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Cetylpyridinium chloride (CPC) is a quaternary ammonium antimicrobial used in numerous personal care products, human food, cosmetic products, and cleaning solutions. Yet, there is minimal published data on CPC effects on eukaryotes, immune signaling, and human health. Previously, we showed that low-micromolar CPC inhibits rat mast cell function by inhibiting antigen (Ag)-stimulated Ca 2+ mobilization, microtubule polymerization, and degranulation. In this study, we extend the findings to human mast cells (LAD2) and present data indicating that CPC's mechanism of action centers on its positively-charged quaternary nitrogen in its pyridinium headgroup. CPC's inhibitory effect is independent of signaling platform receptor architecture. Tyrosine phosphorylation events are a trigger of Ca 2+ mobilization necessary for degranulation. CPC inhibits global tyrosine phosphorylation in Ag-stimulated mast cells. Specifically, CPC inhibits tyrosine phosphorylation of specific key players Syk kinase and LAT, a substrate of Syk. In contrast, CPC does not affect Lyn kinase phosphorylation. Thus, CPC's root mechanism is electrostatic disruption of particular tyrosine phosphorylation events essential for signaling. This work outlines the biochemical mechanisms underlying the effects of CPC on immune signaling and allows the prediction of CPC effects on cell types, like T cells, that share similar signaling elements.
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JC polyomavirus (JCPyV) is a nonenveloped, double-stranded DNA virus that infects the majority of the population. Immunocompetent individuals harbor infection in their kidneys, while severe immunosuppression can result in JCPyV spread to the brain, causing the neurodegenerative disease progressive multifocal leukoencephalopathy (PML). Due to a lack of approved therapies to treat JCPyV and PML, the disease results in rapid deterioration, and is often fatal. In order to identify potential antiviral treatments for JCPyV, a high-throughput, large-scale drug screen was performed using the National Institutes of Health Clinical Collection (NCC). Drugs from the NCC were tested for inhibitory effects on JCPyV infection, and drugs from various classes that reduced JCPyV infection were identified, including receptor agonists and antagonists, calcium signaling modulators, and enzyme inhibitors. Given the role of calcium signaling in viral infection including Merkel cell polyomavirus and simian virus 40 polyomavirus (SV40), calcium signaling inhibitors were further explored for the capacity to impact JCPyV infection. Calcium and calmodulin inhibitors trifluoperazine (TFP), W-7, tetrandrine, and nifedipine reduced JCPyV infection, and TFP specifically reduced viral internalization. Additionally, TFP and W-7 reduced infection by BK polyomavirus, SV40, and SARS-CoV-2. These results highlight specific inhibitors, some FDA-approved, for the possible treatment and prevention of JCPyV and several other viruses, and further illuminate the calcium and calmodulin pathway as a potential target for antiviral drug development.
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Virus JC , Leucoencefalopatía Multifocal Progresiva , Enfermedades Neurodegenerativas , Infecciones por Polyomavirus , Sulfonamidas , Humanos , Calcio , Calmodulina , Leucoencefalopatía Multifocal Progresiva/tratamiento farmacológico , Leucoencefalopatía Multifocal Progresiva/genética , Virus JC/genética , Virus 40 de los Simios , Antivirales/farmacologíaRESUMEN
Viruses rely on host-cell machinery in order to invade host cells and carry out a successful infection. G-protein coupled receptor (GPCR)-mediated signaling pathways are master regulators of cellular physiological processing and are an attractive target for viruses to rewire cells during infection. In particular, the GPCR-associated scaffolding proteins ß-arrestins and GPCR signaling effectors G-protein receptor kinases (GRKs) have been identified as key cellular factors that mediate viral entry and orchestrate signaling pathways that reprogram cells for viral replication. Interestingly, a broad range of viruses have been identified to activate and/or require GPCR-mediated pathways for infection, including polyomaviruses, flaviviruses, influenza virus, and SARS-CoV-2, demonstrating that these viruses may have conserved mechanisms of host-cell invasion. Thus, GPCR-mediated pathways highlight an attractive target for the development of broad antiviral therapies.
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COVID-19 , Quinasas de Receptores Acoplados a Proteína-G , Humanos , Quinasas de Receptores Acoplados a Proteína-G/metabolismo , beta-Arrestinas/metabolismo , Internalización del Virus , SARS-CoV-2 , Receptores Acoplados a Proteínas G/metabolismo , FosforilaciónRESUMEN
The organization and dynamics of plasma membrane receptors are a critical link in virus-receptor interactions, which finetune signaling efficiency and determine cellular responses during infection. Characterizing the mechanisms responsible for the active rearrangement and clustering of receptors may aid in developing novel strategies for the therapeutic treatment of viruses. Virus-receptor interactions are poorly understood at the nanoscale, yet they present an attractive target for the design of drugs and for the illumination of viral infection and pathogenesis. This study utilizes super-resolution microscopy and related techniques, which surpass traditional microscopy resolution limitations, to provide both a spatial and temporal assessment of the interactions of human JC polyomavirus (JCPyV) with 5-hydroxytrypamine 2 receptors (5-HT2Rs) subtypes during viral entry. JCPyV causes asymptomatic kidney infection in the majority of the population and can cause fatal brain disease, and progressive multifocal leukoencephalopathy (PML), in immunocompromised individuals. Using Fluorescence Photoactivation Localization Microscopy (FPALM), the colocalization of JCPyV with 5-HT2 receptor subtypes (5-HT2A, 5-HT2B, and 5-HT2C) during viral attachment and viral entry was analyzed. JCPyV was found to significantly enhance the clustering of 5-HT2 receptors during entry. Cluster analysis of infected cells reveals changes in 5-HT2 receptor cluster attributes, and radial distribution function (RDF) analyses suggest a significant increase in the aggregation of JCPyV particles colocalized with 5-HT2 receptor clusters in JCPyV-infected samples. These findings provide novel insights into receptor patterning during viral entry and highlight improved technologies for the future development of therapies for JCPyV infection as well as therapies for diseases involving 5-HT2 receptors.
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Virus JC , Leucoencefalopatía Multifocal Progresiva , Infecciones por Polyomavirus , Humanos , Virus JC/fisiología , Serotonina , Acoplamiento ViralRESUMEN
The COVID-19 pandemic has revealed the importance of the detection of airborne pathogens. Here, we present composite air filters featuring a bioinspired liquid coating that facilitates the removal of captured aerosolized bacteria and viruses for further analysis. We tested three types of air filters: commercial polytetrafluoroethylene (PTFE), which is well known for creating stable liquid coatings, commercial high-efficiency particulate air (HEPA) filters, which are widely used, and in-house-manufactured cellulose nanofiber mats (CNFMs), which are made from sustainable materials. All filters were coated with omniphobic fluorinated liquid to maximize the release of pathogens. We found that coating both the PTFE and HEPA filters with liquid improved the rate at which Escherichia coli was recovered using a physical removal process compared to uncoated controls. Notably, the coated HEPA filters also increased the total number of recovered cells by 57%. Coating the CNFM filters did not improve either the rate of release or the total number of captured cells. The most promising materials, the liquid-coated HEPA, filters were then evaluated for their ability to facilitate the removal of pathogenic viruses via a chemical removal process. Recovery of infectious JC polyomavirus, a nonenveloped virus that attacks the central nervous system, was increased by 92% over uncoated controls; however, there was no significant difference in the total amount of genomic material recovered compared to that of controls. In contrast, significantly more genomic material was recovered for SARS-CoV-2, the airborne, enveloped virus, which causes COVID-19, from liquid-coated filters. Although the amount of infectious SARS-CoV-2 recovered was 58% higher, these results were not significantly different from uncoated filters due to high variability. These results suggest that the efficient recovery of airborne pathogens from liquid-coated filters could improve air sampling efforts, enhancing biosurveillance and global pathogen early warning.
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Filtros de Aire , COVID-19 , Virus , Humanos , Pandemias , SARS-CoV-2 , COVID-19/prevención & control , Bacterias , Polvo , PolitetrafluoroetilenoRESUMEN
Curriculum guidelines for virology are needed to best guide student learning due to the continuous and ever-increasing volume of virology information, the need to ensure that undergraduate and graduate students have a foundational understanding of key virology concepts, and the importance in being able to communicate that understanding to both other virologists and nonvirologists. Such guidelines, developed by virology educators and the American Society for Virology Education and Career Development Committee, are described herein.
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Curriculum , Universidades , Virología , Educación de Postgrado , Estados Unidos , Virología/educaciónRESUMEN
JC polyomavirus (JCPyV) is the causative agent of the fatal, incurable, neurological disease, progressive multifocal leukoencephalopathy (PML). The virus is present in most of the adult population as a persistent, asymptotic infection in the kidneys. During immunosuppression, JCPyV reactivates and invades the central nervous system. A main predictor of disease outcome is determined by mutations within the hypervariable region of the viral genome. In patients with PML, JCPyV undergoes genetic rearrangements in the noncoding control region (NCCR). The outcome of these rearrangements influences transcription factor binding to the NCCR, orchestrating viral gene transcription. This study examines 989 NCCR sequences from patient isolates deposited in GenBank to determine the frequency of mutations based on patient isolation site and disease status. The transcription factor binding sites (TFBS) were also analyzed to understand how these rearrangements could influence viral transcription. It was determined that the number of TFBS was significantly higher in PML samples compared to non-PML samples. Additionally, TFBS that could promote JCPyV infection were more prevalent in samples isolated from the cerebrospinal fluid compared to other locations. Collectively, this research describes the extent of mutations in the NCCR that alter TFBS and how they correlate with disease outcome.
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Genoma Viral , Virus JC , Leucoencefalopatía Multifocal Progresiva , Adulto , Sitios de Unión , Aberraciones Cromosómicas , Humanos , Virus JC/genética , Leucoencefalopatía Multifocal Progresiva/virología , Factores de Transcripción/genéticaRESUMEN
Astrocytes are a main target of JC polyomavirus (JCPyV) in the central nervous system (CNS), where the destruction of these cells, along with oligodendrocytes, leads to the fatal disease progressive multifocal leukoencephalopathy (PML). There is no cure currently available for PML, so it is essential to discover antivirals for this aggressive disease. Additionally, the lack of a tractable in vivo models for studying JCPyV infection makes primary cells an accurate alternative for elucidating mechanisms of viral infection in the CNS. This research to better understand the signaling pathways activated in response to JCPyV infection reveals and establishes the importance of the PI3K/AKT/mTOR signaling pathway in JCPyV infection in primary human astrocytes compared to transformed cell lines. Using RNA sequencing and chemical inhibitors to target PI3K, AKT, and mTOR, we have demonstrated the importance of this signaling pathway in JCPyV infection of primary astrocytes not observed in transformed cells. Collectively, these findings illuminate the potential for repurposing drugs that are involved with inhibition of the PI3K/AKT/mTOR signaling pathway and cancer treatment as potential therapeutics for PML, caused by this neuroinvasive virus.
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Astrocitos/metabolismo , Astrocitos/virología , Virus JC/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Butadienos/farmacología , Células Cultivadas , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Biológicos , Nitrilos/farmacología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Wortmanina/farmacologíaRESUMEN
JC polyomavirus (JCPyV) is a neuroinvasive pathogen causing a fatal, demyelinating disease of the central nervous system (CNS) known as progressive multifocal leukoencephalopathy (PML). Within the CNS, JCPyV predominately targets two cell types: oligodendrocytes and astrocytes. The underlying mechanisms of astrocytic infection are poorly understood, yet recent findings suggest critical differences in JCPyV infection of primary astrocytes compared to a widely studied immortalized cell model. RNA sequencing was performed in primary normal human astrocytes (NHAs) to analyze the transcriptomic profile that emerges during JCPyV infection. Through a comparative analysis, it was validated that JCPyV requires the mitogen-activated protein kinase, extracellular signal-regulated kinase (MAPK/ERK) pathway, and additionally requires the expression of dual-specificity phosphatases (DUSPs). Specifically, the expression of DUSP1 is needed to establish a successful infection in NHAs, yet this was not observed in an immortalized cell model of JCPyV infection. Additional analyses demonstrated immune activation uniquely observed in NHAs. These results support the hypothesis that DUSPs within the MAPK/ERK pathway impact viral infection and influence potential downstream targets and cellular pathways. Collectively, this research implicates DUSP1 in JCPyV infection of primary human astrocytes, and most importantly, further resolves the signaling events that lead to successful JCPyV infection in the CNS.
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Astrocitos/virología , Fosfatasa 1 de Especificidad Dual/metabolismo , Virus JC/fisiología , Sistema de Señalización de MAP Quinasas , Astrocitos/metabolismo , Línea Celular , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , RNA-SeqRESUMEN
Polyomaviruses are mostly non-pathogenic, yet some can cause human disease especially under conditions of immunosuppression, including JC, BK, and Merkel cell polyomaviruses. Direct interactions between viruses and the host early during infection dictate the outcome of disease, many of which remain enigmatic. However, significant work in recent years has contributed to our understanding of how this virus family establishes an infection, largely due to advances made for animal polyomaviruses murine and SV40. Here we summarize the major findings that have contributed to our understanding of polyomavirus entry, trafficking, disassembly, signaling, and immune evasion during the infectious process and highlight major unknowns in these processes that are open areas of study.
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Poliomavirus/fisiología , Internalización del Virus , Animales , Núcleo Celular/virología , Retículo Endoplásmico/virología , Endosomas/virología , Humanos , Evasión Inmune , Transducción de Señal , Acoplamiento ViralRESUMEN
JC polyomavirus (JCPyV) infects the majority of the population, establishing a lifelong, asymptomatic infection in the kidney of healthy individuals. People that become severely immunocompromised may experience JCPyV reactivation, which can cause progressive multifocal leukoencephalopathy (PML), a neurodegenerative disease. Due to a lack of therapeutic options, PML results in fatality or significant debilitation among affected individuals. Cellular internalization of JCPyV is mediated by serotonin 5-hydroxytryptamine subfamily 2 receptors (5-HT2Rs) via clathrin-mediated endocytosis. The JCPyV entry process requires the clathrin-scaffolding proteins ß-arrestin, adaptor protein 2 (AP2), and dynamin. Further, a ß-arrestin interacting domain, the Ala-Ser-Lys (ASK) motif, within the C-terminus of 5-HT2AR is important for JCPyV internalization and infection. Interestingly, 5-HT2R subtypes A, B, and C equally support JCPyV entry and infection, and all subtypes contain an ASK motif, suggesting a conserved mechanism for viral entry. However, the role of the 5-HT2R ASK motifs and the activation of ß-arrestin-associated proteins during internalization has not been fully elucidated. Through mutagenesis, the ASK motifs within 5-HT2BR and 5-HT2CR were identified as critical for JCPyV internalization and infectivity. Further, utilizing biochemical pulldown techniques, mutagenesis of the ASK motifs in 5-HT2BR and 5-HT2CR resulted in reduced ß-arrestin binding. Utilizing small-molecule chemical inhibitors and RNA interference, G-protein receptor kinase 2 (GRK2) was determined to be required for JCPyV internalization and infection by mediating interactions between ß-arrestin and the ASK motif of 5-HT2Rs. These findings demonstrate that GRK2 and ß-arrestin interactions with 5-HT2Rs are critical for JCPyV entry by clathrin-mediated endocytosis and resultant infection.IMPORTANCE As intracellular parasites, viruses require a host cell to replicate and cause disease. Therefore, virus-host interactions contribute to viral pathogenesis. JC polyomavirus (JCPyV) infects most of the population, establishing a lifelong asymptomatic infection within the kidney. Under conditions of severe immunosuppression JCPyV may spread to the central nervous system, causing the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). Individuals living with HIV or undergoing immunomodulatory therapies are at risk for developing PML. The mechanisms of how JCPyV uses specific receptors on the surface of host cells to initiate internalization and infection is a poorly understood process. We have further identified cellular proteins involved in JCPyV internalization and infection and elucidated their specific interactions that are responsible for activation of receptors. Collectively, these findings illuminate how viruses usurp cellular receptors during infection, contributing to current development efforts for therapeutic options for the treatment or prevention of PML.
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Members of the Polyomaviridae family differ in their host range, pathogenesis, and disease severity. To date, some of the most studied polyomaviruses include human JC, BK, and Merkel cell polyomavirus and non-human subspecies murine and simian virus 40 (SV40) polyomavirus. Although dichotomies in host range and pathogenesis exist, overlapping features of the infectious cycle illuminate the similarities within this virus family. Of particular interest to human health, JC, BK, and Merkel cell polyomavirus have all been linked to critical, often fatal, illnesses, emphasizing the importance of understanding the underlying viral infections that result in the onset of these diseases. As there are significant overlaps in the capacity of polyomaviruses to cause disease in their respective hosts, recent advancements in characterizing the infectious life cycle of non-human murine and SV40 polyomaviruses are key to understanding diseases caused by their human counterparts. This review focuses on the molecular mechanisms by which different polyomaviruses hijack cellular processes to attach to host cells, internalize, traffic within the cytoplasm, and disassemble within the endoplasmic reticulum (ER), prior to delivery to the nucleus for viral replication. Unraveling the fundamental processes that facilitate polyomavirus infection provides deeper insight into the conserved mechanisms of the infectious process shared within this virus family, while also highlighting critical unique viral features.
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Interacciones Microbiota-Huesped/genética , Poliomavirus/genética , Internalización del Virus , Replicación Viral , Animales , Núcleo Celular/virología , Especificidad del Huésped , Humanos , Poliomavirus/patogenicidad , Infecciones por Polyomavirus/virologíaRESUMEN
JC polyomavirus (JCPyV) infects 50 to 80% of the population and is the causative agent of a fatal demyelinating disease of the central nervous system (CNS). JCPyV presents initially as a persistent infection in the kidneys of healthy people, but during immunosuppression, the virus can reactivate and cause progressive multifocal leukoencephalopathy (PML). Within the CNS, JCPyV predominately targets two cell types, oligodendrocytes and astrocytes. Until recently, the role of astrocytes has been masked by the pathology in the myelin-producing oligodendrocytes, which are lytically destroyed by the virus. To better understand how astrocytes are impacted during JCPyV infection, the temporal regulation and infectious cycle of JCPyV were analyzed in primary normal human astrocytes (NHAs). Previous research to define the molecular mechanisms underlying JCPyV infection has mostly relied on the use of cell culture models, such as SVG-A cells (SVGAs), an immortalized, mixed population of glial cells transformed with simian virus 40 (SV40) T antigen. However, SVGAs present several limitations due to their immortalized characteristics, and NHAs represent an innovative approach to study JCPyV infection in vitro Using infectivity assays, quantitative PCR, and immunofluorescence assay approaches, we have further characterized JCPyV infectivity in NHAs. The JCPyV infectious cycle is significantly delayed in NHAs, and the expression of SV40 T antigen alters the cellular environment, which impacts viral infection in immortalized cells. This research establishes a foundation for the use of primary NHAs in future studies and will help unravel the role of astrocytes in PML pathogenesis.IMPORTANCE Animal models are crucial in advancing biomedical research and defining the pathogenesis of human disease. Unfortunately, not all diseases can be easily modeled in a nonhuman host or such models are cost prohibitive to generate, including models for the human-specific virus JC polyomavirus (JCPyV). JCPyV infects most of the population but can cause a rare, fatal disease, progressive multifocal leukoencephalopathy (PML). There have been considerable advancements in understanding the molecular mechanisms of JCPyV infection, but this has mostly been limited to immortalized cell culture models. In contrast, PML pathogenesis research has been greatly hindered because of the lack of an animal model. We have further characterized JCPyV infection in primary human astrocytes to better define the infectious process in a primary cell type. Albeit a cell culture model, primary astrocytes may better recapitulate human disease, are easier to maintain than other primary cells, and are less expensive than using an animal model.
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Astrocitos/virología , Progresión de la Enfermedad , Virus JC/fisiología , Infecciones por Polyomavirus/virología , Animales , Antígenos Virales de Tumores , Técnicas de Cultivo de Célula/métodos , Línea Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Virus JC/genética , Virus JC/patogenicidad , Leucoencefalopatía Multifocal Progresiva/virología , Neuroglía , Virus 40 de los Simios , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
JC polyomavirus (JCPyV), a ubiquitous human pathogen, is the etiological agent of the fatal neurodegenerative disease progressive multifocal leukoencephalopathy (PML). Like most viruses, JCPyV infection requires the activation of host-cell signaling pathways in order to promote viral replication processes. Previous works have established the necessity of the extracellular signal-regulated kinase (ERK), the terminal core kinase of the mitogen-activated protein kinase (MAPK) cascade (MAPK-ERK) for facilitating transcription of the JCPyV genome. However, the underlying mechanisms by which the MAPK-ERK pathway becomes activated and induces viral transcription are poorly understood. Treatment of cells with siRNAs specific for Raf and MAP kinase kinase (MEK) targets proteins in the MAPK-ERK cascade, significantly reducing JCPyV infection. MEK, the dual-specificity kinase responsible for the phosphorylation of ERK, is phosphorylated at times congruent with early events in the virus infectious cycle. Moreover, a MAPK-specific signaling array revealed that transcription factors downstream of the MAPK cascade, including cMyc and SMAD4, are upregulated within infected cells. Confocal microscopy analysis demonstrated that cMyc and SMAD4 shuttle to the nucleus during infection, and nuclear localization is reduced when ERK is inhibited. These findings suggest that JCPyV induction of the MAPK-ERK pathway is mediated by Raf and MEK and leads to the activation of downstream transcription factors during infection. This study further defines the role of the MAPK cascade during JCPyV infection and the downstream signaling consequences, illuminating kinases as potential therapeutic targets for viral infection.
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Interacciones Huésped-Patógeno , Virus JC/fisiología , Sistema de Señalización de MAP Quinasas , Infecciones por Polyomavirus/metabolismo , Infecciones por Polyomavirus/virología , Factores de Transcripción/metabolismo , Biomarcadores , Células Cultivadas , Resistencia a la Enfermedad/genética , Susceptibilidad a Enfermedades , Técnicas de Silenciamiento del Gen , Interacciones Huésped-Patógeno/genética , Humanos , Infecciones por Polyomavirus/genética , Unión Proteica , Transporte de Proteínas , Quinasas raf/genética , Quinasas raf/metabolismoRESUMEN
The extracellular signal-regulated kinases (ERKs) comprise a particular branch of the mitogen-activated protein kinase cascades (MAPK) that transmits extracellular signals into the intracellular environment to trigger cellular growth responses. Similar to other MAPK cascades, the MAPK-ERK pathway signals through three core kinases-Raf, MAPK/ERK kinase (MEK), and ERK-which drive the signaling mechanisms responsible for the induction of cellular responses from extracellular stimuli including differentiation, proliferation, and cellular survival. However, pathogens like DNA viruses alter MAPK-ERK signaling in order to access DNA replication machineries, induce a proliferative state in the cell, or even prevent cell death mechanisms in response to pathogen recognition. Differential utilization of this pathway by multiple DNA viruses highlights the dynamic nature of the MAPK-ERK pathway within the cell and the importance of its function in regulating a wide variety of cellular fates that ultimately influence viral infection and, in some cases, result in tumorigenesis.
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Infecciones por Virus ADN/metabolismo , Infecciones por Virus ADN/virología , Virus ADN/fisiología , Interacciones Huésped-Patógeno , Sistema de Señalización de MAP Quinasas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Unión ProteicaRESUMEN
JC polyomavirus (JCPyV) is a ubiquitous human pathogen that causes progressive multifocal leukoencephalopathy (PML). The entry receptors for JCPyV belong to the 5-hydroxytryptamine 2 receptor (5-HT2R) family, but how individual members of the family function to facilitate infection is not known. We used proximity ligation assay (PLA) to determine that JCPyV interacts with each of the 5-HT2 receptors (5-HT2Rs) in a narrow window of time during entry. We used CRISPR-Cas9 to randomly introduce stop codons in the gene for each receptor and discovered that the second intracellular loop of each was necessary for infection. This loop contains a motif possibly involved in receptor internalization by ß-arrestin. Mutation of this motif and small interfering RNA (siRNA) knockdown of ß-arrestin recapitulated the results of our CRISPR-Cas9 screen, showing that this motif is critical. Our results have implications for the role these receptors play in virus infection and for their normal functioning as receptors for serotonin.
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Virus JC/genética , Receptores de Serotonina 5-HT2/genética , Receptores de Serotonina 5-HT2/metabolismo , Receptores Virales/genética , Receptores Virales/metabolismo , Internalización del Virus , Células HEK293 , Interacciones Huésped-Patógeno/genética , Humanos , Virus JC/patogenicidad , beta-Arrestinas/genética , beta-Arrestinas/metabolismoRESUMEN
JC polyomavirus (JCPyV) is a ubiquitous human pathogen and the causative agent of a fatal demyelinating disease in severely immunocompromised individuals. Due to the lack of successful pharmacological interventions, the study of JCPyV infection strategies in a rapid and highly sensitive manner is critical for the characterization of potential antiviral therapeutics. Conventional methodologies for studying viral infectivity often utilize the detection of viral proteins through immunofluorescence microscopy-based techniques. While these methodologies are well established in the field, they require significant time investments and lack a high-throughput modality. Scanning imager-based detection methods like the In-cell Western (ICW)TM have been previously utilized to overcome these challenges incurred by traditional microscopy-based infectivity assays. This automated technique provides not only rapid detection of viral infection status, but can also be optimized to detect changes in host-cell protein expression during JCPyV challenge. Compared to traditional manual determinations of infectivity through microscopy-based techniques, the ICW provides an expeditious and robust determination of JCPyV infection. The optimization of the ICW for the detection of viral and cellular proteins during JCPyV infection provides significant time and cost savings by diminishing sample preparation time and increasing resource utilization. While the ICW cannot provide single-cell analysis information and is limited in the detection of quantitation of low-expressing proteins, this assay provides a high-throughput system to study JCPyV, previously unavailable to the field. Thus, the high-throughput nature and dynamic experimental range of the ICW can be applied to the study of JCPyV infection.
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The lipid phosphatidylinositol 4,5-bisphosphate (PIP2) forms nanoscopic clusters in cell plasma membranes; however, the processes determining PIP2 mobility and thus its spatial patterns are not fully understood. Using super-resolution imaging of living cells, we find that PIP2 is tightly colocalized with and modulated by overexpression of the influenza viral protein hemagglutinin (HA). Within and near clusters, HA and PIP2 follow a similar spatial dependence, which can be described by an HA-dependent potential gradient; PIP2 molecules move as if they are attracted to the center of clusters by a radial force of 0.079 ± 0.002 pN in HAb2 cells. The measured clustering and dynamics of PIP2 are inconsistent with the unmodified forms of the raft, tether, and fence models. Rather, we found that the spatial PIP2 distributions and how they change in time are explained via a novel, to our knowledge, dynamic mechanism: a radial gradient of PIP2 binding sites that are themselves mobile. This model may be useful for understanding other biological membrane domains whose distributions display gradients in density while maintaining their mobility.
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Membrana Celular/química , Membrana Celular/metabolismo , Colorantes Fluorescentes/metabolismo , Hemaglutininas Virales/metabolismo , Orthomyxoviridae , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animales , Supervivencia Celular , Ratones , Modelos Biológicos , Células 3T3 NIHRESUMEN
JC polyomavirus (JCPyV) establishes a persistent, lifelong, asymptomatic infection within the kidney of the majority of the human population. Under conditions of severe immunosuppression or immune modulation, JCPyV can reactivate in the central nervous system (CNS) and cause progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease. Initiation of infection is mediated through viral attachment to α2,6-sialic acid-containing lactoseries tetrasaccharide c (LSTc) on the surface of host cells. JCPyV internalization is dependent on serotonin 5-hydroxytryptamine subfamily 2 receptors (5-HT2Rs), and entry is thought to occur by clathrin-mediated endocytosis (CME). However, the JCPyV entry process and the cellular factors involved in viral internalization remain poorly understood. Treatment of cells with small-molecule chemical inhibitors and RNA interference of 5-HT2R endocytic machinery, including ß-arrestin, clathrin, AP2, and dynamin, significantly reduced JCPyV infection. However, infectivity of the polyomavirus simian virus 40 (SV40) was not affected by CME-specific treatments. Inhibition of clathrin or ß-arrestin specifically reduced JCPyV internalization but did not affect viral attachment. Furthermore, mutagenesis of a ß-arrestin binding domain (Ala-Ser-Lys) within the intracellular C terminus of 5-HT2AR severely diminished internalization and infection, suggesting that ß-arrestin interactions with 5-HT2AR are critical for JCPyV infection and entry. These conclusions illuminate key host factors that regulate clathrin-mediated endocytosis of JCPyV, which is necessary for viral internalization and productive infection.IMPORTANCE Viruses usurp cellular factors to invade host cells. Activation and utilization of these proteins upon initiation of viral infection are therefore required for productive infection and resultant viral disease. The majority of healthy individuals are asymptomatically infected by JC polyomavirus (JCPyV), but if the host immune system is compromised, JCPyV can cause progressive multifocal leukoencephalopathy (PML), a rare, fatal, demyelinating disease. Individuals infected with HIV or taking prolonged immunomodulatory therapies have a heightened risk for developing PML. The cellular proteins and pathways utilized by JCPyV to mediate viral entry are poorly understood. Our findings further characterize how JCPyV utilizes the clathrin-mediated endocytosis pathway to invade host cells. We have identified specific components of this pathway that are necessary for the viral entry process and infection. Collectively, the conclusions increase our understanding of JCPyV infection and pathogenesis and may contribute to the future development of novel therapeutic strategies for PML.