RESUMEN
Regulatory myeloid immune cells, such as myeloid-derived suppressor cells (MDSCs), populate inflamed or cancerous tissue and block immune cell effector functions. The lack of mechanistic insight into MDSC suppressive activity and a marker for their identification has hampered attempts to overcome T cell inhibition and unleash anti-cancer immunity. Here, we report that human MDSCs were characterized by strongly reduced metabolism and conferred this compromised metabolic state to CD8+ T cells, thereby paralyzing their effector functions. We identified accumulation of the dicarbonyl radical methylglyoxal, generated by semicarbazide-sensitive amine oxidase, to cause the metabolic phenotype of MDSCs and MDSC-mediated paralysis of CD8+ T cells. In a murine cancer model, neutralization of dicarbonyl activity overcame MDSC-mediated T cell suppression and, together with checkpoint inhibition, improved the efficacy of cancer immune therapy. Our results identify the dicarbonyl methylglyoxal as a marker metabolite for MDSCs that mediates T cell paralysis and can serve as a target to improve cancer immune therapy.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inmunoterapia/métodos , Melanoma/inmunología , Células Supresoras de Origen Mieloide/inmunología , Piruvaldehído/metabolismo , Amina Oxidasa (conteniendo Cobre)/metabolismo , Animales , Linfocitos T CD8-positivos/trasplante , Comunicación Celular , Proliferación Celular , Humanos , Tolerancia Inmunológica , Activación de Linfocitos , Melanoma Experimental , Ratones , Ratones Transgénicos , Neoplasias Experimentales , Receptor de Muerte Celular Programada 1/metabolismoRESUMEN
BACKGROUND & AIMS: High expression of phosphatidylinositol 4-kinase III alpha (PI4KIIIα) correlates with poor survival rates in patients with hepatocellular carcinoma. In addition, hepatitis C virus (HCV) infections activate PI4KIIIα and contribute to hepatocellular carcinoma progression. We aimed at mechanistically understanding the impact of PI4KIIIα on the progression of liver cancer and the potential contribution of HCV in this process. METHODS: Several hepatic cell culture and mouse models were used to study the functional importance of PI4KIIIα on liver pathogenesis. Antibody arrays, gene silencing, and PI4KIIIα-specific inhibitor were applied to identify the involved signaling pathways. The contribution of HCV was examined by using HCV infection or overexpression of its nonstructural protein. RESULTS: High PI4KIIIα expression and/or activity induced cytoskeletal rearrangements via increased phosphorylation of paxillin and cofilin. This led to morphologic alterations and higher migratory and invasive properties of liver cancer cells. We further identified the liver-specific lipid kinase phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2γ) working downstream of PI4KIIIα in regulation of the cytoskeleton. PIK3C2γ generates plasma membrane phosphatidylinositol 3,4-bisphosphate-enriched, invadopodia-like structures that regulate cytoskeletal reorganization by promoting Akt2 phosphorylation. CONCLUSIONS: PI4KIIIα regulates cytoskeleton organization via PIK3C2γ/Akt2/paxillin-cofilin to favor migration and invasion of liver cancer cells. These findings provide mechanistic insight into the contribution of PI4KIIIα and HCV to the progression of liver cancer and identify promising targets for therapeutic intervention.
Asunto(s)
Factores Despolimerizantes de la Actina , Carcinoma Hepatocelular , Movimiento Celular , Citoesqueleto , Neoplasias Hepáticas , Invasividad Neoplásica , Paxillin , Transducción de Señal , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Humanos , Animales , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Citoesqueleto/metabolismo , Citoesqueleto/patología , Paxillin/metabolismo , Ratones , Factores Despolimerizantes de la Actina/metabolismo , Factores Despolimerizantes de la Actina/genética , Fosforilación , Hepacivirus , Línea Celular Tumoral , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Células Hep G2 , Hepatitis C/patología , Hepatitis C/metabolismo , Hepatitis C/virología , Interferencia de ARNRESUMEN
Human immunodeficiency virus (HIV)-1 assembly is initiated by Gag binding to the inner leaflet of the plasma membrane (PM). Gag targeting is mediated by its N-terminally myristoylated matrix (MA) domain and PM phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Upon Gag assembly, envelope (Env) glycoproteins are recruited to assembly sites; this process depends on the MA domain of Gag and the Env cytoplasmic tail. To investigate the dynamics of Env recruitment, we applied a chemical dimerizer system to manipulate HIV-1 assembly by reversible PI(4,5)P2 depletion in combination with super resolution and live-cell microscopy. This approach enabled us to control and synchronize HIV-1 assembly and track Env recruitment to individual nascent assembly sites in real time. Single virion tracking revealed that Gag and Env are accumulating at HIV-1 assembly sites with similar kinetics. PI(4,5)P2 depletion prevented Gag PM targeting and Env cluster formation, confirming Gag dependence of Env recruitment. In cells displaying pre-assembled Gag lattices, PI(4,5)P2 depletion resulted in the disintegration of the complete assembly domain, as not only Gag but also Env clusters were rapidly lost from the PM. These results argue for the existence of a Gag-induced and -maintained membrane micro-environment, which attracts Env. Gag cluster dissociation by PI(4,5)P2 depletion apparently disrupts this micro-environment, resulting in the loss of Env from the former assembly domain.IMPORTANCEHuman immunodeficiency virus (HIV)-1 assembles at the plasma membrane of infected cells, resulting in the budding of membrane-enveloped virions. HIV-1 assembly is a complex process initiated by the main structural protein of HIV-1, Gag. Interestingly, HIV-1 incorporates only a few envelope (Env) glycoproteins into budding virions, although large Env accumulations surrounding nascent Gag assemblies are detected at the plasma membrane of HIV-expressing cells. The matrix domain of Gag and the Env cytoplasmatic tail play a role in Env recruitment to HIV-1 assembly sites and its incorporation into nascent virions. However, the regulation of these processes is incompletely understood. By combining a chemical dimerizer system to manipulate HIV-1 assembly with super resolution and live-cell microscopy, our study provides new insights into the interplay between Gag, Env, and host cell membranes during viral assembly and into Env incorporation into HIV-1 virions.
RESUMEN
Liver-generated plasma Apolipoprotein E (ApoE)-containing lipoproteins (LPs) (ApoE-LPs) play central roles in lipid transport and metabolism. Perturbations of ApoE can result in several metabolic disorders and ApoE genotypes have been associated with multiple diseases. ApoE is synthesized at the endoplasmic reticulum and transported to the Golgi apparatus for LP assembly; however, the ApoE-LPs transport pathway from there to the plasma membrane is largely unknown. Here, we established an integrative imaging approach based on a fully functional fluorescently tagged ApoE. We found that newly synthesized ApoE-LPs accumulate in CD63-positive endosomes of hepatocytes. In addition, we observed the co-egress of ApoE-LPs and CD63-positive intraluminal vesicles (ILVs), which are precursors of extracellular vesicles (EVs), along the late endosomal trafficking route in a microtubule-dependent manner. A fraction of ApoE-LPs associated with CD63-positive EVs appears to be co-transmitted from cell to cell. Given the important role of ApoE in viral infections, we employed as well-studied model the hepatitis C virus (HCV) and found that the viral replicase component nonstructural protein 5A (NS5A) is enriched in ApoE-containing ILVs. Interaction between NS5A and ApoE is required for the efficient release of ILVs containing HCV RNA. These vesicles are transported along the endosomal ApoE egress pathway. Taken together, our data argue for endosomal egress and transmission of hepatic ApoE-LPs, a pathway that is hijacked by HCV. Given the more general role of EV-mediated cell-to-cell communication, these insights provide new starting points for research into the pathophysiology of ApoE-related metabolic and infection-related disorders.
Asunto(s)
Hepacivirus , Hepatitis C , Humanos , Hepacivirus/fisiología , Lipopolisacáridos/metabolismo , Ensamble de Virus/fisiología , Endosomas/metabolismo , Apolipoproteínas E/metabolismoRESUMEN
Immature dendritic cells (iDCs) migrate in microenvironments with distinct cell and extracellular matrix densities in vivo and contribute to HIV-1 dissemination and mounting of antiviral immune responses. Here, we find that, compared to standard 2D suspension cultures, 3D collagen as tissue-like environment alters iDC properties and their response to HIV-1 infection. iDCs adopt an elongated morphology with increased deformability in 3D collagen at unaltered activation, differentiation, cytokine secretion, or responsiveness to LPS. While 3D collagen reduces HIV-1 particle uptake by iDCs, fusion efficiency is increased to elevate productive infection rates due to elevated cell surface exposure of the HIV-1-binding receptor DC-SIGN. In contrast, 3D collagen reduces HIV transfer to CD4 T cells from iDCs. iDC adaptations to 3D collagen include increased pro-inflammatory cytokine production and reduced antiviral gene expression in response to HIV-1 infection. Adhesion to a 2D collagen matrix is sufficient to increase iDC deformability, DC-SIGN exposure, and permissivity to HIV-1 infection. Thus, mechano-physical cues of 2D and 3D tissue-like collagen environments regulate iDC function and shape divergent roles during HIV-1 infection.
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Infecciones por VIH , VIH-1 , Humanos , Citocinas/metabolismo , Colágeno/metabolismo , Antivirales , Células DendríticasRESUMEN
HIV-1 persists in a latent form during antiretroviral therapy, mainly in CD4+ T cells, thus hampering efforts for a cure. HIV-1 infection is accompanied by metabolic alterations, such as oxidative stress, but the effect of cellular antioxidant responses on viral replication and latency is unknown. Here, we show that cells survive retroviral replication, both in vitro and in vivo in SIVmac-infected macaques, by upregulating antioxidant pathways and the intertwined iron import pathway. These changes are associated with remodeling of promyelocytic leukemia protein nuclear bodies (PML NBs), an important constituent of nuclear architecture and a marker of HIV-1 latency. We found that PML NBs are hyper-SUMOylated and that PML protein is degraded via the ubiquitin-proteasome pathway in productively infected cells, before latency establishment and after reactivation. Conversely, normal numbers of PML NBs were restored upon transition to latency or by decreasing oxidative stress or iron content. Our results highlight antioxidant and iron import pathways as determinants of HIV-1 latency and support their pharmacologic inhibition as tools to regulate PML stability and impair latency establishment.
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Redes Reguladoras de Genes , Infecciones por VIH/virología , VIH-1/fisiología , Hierro/metabolismo , Proteína de la Leucemia Promielocítica/metabolismo , Animales , Línea Celular , Modelos Animales de Enfermedad , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Humanos , Macaca , Oxidación-Reducción , Proteolisis , Análisis de Secuencia de ARN , Sumoilación , Regulación hacia Arriba , Latencia del VirusRESUMEN
Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes and isolates, hampering the development of prophylactic vaccines. A consensus gt1b genome (termed GLT1) was generated from an HCV infected liver-transplanted patient. GLT1 replicated to an outstanding efficiency in Huh7 cells upon SEC14L2 expression, by use of replication enhancing mutations or with a previously developed inhibitor-based regimen. RNA replication levels almost reached JFH-1, but full-length genomes failed to produce detectable amounts of infectious virus. Long-term passaging led to the adaptation of a genome carrying 21 mutations and concomitant production of high levels of transmissible infectivity (GLT1cc). During the adaptation, GLT1 spread in the culture even in absence of detectable amounts of free virus, likely due to cell-to-cell transmission, which appeared to substantially contribute to spreading of other isolates as well. Mechanistically, genome replication and particle production efficiency were enhanced by adaptation, while cell entry competence of HCV pseudoparticles was not affected. Furthermore, GLT1cc retained the ability to replicate in human liver chimeric mice, which was critically dependent on a mutation in domain 3 of nonstructural protein NS5A. Over the course of infection, only one mutation in the surface glycoprotein E2 consistently reverted to wildtype, facilitating assembly in cell culture but potentially affecting CD81 interaction in vivo. Overall, GLT1cc is an efficient gt1b infectious cell culture model, paving the road to a rationale-based establishment of new infectious HCV isolates and represents an important novel tool for the development of prophylactic HCV vaccines.
Asunto(s)
Hepacivirus , Hepatitis C , Animales , Técnicas de Cultivo de Célula , Genotipo , Humanos , Ratones , Mutación , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
Emergence of the novel pathogenic coronavirus SARS-CoV-2 and its rapid pandemic spread presents challenges that demand immediate attention. Here, we describe the development of a semi-quantitative high-content microscopy-based assay for detection of three major classes (IgG, IgA, and IgM) of SARS-CoV-2 specific antibodies in human samples. The possibility to detect antibodies against the entire viral proteome together with a robust semi-automated image analysis workflow resulted in specific, sensitive and unbiased assay that complements the portfolio of SARS-CoV-2 serological assays. Sensitive, specific and quantitative serological assays are urgently needed for a better understanding of humoral immune response against the virus as a basis for developing public health strategies to control viral spread. The procedure described here has been used for clinical studies and provides a general framework for the application of quantitative high-throughput microscopy to rapidly develop serological assays for emerging virus infections.
Asunto(s)
Anticuerpos Antivirales/sangre , COVID-19/diagnóstico , Inmunoensayo , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Microscopía/métodos , SARS-CoV-2/inmunología , COVID-19/inmunología , COVID-19/virología , Prueba de COVID-19/métodos , Técnica del Anticuerpo Fluorescente , Ensayos Analíticos de Alto Rendimiento , Humanos , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Sueros Inmunes/química , Aprendizaje Automático , Sensibilidad y EspecificidadRESUMEN
Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of this include flaviviruses, such as dengue virus and Zika virus, which cause millions of yearly infections around the globe, and coronaviruses, such as SARS-CoV-2, the source of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of research aimed at determining methods to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective treatments. Here, we describe the generation and characterization of a reporter system that can be used to visualize and identify cells infected with dengue virus or SARS-CoV-2. This system is based on viral protease activity that mediates cleavage and nuclear translocation of an engineered fluorescent protein stably expressed in cells. We show the suitability of this system for live cell imaging, for visualization of single infected cells, and for screening and testing of antiviral compounds. With the integrated modular building blocks, this system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility.IMPORTANCE Reporter systems are useful tools for fast and quantitative visualization of virus-infected cells within a host cell population. Here, we describe a reporter system that takes advantage of virus-encoded proteases expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the GFP moiety translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.
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COVID-19/virología , Virus del Dengue/aislamiento & purificación , Dengue/virología , SARS-CoV-2/aislamiento & purificación , Células A549 , Animales , COVID-19/diagnóstico , COVID-19/metabolismo , COVID-19/patología , Línea Celular , Chlorocebus aethiops , Dengue/diagnóstico , Dengue/metabolismo , Dengue/patología , Virus del Dengue/genética , Virus del Dengue/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Señales de Localización Nuclear/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Células Vero , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
The ongoing SARS-CoV-2 pandemic with over 80 million infections and more than a million deaths worldwide represents the worst global health crisis of the 21th century. Beyond the health crisis, the disruptions caused by the COVID-19 pandemic have serious global socio-economic consequences. It has also placed a significant pressure on the scientific community to understand the virus and its pathophysiology and rapidly provide anti-viral treatments and procedures in order to help the society and stop the virus spread. Here, we outline how advanced microscopy technologies such as high-throughput microscopy and electron microscopy played a major role in rapid response against SARS-CoV-2. General applicability of developed microscopy technologies makes them uniquely positioned to act as the first line of defence against any emerging infection in the future.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , COVID-19 , Microscopía/métodos , SARS-CoV-2 , Anticuerpos Antivirales/sangre , Antivirales/farmacología , COVID-19/diagnóstico , COVID-19/patología , COVID-19/virología , Prueba Serológica para COVID-19 , Vacunas contra la COVID-19 , Microscopía por Crioelectrón , Desarrollo de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Microscopía Electrónica , Pandemias , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/inmunología , SARS-CoV-2/fisiología , SARS-CoV-2/ultraestructura , Replicación ViralRESUMEN
The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids.IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans-complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production.
Asunto(s)
Proteínas de la Cápside/metabolismo , Dependovirus/genética , Vectores Genéticos , Ensamble de Virus , Animales , Proteínas de la Cápside/genética , Dependovirus/efectos de los fármacos , Dependovirus/metabolismo , Células HeLa , Humanos , Insectos , Mamíferos , Parvovirus/genética , Parvovirus/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Estabilidad Proteica , Células Sf9 , Virión/metabolismoRESUMEN
UNLABELLED: The host cell restriction factor CD317/tetherin traps virions at the surface of producer cells to prevent their release. The HIV-1 accessory protein Vpu antagonizes this restriction. Vpu reduces the cell surface density of the restriction factor and targets it for degradation; however, these activities are dispensable for enhancing particle release. Instead, Vpu has been suggested to antagonize CD317/tetherin by preventing recycling of internalized CD317/tetherin to the cell surface, blocking anterograde transport of newly synthesized CD317/tetherin, and/or displacing the restriction factor from virus assembly sites at the plasma membrane. At the molecular level, antagonism relies on the physical interaction of Vpu with CD317/tetherin. Recent findings suggested that phosphorylation of a diserine motif enables Vpu to bind to adaptor protein 1 (AP-1) trafficking complexes via two independent interaction motifs and to couple CD317/tetherin to the endocytic machinery. Here, we used a panel of Vpu proteins with specific mutations in individual interaction motifs to define which interactions are required for antagonism of CD317/tetherin. Impairing recycling or anterograde transport of CD317/tetherin to the plasma membrane was insufficient for antagonism. In contrast, excluding CD317/tetherin from HIV-1 assembly sites depended on Vpu motifs for interaction with AP-1 and CD317/tetherin and correlated with antagonism of the particle release restriction. Consistently, interference with AP-1 function or its expression blocked these Vpu activities. Our results define displacement from HIV-1 assembly sites as active principle of CD317/tetherin antagonism by Vpu and support a role of tripartite complexes between Vpu, AP-1, and CD317/tetherin in this process. IMPORTANCE: CD317/tetherin poses an intrinsic barrier to human immunodeficiency virus type 1 (HIV-1) replication in human cells by trapping virus particles at the surface of producer cells and thereby preventing their release. The viral protein Vpu antagonizes this restriction, and molecular interactions with the restriction factor and adaptor protein complex 1 (AP-1) were suggested to mediate this activity. Vpu modulates intracellular trafficking of CD317/tetherin and excludes the restriction factor from HIV-1 assembly sites at the plasma membrane, but the relative contribution of these effects to antagonism remain elusive. Using a panel of Vpu mutants, as well as interference with AP-1 function and expression, we show here that Vpu antagonizes CD317/tetherin by blocking its recruitment to viral assembly sites in an AP-1-dependent manner. These results refine our understanding of the molecular mechanisms of CD317/tetherin antagonism and suggest complexes of Vpu with the restriction factor and AP-1 as targets for potential therapeutic intervention.
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Complejo 1 de Proteína Adaptadora/metabolismo , Infecciones por VIH/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Ensamble de Virus/fisiología , Secuencia de Aminoácidos , Antígenos CD/metabolismo , Membrana Celular/metabolismo , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/metabolismo , Infecciones por VIH/virología , VIH-1/patogenicidad , Células HeLa , Humanos , Transporte de Proteínas , Homología de Secuencia de Aminoácido , Virión/fisiología , Liberación del VirusRESUMEN
UNLABELLED: Transcription factors of the far-upstream element-binding protein (FBP) family represent cellular pathway hubs, and their overexpression in liver cancer (hepatocellular carcinoma [HCC]) stimulates tumor cell proliferation and correlates with poor prognosis. Here we determine the mode of oncogenic FBP overexpression in HCC cells. Using perturbation approaches (kinase inhibitors, small interfering RNAs) and a novel system for rapalog-dependent activation of AKT isoforms, we demonstrate that activity of the phosphatidylinositol-4,5-biphosphate 3-kinase/AKT pathway is involved in the enrichment of nuclear FBP1 and FBP2 in liver cancer cells. In human HCC tissues, phospho-AKT significantly correlates with nuclear FBP1/2 accumulation and expression of the proliferation marker KI67. Mechanistic target of rapamycin (mTOR) inhibition or blockade of its downstream effector eukaryotic translation initiation factor 4E activity equally reduced FBP1/2 concentrations. The mTORC1 inhibitor rapamycin diminishes FBP enrichment in liver tumors after hydrodynamic gene delivery of AKT plasmids. In addition, the multikinase inhibitor sorafenib significantly reduces FBP levels in HCC cells and in multidrug resistance 2-deficient mice that develop HCC due to severe inflammation. Both FBP1/2 messenger RNAs are highly stable, with FBP2 being more stable than FBP1. Importantly, inhibition of phosphatidylinositol-4,5-biphosphate 3-kinase/AKT/mTOR signaling significantly diminishes FBP1/2 protein stability in a caspase-3/-7-dependent manner. CONCLUSION: These data provide insight into a transcription-independent mechanism of FBP protein enrichment in liver cancer; further studies will have to show whether this previously unknown interaction between phosphatidylinositol-4,5-biphosphate 3-kinase/AKT/mTOR pathway activity and caspase-mediated FBP stabilization allows the establishment of interventional strategies in FBP-positive HCCs.
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Carcinoma Hepatocelular/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias Hepáticas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Femenino , Humanos , Masculino , Estabilidad Proteica , Proteínas de Unión al ARNRESUMEN
Chemical dimerizers are powerful tools for non-invasive manipulation of enzyme activities in intact cells. Here we introduce the first rapidly reversible small-molecule-based dimerization system and demonstrate a sufficiently fast switch-off to determine kinetics of lipid metabolizing enzymes in living cells. We applied this new method to induce and stop phosphatidylinositol 3-kinase (PI3K) activity, allowing us to quantitatively measure the turnover of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and its downstream effectors by confocal fluorescence microscopy as well as standard biochemical methods.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Dimerización , Células HeLa , Humanos , Cinética , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Tacrolimus/farmacologíaRESUMEN
Immune cell phenotyping frequently detects lineage-unrelated receptors. Here, we report that surface receptors can be transferred from primary macrophages to CD4 T cells and identify the Fcγ receptor CD32 as driver and cargo of this trogocytotic transfer. Filamentous CD32+ nanoprotrusions deposit distinct plasma membrane patches onto target T cells. Transferred receptors confer cell migration and adhesion properties, and macrophage-derived membrane patches render resting CD4 T cells susceptible to infection by serving as hotspots for HIV-1 binding. Antibodies that recognize T cell epitopes enhance CD32-mediated trogocytosis. Such autoreactive anti-HIV-1 envelope antibodies can be found in the blood of HIV-1 patients and, consistently, the percentage of CD32+ CD4 T cells is increased in their blood. This CD32-mediated, antigen-independent cell communication mode transiently expands the receptor repertoire and functionality of immune cells. HIV-1 hijacks this mechanism by triggering the generation of trogocytosis-promoting autoantibodies to gain access to immune cells critical to its persistence.
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Infecciones por VIH , Seropositividad para VIH , VIH-1 , Humanos , Linfocitos T CD4-Positivos , Receptores de IgG/metabolismo , Autoanticuerpos/metabolismo , TrogocitosisRESUMEN
CD4 T cell activation induces nuclear and cytoplasmic actin polymerization via the Arp2/3 complex to activate cytokine expression and strengthen T cell receptor (TCR) signaling. Actin polymerization dynamics and filament morphology differ between nucleus and cytoplasm. However, it is unclear how the Arp2/3 complex mediates distinct nuclear and cytoplasmic actin polymerization in response to a common stimulus. In humans, the ARP3, ARPC1, and ARPC5 subunits of the Arp2/3 complex exist as two different isoforms, resulting in complexes with different properties. Here, we show that the Arp2/3 subunit isoforms ARPC5 and ARPC5L play a central role in coordinating distinct actin polymerization events in CD4 T cells. While ARPC5L is heterogeneously expressed in individual CD4 T cells, it specifically drives nuclear actin polymerization upon T cell activation. In contrast, ARPC5 is evenly expressed in CD4 T cell populations and is required for cytoplasmic actin dynamics. Interestingly, nuclear actin polymerization triggered by a different stimulus, DNA replication stress, specifically requires ARPC5 but not ARPC5L. TCR signaling but not DNA replication stress induces nuclear actin polymerization via nuclear calcium-calmodulin signaling and N-WASP. Diversity in the molecular properties and individual expression patterns of ARPC5 subunit isoforms thus tailors Arp2/3-mediated actin polymerization to different physiological stimuli.
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Actinas , Calmodulina , Humanos , Proteína 2 Relacionada con la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a phospholipid residing on early endosomes, where it is proposed to be involved in endosomal fusion. We synthesized membrane-permeant derivatives of PtdIns(3)P, including a caged version that is to our knowledge the first photoactivatable phosphoinositide derivative developed so far. In living cells, photoactivation of caged PtdIns(3)P induced rapid endosomal fusion in an EEA1-dependent fashion, thus providing in vivo evidence that PtdIns(3)P is a sufficient signal for driving this process.
Asunto(s)
Fusión Celular , Permeabilidad de la Membrana Celular , Endosomas/ultraestructura , Fosfatos de Fosfatidilinositol/metabolismoRESUMEN
The cone-shaped mature HIV-1 capsid is the main orchestrator of early viral replication. After cytosolic entry, it transports the viral replication complex along microtubules toward the nucleus. While it was initially believed that the reverse transcribed genome is released from the capsid in the cytosol, recent observations indicate that a high amount of capsid protein (CA) remains associated with subviral complexes during import through the nuclear pore complex (NPC). Observation of postentry events via microscopic detection of HIV-1 CA is challenging, since epitope shielding limits immunodetection and the genetic fragility of CA hampers direct labeling approaches. Here, we present a minimally invasive strategy based on genetic code expansion and click chemistry that allows for site-directed fluorescent labeling of HIV-1 CA, while retaining virus morphology and infectivity. Thereby, we could directly visualize virions and subviral complexes using advanced microscopy, including nanoscopy and correlative imaging. Quantification of signal intensities of subviral complexes revealed an amount of CA associated with nuclear complexes in HeLa-derived cells and primary T cells consistent with a complete capsid and showed that treatment with the small molecule inhibitor PF74 did not result in capsid dissociation from nuclear complexes. Cone-shaped objects detected in the nucleus by electron tomography were clearly identified as capsid-derived structures by correlative microscopy. High-resolution imaging revealed dose-dependent clustering of nuclear capsids, suggesting that incoming particles may follow common entry routes. IMPORTANCE The cone-shaped capsid of HIV-1 has recently been recognized as a master organizer of events from cell entry of the virus to the integration of the viral genome into the host cell DNA. Fluorescent labeling of the capsid is essential to study its role in these dynamic events by microscopy, but viral capsid proteins are extremely challenging targets for the introduction of labels. Here we describe a minimally invasive strategy that allows us to visualize the HIV-1 capsid protein in infected cells by live-cell imaging and superresolution microscopy. Applying this strategy, we confirmed that, contrary to earlier assumptions, an equivalent of a complete capsid can enter the host cell nucleus through nuclear pores. We also observed that entering capsids cluster in the nucleus in a dose-dependent manner, suggesting that they may have followed a common entry route to a site suitable for viral genome release.
Asunto(s)
Seropositividad para VIH , VIH-1 , Humanos , VIH-1/genética , VIH-1/metabolismo , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Replicación Viral/genética , Núcleo Celular/metabolismo , Seropositividad para VIH/metabolismo , Código Genético , Epítopos/metabolismoRESUMEN
Malaria-causing parasites proliferate within erythrocytes through schizogony, forming multinucleated stages before cellularization. Nuclear multiplication does not follow a strict geometric 2n progression, and each proliferative cycle produces a variable number of progeny. Here, by tracking nuclei and DNA replication, we show that individual nuclei replicate their DNA at different times, despite residing in a shared cytoplasm. Extrapolating from experimental data using mathematical modeling, we provide strong indication that a limiting factor exists, which slows down the nuclear multiplication rate. Consistent with this prediction, our data show that temporally overlapping DNA replication events were significantly slower than partially overlapping or nonoverlapping events. Our findings suggest the existence of evolutionary pressure that selects for asynchronous DNA replication, balancing available resources with rapid pathogen proliferation.
Asunto(s)
Núcleo Celular , Plasmodium falciparum , División Celular , Replicación del ADN , Eritrocitos/parasitología , Plasmodium falciparum/genéticaRESUMEN
Combinations of direct-acting antivirals are needed to minimize drug resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and testing of a number of drug regimens has led to conflicting results. Here, we show that cobicistat, which is an FDA-approved drug booster that blocks the activity of the drug-metabolizing proteins cytochrome P450-3As (CYP3As) and P-glycoprotein (P-gp), inhibits SARS-CoV-2 replication. Two independent cell-to-cell membrane fusion assays showed that the antiviral effect of cobicistat is exerted through inhibition of spike protein-mediated membrane fusion. In line with this, incubation with low-micromolar concentrations of cobicistat decreased viral replication in three different cell lines including cells of lung and gut origin. When cobicistat was used in combination with remdesivir, a synergistic effect on the inhibition of viral replication was observed in cell lines and in a primary human colon organoid. This was consistent with the effects of cobicistat on two of its known targets, CYP3A4 and P-gp, the silencing of which boosted the in vitro antiviral activity of remdesivir in a cobicistat-like manner. When administered in vivo to Syrian hamsters at a high dose, cobicistat decreased viral load and mitigated clinical progression. These data highlight cobicistat as a therapeutic candidate for treating SARS-CoV-2 infection and as a potential building block of combination therapies for COVID-19. IMPORTANCE The lack of effective antiviral treatments against SARS-CoV-2 is a significant limitation in the fight against the COVID-19 pandemic. Single-drug regimens have so far yielded limited results, indicating that combinations of antivirals might be required, as previously seen for other RNA viruses. Our work introduces the drug booster cobicistat, which is approved by the FDA and typically used to potentiate the effect of anti-HIV protease inhibitors, as a candidate inhibitor of SARS-CoV-2 replication. Beyond its direct activity as an antiviral, we show that cobicistat can enhance the effect of remdesivir, which was one of the first drugs proposed for treatment of SARS-CoV-2. Overall, the dual action of cobicistat as a direct antiviral and a drug booster can provide a new approach to design combination therapies and rescue the activity of compounds that are only partially effective in monotherapy.