-1 infection into acquired immunodeficiency syndrome (AIDS). cART has extended many lives, but people living with HIV-1 (PLWH) face lifelong ailments such as HIV-associated neurocognitive disorders (HAND) that range from asymptomatic HAND to HIV-1-associated dementia. HAND has been attributed to chronic inflammation and low-level infection within the central nervous system (CNS) caused by proinflammatory cytokines and viral products. These molecules are shuttled into the CNS within extracellular vesicles (EVs), lipid bound nanoparticles, and are released from cells as a form of intercellular communication. This study investigates the impact of cannabidiol (CBD), as a promising and potential therapeutic for HAND patients, and a similar synthetic molecule, HU308, on the EVs released from HIV-1-infected myeloid cells as well as HIV-1-infected 3D neurospheres. The data shows that both CBD and HU308 decrease non-coding and coding viral RNA (TAR and env) as well as proinflammatory cytokines as IL-1ß and TNF-α mRNA. This decrease in viral RNA occurs in in vitro differentiated primary macrophages, in EVs released from HIV-1-infected cells monocytes, and infected neurospheres. Furthermore, a 3D neurosphere model shows an overall decrease in proinflammatory mRNA with HU308. Finally, using a humanized mouse model of HIV-1 infection, plasma viral RNA was shown to significantly decrease with HU308 alone and was most effective in combination with cART, even when compared to the typical cART treatment. Overall, CBD or HU308 may be a viable option to decrease EV release and associated cytokines which would dampen the virus spread and may be used in effective treatment of HAND in combination with cART.
RESUMO
We characterized the in vivo interstitial fluid (IF) content of extracellular vesicles (EVs) using the GFP-4T1 syngeneic murine cancer model to study EVs in-transit to the draining lymph node. GFP labelling confirmed the IF EV tumour cell origin. Molecular analysis revealed an abundance of IF EV-associated proteins specifically involved in mitophagy and secretory autophagy. A set of proteins required for sequential steps of fission-induced mitophagy preferentially populated the CD81+/PD-L1+ IF EVs; PINK1, TOM20, and ARIH1 E3 ubiquitin ligase (required for Parkin-independent mitophagy), DRP1 and FIS1 (mitochondrial peripheral fission), VDAC-1 (ubiquitination state triggers mitophagy away from apoptosis), VPS35, SEC22b, and Rab33b (vacuolar sorting). Comparing in vivo IF EVs to in vitro EVs revealed 40% concordance, with an elevation of mitophagy proteins in the CD81+ EVs for both murine and human cell lines subjected to metabolic stress. The export of cellular mitochondria proteins to CD81+ EVs was confirmed by density gradient isolation from the bulk EV isolate followed by anti-CD81 immunoprecipitation, molecular sieve chromatography, and MitoTracker export into CD81+ EVs. We propose the 4T1 in vivo model as a versatile tool to functionally characterize IF EVs. IF EV export of fission mitophagy proteins has broad implications for mitochondrial function and cellular immunology.
Assuntos
Vesículas Extracelulares , Neoplasias , Animais , Líquido Extracelular/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Camundongos , Mitofagia , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte VesicularRESUMO
HIV-1 remains an incurable infection that is associated with substantial economic and epidemiologic impacts. HIV-associated neurocognitive disorders (HAND) are commonly linked with HIV-1 infection; despite the development of combination antiretroviral therapy (cART), HAND is still reported to affect at least 50% of HIV-1 infected individuals. It is believed that the over-amplification of inflammatory pathways, along with release of toxic viral proteins from infected cells, are primarily responsible for the neurological damage that is observed in HAND; however, the underlying mechanisms are not well-defined. Therefore, there is an unmet need to develop more physiologically relevant and reliable platforms for studying these pathologies. In recent years, neurospheres derived from induced pluripotent stem cells (iPSCs) have been utilized to model the effects of different neurotropic viruses. Here, we report the generation of neurospheres from iPSC-derived neural progenitor cells (NPCs) and we show that these cultures are permissive to retroviral (e.g. HIV-1, HTLV-1) replication. In addition, we also examine the potential effects of stem cell derived extracellular vesicles (EVs) on HIV-1 damaged cells as there is abundant literature supporting the reparative and regenerative properties of stem cell EVs in the context of various CNS pathologies. Consistent with the literature, our data suggests that stem cell EVs may modulate neuroprotective and anti-inflammatory properties in damaged cells. Collectively, this study demonstrates the feasibility of NPC-derived neurospheres for modeling HIV-1 infection and, subsequently, highlights the potential of stem cell EVs for rescuing cellular damage induced by HIV-1 infection.
Assuntos
Vesículas Extracelulares , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1 , Células-Tronco Pluripotentes Induzidas/virologia , Células-Tronco Neurais/virologia , Células Cultivadas , Vesículas Extracelulares/fisiologia , Infecções por HIV/complicações , HIV-1/fisiologia , Humanos , Transtornos Neurocognitivos/etiologia , Neuroproteção , Replicação ViralRESUMO
Sex-lethal (Sxl), the master regulatory gene of Drosophila somatic sex determination, is stably maintained in an on or an off state by autoregulatory control of Sxl premRNA processing. Establishment of the correct Sxl splicing pattern requires the coordinate regulation of two Sxl promoters. The first of these promoters, SxlPe, responds to the female dose of two X chromosomes to produce a pulse of Sxl protein that acts on the premRNA products from the second promoter, SxlPm, to establish the splicing loop. SxlPm is active in both sexes throughout most of development, but nothing is known about how SxlPm is expressed during the transition from X signal assessment to maintenance splicing. We found that SxlPm is activated earlier in females than in males in a range of Drosophila species, and that its expression overlaps briefly with that of SxlPe during the syncytial blastoderm stage. Activation of SxlPm depends on the scute, daughterless, and runt transcription factors, which communicate X chromosome dose to SxlPe, but is independent of the X signal element sisA and the maternal co-repressor groucho. We show that DNA sequences regulating the response of SxlPe to the X chromosome dose also control the sex-differential response of SxlPm. We propose that co-expression of Sxl protein and its premRNA substrate facilitates the transition from transcriptional to splicing control, and that delayed activation of SxlPm in males buffers against the inappropriate activation of Sxl by fluctuations in the strength of the X chromosome signal.
Assuntos
Drosophila/genética , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Processos de Determinação Sexual , Animais , Proteínas de Drosophila/genética , Feminino , Masculino , Splicing de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Cromossomo XRESUMO
Here, we have attempted to address the timing of EV and virion release from virally infected cells. Uninfected (CEM), HIV-1-infected (J1.1), and human T cell leukemia virus-1 (HTLV-1)-infected (HUT102) cells were synchronized in G0. Viral latency was reversed by increasing gene expression with the addition of serum-rich media and inducers. Supernatants and cell pellets were collected post-induction at different timepoints and assayed for extracellular vesicle (EV) and autophagy markers; and for viral proteins and RNAs. Tetraspanins and autophagy-related proteins were found to be differentially secreted in HIV-1- and HTLV-1-infected cells when compared with uninfected controls. HIV-1 proteins were present at 6 h and their production increased up to 24 h. HTLV-1 proteins peaked at 6 h and plateaued. HIV-1 and HTLV-1 RNA production correlated with viral protein expression. Nanoparticle tracking analysis (NTA) showed increase of EV concentration over time in both uninfected and infected samples. Finally, the HIV-1 supernatant from the 6-h samples was found not to be infectious; however, the virus from the 24-h samples was successfully rescued and infectious. Overall, our data indicate that EV release may occur prior to viral release from infected cells, thereby implicating a potentially significant effect of EVs on uninfected recipient cells prior to subsequent viral infection and spread.
Assuntos
Vesículas Extracelulares/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Infecções por HTLV-I/metabolismo , Infecções por HTLV-I/patologia , Vírion/metabolismo , Apoptose , Biomarcadores/metabolismo , Linhagem Celular , Meios de Cultivo Condicionados , Citocinas/metabolismo , HIV-1/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Humanos , Modelos Biológicos , Células Mieloides/metabolismo , RNA Viral/metabolismo , Linfócitos T/metabolismoRESUMO
X-linked signal elements (XSEs) communicate the dose of X chromosomes to the regulatory-switch gene Sex-lethal (Sxl) during Drosophila sex determination. Unequal XSE expression in precellular XX and XY nuclei ensures that only XX embryos will activate the establishment promoter, SxlPe, to produce a pulse of the RNA-binding protein, SXL [1]. Once XSE protein concentrations have been assessed, SxlPe is inactivated and the maintenance promoter, SxlPm, is turned on in both sexes; however, only in females is SXL present to direct the SxlPm-derived transcripts to be spliced into functional mRNA [2, 3]. Thereafter, Sxl is maintained in the on state by positive autoregulatory RNA splicing [2]. Once set in the stable on (female) or off (male) state, Sxl controls somatic sexual development through control of downstream effectors of sexual differentiation and dosage compensation [1, 4]. Most XSEs encode transcription factors that bind SxlPe, but the XSE unpaired (upd) encodes a secreted ligand for the JAK/STAT pathway [5-7]. We show that although STAT directly regulates SxlPe, it is dispensable for promoter activation. Instead, JAK/STAT is needed to maintain high-level SxlPe expression in order to ensure Sxl autoregulation in XX embryos. Thus, upd is a unique XSE that augments, rather than defines, the initial sex-determination signal.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Janus Quinases/metabolismo , Proteínas de Ligação a RNA/genética , Fatores de Transcrição STAT/metabolismo , Processos de Determinação Sexual , Fatores de Transcrição/metabolismo , Animais , Drosophila/genética , Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Fatores de Transcrição/genética , Transcrição Gênica , Cromossomo XRESUMO
In the textbook view, the ratio of X chromosomes to autosome sets, X:A, is the primary signal specifying sexual fate in Drosophila. An alternative idea is that X chromosome number signals sex through the direct actions of several X-encoded signal element (XSE) proteins. In this alternative, the influence of autosome dose on X chromosome counting is largely indirect. Haploids (1X;1A), which possess the male number of X chromosomes but the female X:A of 1.0, and triploid intersexes (XX;AAA), which possess a female dose of two X chromosomes and the ambiguous X:A ratio of 0.67, represent critical tests of these hypotheses. To directly address the effects of ploidy in primary sex determination, we compared the responses of the signal target, the female-specific SxlPe promoter of the switch gene Sex-lethal, in haploid, diploid, and triploid embryos. We found that haploids activate SxlPe because an extra precellular nuclear division elevates total X chromosome numbers and XSE levels beyond those in diploid males. Conversely, triploid embryos cellularize one cycle earlier than diploids, causing premature cessation of SxlPe expression. This prevents XX;AAA embryos from fully engaging the autoregulatory mechanism that maintains subsequent Sxl expression, causing them to develop as sexual mosaics. We conclude that the X:A ratio predicts sexual fate, but does not actively specify it. Instead, the instructive X chromosome signal is more appropriately seen as collective XSE dose in the early embryo. Our findings reiterate that correlations between X:A ratios and cell fates in other organisms need not implicate the value of the ratio as an active signal.
Assuntos
Drosophila melanogaster/genética , Dosagem de Genes/genética , Ploidias , Cromossomos Sexuais/genética , Processos de Determinação Sexual , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Modelos Biológicos , Fenótipo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Tempo , Transcrição Gênica/genéticaRESUMO
Runx proteins are bifunctional transcription factors that both repress and activate transcription in animal cells. Typically, Runx proteins work in concert with other transcriptional regulators, including co-activators and co-repressors to mediate their biological effects. In Drosophila melanogaster the archetypal Runx protein, Runt, functions in numerous processes including segmentation, neurogenesis and sex determination. During primary sex determination Runt acts as one of four X-linked signal element (XSE) proteins that direct female-specific activation of the establishment promoter (Pe) of the master regulatory gene Sex-lethal (Sxl). Successful activation of SxlPe requires that the XSE proteins overcome the repressive effects of maternally deposited Groucho (Gro), a potent co-repressor of the Gro/TLE family. Runx proteins, including Runt, contain a C-terminal peptide, VWRPY, known to bind to Gro/TLE proteins to mediate transcriptional repression. We show that Runt's VWRPY co-repressor-interaction domain is needed for Runt to activate SxlPe Deletion of the Gro-interaction domain eliminates Runt-ability to activate SxlPe, whereas replacement with a higher affinity, VWRPW, sequence promotes Runt-mediated transcription. This suggests that Runt may activate SxlPe by antagonizing Gro function, a conclusion consistent with earlier findings that Runt is needed for Sxl expression only in embryonic regions with high Gro activity. Surprisingly we found that Runt is not required for the initial activation of SxlPe Instead, Runt is needed to keep SxlPe active during the subsequent period of high-level Sxl transcription suggesting that Runt helps amplify the difference between female and male XSE signals by counter-repressing Gro in female, but not in male, embryos.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
HIV-1 is a global health crisis that has infected more than 37 million people. Latent reservoirs throughout the body are a major hurdle when it comes to eradicating the virus. In our previous study, we found that exosomes, a type of extracellular vesicle (EV), from uninfected cells activate the transcription of HIV-1 in latent infected cells, regardless of combination antiretroviral therapy (cART). In this study, we investigated the specific mechanism behind the EV activation of latent HIV-1. We found that phosphorylated c-Src is present in EVs of various cell lines and has the ability to activate downstream proteins such as EGFR, initiating a signal cascade. EGFR is then able to activate the PI3K/AKT/mTOR pathway, resulting in the activation of STAT3 and SRC-1, culminating in the reversal of HIV-1 latency. This was verified by examining levels of HIV-1 TAR, genomic RNA and HIV-1 Gag p24 protein in cell lines and primary cells. We found that EVs containing c-Src rescued HIV-1 despite the presence of inhibitors, validating the importance of EV-associated c-Src in latent HIV-1 activation. Lastly, we discovered an increased recruitment of p300 and NF-κB in the nucleus of EV-treated infected cells. Collectively, our data suggest that EV-associated c-Src is able to activate latent HIV-1 via the PI3K/AKT/mTOR pathway and SRC-1/p300-driven chromatin remodeling. These findings could aid in designing new strategies to prevent the reactivation of latent HIV-1 in patients under cART.
Assuntos
Exossomos/metabolismo , HIV-1/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Ativação Viral/fisiologia , Latência Viral/fisiologia , Linhagem Celular Tumoral , Proteína p300 Associada a E1A/metabolismo , Receptores ErbB/metabolismo , Vesículas Extracelulares/metabolismo , Proteína do Núcleo p24 do HIV/metabolismo , Infecções por HIV , Humanos , Células Jurkat , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator de Transcrição STAT3/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transcrição Gênica/genética , Ativação Transcricional/genética , Células U937RESUMO
HIV-1 viral transcription persists in patients despite antiretroviral treatment, potentially due to intermittent HIV-1 LTR activation. While several mathematical models have been explored in the context of LTR-protein interactions, in this work for the first time HIV-1 LTR model featuring repressed, intermediate, and activated LTR states is integrated with generation of long (env) and short (TAR) RNAs and proteins (Tat, Pr55, and p24) in T-cells and macrophages using both cell lines and infected primary cells. This type of extended modeling framework allows us to compare and contrast behavior of these two cell types. We demonstrate that they exhibit unique LTR dynamics, which ultimately results in differences in the magnitude of viral products generated. One of the distinctive features of this work is that it relies on experimental data in reaction rate computations. Two RNA transcription rates from the activated promoter states are fit by comparison of experimental data to model predictions. Fitting to the data also provides estimates for the degradation/exit rates for long and short viral RNA. Our experimentally generated data is in reasonable agreement for the T-cell as well macrophage population and gives strong evidence in support of using the proposed integrated modeling paradigm. Sensitivity analysis performed using Latin hypercube sampling method confirms robustness of the model with respect to small parameter perturbations. Finally, incorporation of a transcription inhibitor (F07#13) into the governing equations demonstrates how the model can be used to assess drug efficacy. Collectively, our model indicates transcriptional differences between latently HIV-1 infected T-cells and macrophages and provides a novel platform to study various transcriptional dynamics leading to latency or activation in numerous cell types and physiological conditions.
Assuntos
Fármacos Anti-HIV/farmacologia , Regulação Viral da Expressão Gênica/imunologia , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Macrófagos/imunologia , Linfócitos T/imunologia , Fármacos Anti-HIV/uso terapêutico , Linhagem Celular , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Farmacorresistência Viral/imunologia , Infecções por HIV/sangue , Infecções por HIV/imunologia , Repetição Terminal Longa de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Humanos , Macrófagos/virologia , Modelos Genéticos , Modelos Imunológicos , Cultura Primária de Células , RNA Viral/genética , RNA Viral/metabolismo , Linfócitos T/virologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/imunologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética , Replicação Viral/imunologiaRESUMO
In Drosophila, XX embryos are fated to develop as females, and XY embryos as males, because the diplo-X dose of four X-linked signal element genes, XSEs, activates the Sex-lethal establishment promoter, SxlPe, whereas the haplo-X XSE dose leaves SxlPe off. The threshold response of SxlPe to XSE concentrations depends in part on the bHLH repressor, Deadpan, present in equal amounts in XX and XY embryos. We identified canonical and non-canonical DNA-binding sites for Dpn at SxlPe and found that cis-acting mutations in the Dpn-binding sites caused stronger and earlier Sxl expression than did deletion of dpn implicating other bHLH repressors in Sxl regulation. Maternal Hey encodes one such bHLH regulator but the E(spl) locus does not. Elimination of the maternal corepressor Groucho also caused strong ectopic Sxl expression in XY, and premature Sxl activation in XX embryos, but Sxl was still expressed differently in the sexes. Our findings suggest that Groucho and associated maternal and zygotic bHLH repressors define the threshold XSE concentrations needed to activate SxlPe and that they participate directly in sex signal amplification. We present a model in which the XSE signal is amplified by a feedback mechanism that interferes with Gro-mediated repression in XX, but not XY embryos.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Processos de Determinação Sexual , Cromossomo X/genética , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a DNA , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Modelos Biológicos , Dados de Sequência Molecular , Mutação/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Tempo , TransgenesRESUMO
Maternally-transmitted endosymbiotic bacteria are ubiquitous in insects. Among other influential phenotypes, many heritable symbionts of arthropods are notorious for manipulating host reproduction through one of four reproductive syndromes, which are generally exerted during early developmental stages of the host: male feminization; parthenogenesis induction; male killing; and cytoplasmic incompatibility (CI). Major advances have been achieved in understanding mechanisms and identifying symbiont factors involved in reproductive manipulation, particularly male killing and cytoplasmic incompatibility. Nonetheless, whether cytoplasmically-transmitted bacteria influence the maternally-loaded components of the egg or early embryo has not been examined. In the present study, we investigated whether heritable endosymbionts that cause different reproductive phenotypes in Drosophila melanogaster influence the mRNA transcriptome of early embryos. We used mRNA-seq to evaluate differential expression in Drosophila embryos lacking endosymbionts (control) to those harbouring the male-killing Spiroplasma poulsonii strain MSRO-Br, the CI-inducing Wolbachia strain wMel, or Spiroplasma poulsonii strain Hyd1; a strain that lacks a reproductive phenotype and is naturally associated with Drosophila hydei. We found no consistent evidence of influence of symbiont on mRNA composition of early embryos, suggesting that the reproductive manipulation mechanism does not involve alteration of maternally-loaded transcripts. In addition, we capitalized on several available mRNA-seq datasets derived from Spiroplasma-infected Drosophila melanogaster embryos, to search for signals of depurination of rRNA, consistent with the activity of Ribosome Inactivating Proteins (RIPs) encoded by Spiroplasma poulsonii. We found small but statistically significant signals of depurination of Drosophila rRNA in the Spiroplasma treatments (both strains), but not in the symbiont-free control or Wolbachia treatment, consistent with the action of RIPs. The depurination signal was slightly stronger in the treatment with the male-killing strain. This result supports a recent report that RIP-induced damage contributes to male embryo death.
Assuntos
Drosophila melanogaster/embriologia , Drosophila melanogaster/microbiologia , Embrião não Mamífero/microbiologia , Simbiose , Transcriptoma/genética , Animais , Drosophila melanogaster/genética , Feminino , Genes de Insetos/genética , Interações Hospedeiro-Patógeno/genética , Masculino , Fenótipo , RNA Ribossômico , Reprodução/genética , Proteínas Inativadoras de Ribossomos/genética , Proteínas Inativadoras de Ribossomos/fisiologia , Análise de Sequência de RNA , Spiroplasma/enzimologia , WolbachiaRESUMO
BACKGROUND: HTLV-1 infects over 20 million people worldwide and causes a progressive neuroinflammatory disorder in a subset of infected individuals called HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The detection of HTLV-1 specific T cells in the cerebrospinal fluid (CSF) suggests this disease is immunopathologically mediated and that it may be driven by viral antigens. Exosomes are microvesicles originating from the endosomal compartment that are shed into the extracellular space by various cell types. It is now understood that several viruses take advantage of this mode of intercellular communication for packaging of viral components as well. We sought to understand if this is the case in HTLV-1 infection, and specifically if HTLV-1 proteins can be found in the CSF of HAM/TSP patients where we know free virus is absent, and furthermore, if exosomes containing HTLV-1 Tax have functional consequences. RESULTS: Exosomes that were positive for HTLV-1 Tax by Western blot were isolated from HAM/TSP patient PBMCs (25/36) in ex vivo cultures by trapping exosomes from culture supernatants. HTLV-1 seronegative PBMCs did not have exosomes with Tax (0/12), (Fisher exact test, p = 0.0001). We were able to observe HAM/TSP patient CSF (12/20) containing Tax+ exosomes but not in HTLV-1 seronegative MS donors (0/5), despite the absence of viral detection in the CSF supernatant (Fisher exact test p = 0.0391). Furthermore, exosomes cultivated from HAM/TSP PBMCs were capable of sensitizing target cells for HTLV-1 specific CTL lysis. CONCLUSION: Cumulatively, these results show that there are HTLV-1 proteins present in exosomes found in virus-free CSF. HAM/TSP PBMCs, particularly CD4+CD25+ T cells, can excrete these exosomes containing HTLV-1 Tax and may be a source of the exosomes found in patient CSF. Importantly, these exosomes are capable of sensitizing an HTLV-1 specific immune response, suggesting that they may play a role in the immunopathology observed in HAM/TSP. Given the infiltration of HTLV-1 Tax-specific CTLs into the CNS of HAM/TSP patients, it is likely that exosomes may also contribute to the continuous activation and inflammation observed in HAM/TSP, and may suggest future targeted therapies in this disorder.
RESUMO
To date, the most effective treatment of HIV-1 is a combination antiretroviral therapy (cART), which reduces viral replication and reverses pathology. We investigated the effect of cART (RT and protease inhibitors) on the content of extracellular vesicles (EVs) released from HIV-1-infected cells. We have previously shown that EVs contain non-coding HIV-1 RNA, which can elicit responses in recipient cells. In this manuscript, we show that TAR RNA levels demonstrate little change with the addition of cART treatment in cell lines, primary macrophages, and patient biofluids. We determined possible mechanisms involved in the selective packaging of HIV-1 RNA into EVs, specifically an increase in EV-associated hnRNP A2/B1. More recent experiments have shown that several other FDA-approved drugs have the ability to alter the content of exosomes released from HIV-1-infected cells. These findings on cART-altered EV content can also be applied to general viral inhibitors (interferons) which are used to treat other chronic infections. Additionally, we describe unique mechanisms of ESCRT pathway manipulation by antivirals, specifically the targeting of VPS4. Collectively, these data imply that, despite antiretroviral therapy, EVs containing viral products are continually released and may cause neurocognitive and immunological dysfunction.
Assuntos
Antirretrovirais/farmacologia , Vesículas Extracelulares/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Adulto , Estudos de Coortes , Vesículas Extracelulares/efeitos dos fármacos , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/patogenicidade , Humanos , Masculino , RNA Viral/genética , Replicação Viral , Adulto Jovem , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMO
BACKGROUND: Kinesin II-mediated anterograde intraflagellar transport (IFT) is essential for the assembly and maintenance of flagella and cilia in various cell types. Kinesin associated protein (KAP) is identified as the non-motor accessory subunit of Kinesin II, but its role in the corresponding motor function is not understood. RESULTS: We show that mutations in the Drosophila KAP (DmKap) gene could eliminate the sensory cilia as well as the sound-evoked potentials of Johnston's organ (JO) neurons. Ultrastructure analysis of these mutants revealed that the ciliary axonemes are absent. Mutations in Klp64D, which codes for a Kinesin II motor subunit in Drosophila, show similar ciliary defects. All these defects are rescued by exclusive expression of DmKAP and KLP64D/KIF3A in the JO neurons of respective mutants. Furthermore, reduced copy number of the DmKap gene was found to enhance the defects of hypomorphic Klp64D alleles. Unexpectedly, however, both the DmKap and the Klp64D mutant adults produce vigorously motile sperm with normal axonemes. CONCLUSIONS: KAP plays an essential role in Kinesin II function, which is required for the axoneme growth and maintenance of the cilia in Drosophila type I sensory neurons. However, the flagellar assembly in Drosophila spermatids does not require Kinesin II and is independent of IFT.