RESUMO
Merkel cell polyomavirus (MCPyV) is the causative agent of the majority of Merkel cell carcinomas (MCC). The virus has limited coding capacity, with its early viral proteins, large T (LT) and small T (sT), being multifunctional and contributing to infection and transformation. A fundamental difference in early viral gene expression between infection and MCPyV-driven tumorigenesis is the expression of a truncated LT (LTtr) in the tumor. In contrast, sT is expressed in both conditions and contributes significantly to oncogenesis. Here, we identified novel functions of early viral proteins by performing genome-wide transcriptome and chromatin studies in primary human fibroblasts. Due to current limitations in infection and tumorigenesis models, we mimic these conditions by ectopically expressing sT, LT or LTtr, individually or in combination, at different time points. In addition to its known function in cell cycle and inflammation modulation, we reveal a fundamentally new function of sT. We show that sT regulates the type I interferon (IFN) response downstream of the type I interferon receptor (IFNAR) by interfering with the interferon-stimulated gene factor 3 (ISGF3)-induced interferon-stimulated gene (ISG) response. Expression of sT leads to a reduction in the expression of interferon regulatory factor 9 (IRF9) which is a central component of the ISGF3 complex. We further show that this function of sT is conserved in BKPyV. We provide a first mechanistic understanding of which early viral proteins trigger and control the type I IFN response, which may influence MCPyV infection, persistence and, during MCC progression, regulation of the tumor microenvironment.
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Induction of type I interferon (IFN) gene expression is among the first lines of cellular defense a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system, showing that M35 interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35's function. Determination of M35's crystal structure combined with reverse genetics revealed that homodimerization is a key feature for M35's immunomodulatory activity. In electrophoretic mobility shift assays (EMSAs), purified M35 protein specifically bound to the regulatory DNA element that governs transcription of the first type I IFN gene induced in nonimmune cells, Ifnb1. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signaling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signaling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labeled transcripts (SLAM-seq) and assessed M35's global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically downregulated basal expression of IRF3-dependent genes. During MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1. Our results suggest that M35-DNA binding directly antagonizes gene induction mediated by IRF3 and impairs the antiviral response more broadly than formerly recognized. IMPORTANCE Replication of the ubiquitous human cytomegalovirus (HCMV) in healthy individuals mostly goes unnoticed but can impair fetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry into host cells, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here, we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key cellular factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.
Assuntos
Infecções por Citomegalovirus , Elementos Facilitadores Genéticos , Fator Regulador 3 de Interferon , Interferon Tipo I , Proteínas da Matriz Viral , Animais , Humanos , Camundongos , Infecções por Citomegalovirus/genética , DNA/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , Muromegalovirus/genética , Muromegalovirus/metabolismo , Proteínas da Matriz Viral/metabolismoRESUMO
BACKGROUND: The ongoing coronavirus disease 2019 pandemic significantly burdens hospitals and other healthcare facilities. Therefore, understanding the entry and transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for effective prevention and preparedness measures. We performed surveillance and analysis of testing and transmission of SARS-CoV-2 infections in a tertiary-care hospital in Germany during the second and third pandemic waves in fall/winter 2020. METHODS: Between calendar week 41 in 2020 and calendar week 1 in 2021, 40%, of all positive patient and staff samples (284 total) were subjected to full-length viral genome sequencing. Clusters were defined based on similar genotypes indicating common sources of infection. We integrated phylogenetic, spatial, and temporal metadata to detect nosocomial infections and outbreaks, uncover transmission chains, and evaluate containment measures' effectiveness. RESULTS: Epidemiologic data and contact tracing readily recognize most healthcare-associated (HA) patient infections. However, sequencing data reveal that temporally preceding index cases and transmission routes can be missed using epidemiologic methods, resulting in delayed interventions and serially linked outbreaks being counted as independent events. While hospital-associated transmissions were significantly elevated at a moderate rate of community transmission during the second wave, systematic testing and high vaccination rates among staff have led to a substantial decrease in HA infections at the end of the second/beginning of the third wave despite high community transmissions. CONCLUSIONS: While epidemiologic analysis is critical for immediate containment of HA SARS-CoV-2 outbreaks, integration of genomic surveillance revealed weaknesses in identifying staff contacts. Our study underscores the importance of high testing frequency and genomic surveillance to detect, contain and prevent SARS-CoV-2-associated infections in healthcare settings.
Assuntos
COVID-19 , Infecção Hospitalar , Humanos , SARS-CoV-2/genética , COVID-19/epidemiologia , Filogenia , Centros de Atenção Terciária , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/prevenção & controleRESUMO
Various pathogens systematically reprogram gene expression in macrophages, but the underlying mechanisms are largely unknown. We investigated whether the enteropathogen Yersinia enterocolitica alters chromatin states to reprogram gene expression in primary human macrophages. Genome-wide chromatin immunoprecipitation (ChIP) seq analyses showed that pathogen-associated molecular patterns (PAMPs) induced up- or down-regulation of histone modifications (HMod) at approximately 14500 loci in promoters and enhancers. Effectors of Y. enterocolitica reorganized about half of these dynamic HMod, with the effector YopP being responsible for about half of these modulatory activities. The reorganized HMod were associated with genes involved in immune response and metabolism. Remarkably, the altered HMod also associated with 61% of all 534 known Rho GTPase pathway genes, revealing a new level in Rho GTPase regulation and a new aspect of bacterial pathogenicity. Changes in HMod were associated to varying degrees with corresponding gene expression, e. g. depending on chromatin localization and cooperation of the HMod. In summary, infection with Y. enterocolitica remodels HMod in human macrophages to modulate key gene expression programs of the innate immune response.
Assuntos
Epigênese Genética , Código das Histonas , Imunidade Inata , Macrófagos/microbiologia , Yersiniose/microbiologia , Yersinia enterocolitica/patogenicidade , Proteínas rho de Ligação ao GTP/metabolismo , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Yersiniose/genética , Yersiniose/imunologia , Yersiniose/metabolismo , Proteínas rho de Ligação ao GTP/genéticaRESUMO
The ubiquitous host protein, CCCTC-binding factor (CTCF), is an essential regulator of cellular transcription and functions to maintain epigenetic boundaries, stabilise chromatin loops and regulate splicing of alternative exons. We have previously demonstrated that CTCF binds to the E2 open reading frame (ORF) of human papillomavirus (HPV) 18 and functions to repress viral oncogene expression in undifferentiated keratinocytes by co-ordinating an epigenetically repressed chromatin loop within HPV episomes. Keratinocyte differentiation disrupts CTCF-dependent chromatin looping of HPV18 episomes promoting induction of enhanced viral oncogene expression. To further characterise CTCF function in HPV transcription control we utilised direct, long-read Nanopore RNA-sequencing which provides information on the structure and abundance of full-length transcripts. Nanopore analysis of primary human keratinocytes containing HPV18 episomes before and after synchronous differentiation allowed quantification of viral transcript species, including the identification of low abundance novel transcripts. Comparison of transcripts produced in wild type HPV18 genome-containing cells to those identified in CTCF-binding deficient genome-containing cells identifies CTCF as a key regulator of differentiation-dependent late promoter activation, required for efficient E1^E4 and L1 protein expression. Furthermore, our data show that CTCF binding at the E2 ORF promotes usage of the downstream weak splice donor (SD) sites SD3165 and SD3284, to the dominant E4 splice acceptor site at nucleotide 3434. These findings demonstrate that in the HPV life cycle both early and late virus transcription programmes are facilitated by recruitment of CTCF to the E2 ORF.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Diferenciação Celular , Regulação Viral da Expressão Gênica , Papillomavirus Humano 18/genética , Infecções por Papillomavirus/virologia , Splicing de RNA , Proteínas Virais/genética , Fator de Ligação a CCCTC/genética , Cromatina/genética , Cromatina/metabolismo , Genoma Viral , Humanos , Queratinócitos/metabolismo , Queratinócitos/virologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Regiões Promotoras Genéticas , Replicação ViralRESUMO
Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas (MCC). MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes. Which molecular mechanisms promote viral integration, if distinct integration patterns exist, and if integration occurs preferentially at loci with specific chromatin states is unknown. We here combined short and long-read (nanopore) next-generation sequencing and present the first high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material. We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that map closely together, and complex integration loci that exhibit local amplification of genomic sequences flanking the viral DNA. Sequence analysis suggests that linear patterns are produced during viral replication by integration of defective/linear genomes into host DNA double strand breaks via non-homologous end joining, NHEJ. In contrast, our data strongly suggest that complex integration patterns are mediated by microhomology-mediated break-induced replication, MMBIR. Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, rather than transcription from juxtaposed host promoters. Taken together, our data explain the characteristics of MCPyV integration and may also provide a model for integration of other oncogenic DNA viruses such as papillomaviruses.
Assuntos
Carcinoma de Célula de Merkel/patologia , Reparo do DNA por Junção de Extremidades , Poliomavírus das Células de Merkel/genética , Infecções por Polyomavirus/complicações , Infecções Tumorais por Vírus/complicações , Integração Viral , Replicação Viral , Antígenos Virais de Tumores , Neoplasias Ósseas/genética , Neoplasias Ósseas/secundário , Neoplasias Ósseas/virologia , Carcinoma de Célula de Merkel/genética , Carcinoma de Célula de Merkel/virologia , Humanos , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/virologia , Recombinação Genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/virologia , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/virologia , Proteínas Virais/genéticaRESUMO
The current pandemic with Severe acute respiratory syndrome-coronavirus-2 has been taking on new dynamics since the emergence of new variants last fall, some of them spreading more rapidly. Many countries currently find themselves in a race to ramp up vaccination strategies that have been initiated and a possible third wave of the pandemic from new variants, such as the Variant of Concern-202012/01 from the B.1.1.7 lineage. Until today, many investigations in death cases of Coronavirus-disease-19 have been conducted, revealing pulmonary damage to be the predominant feature of the disease. Thereby, different degrees of macroscopic and microscopic lung damage have been reported, most of them resembling an Acute Respiratory Distress Syndrome. Far more, systemic complications of the disease such as pulmonary embolisms have been described. However, neither morphologic nor virologic findings of patients dying of the new variants have yet been reported. Here, we report on a comprehensive analysis of radiologic, morphologic, and virologic findings in a fatal case of this variant.
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COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , COVID-19/virologia , Evolução Fatal , Humanos , PandemiasRESUMO
BK polyomavirus-associated nephropathy is a common complication after kidney transplantation leading to reduced graft function or loss. The molecular pathogenesis of BK polyomavirus-induced nephropathy is not well understood. A recent study had described a protective effect of the activating natural killer cell receptor KIR3DS1 in BK polyomavirus-associated nephropathy, suggesting a role of NK cells in modulating disease progression. Using an in vitro cell culture model of human BK polyomavirus infection and kidney biopsy samples from patients with BK polyomavirus-associated nephropathy, we observed significantly increased surface expression of the ligand for KIR3DS1, HLA-F, on BK polyomavirus-infected kidney tubular cells. Upregulation of HLA-F expression resulted in significantly increased binding of KIR3DS1 to BK polyomavirus-infected cells and activation of primary KIR3DS-positive natural killer cells. Thus, our data provide a mechanism by which KIR3DS-positive natural killer cells can control BK polyomavirus infection of the kidney, and rationale for exploring HLA-F/KIR3DS1 interactions for immunotherapeutic approaches in BK polyomavirus-associated nephropathy.
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Vírus BK , Nefropatias , Infecções por Polyomavirus , Infecções Tumorais por Vírus , Humanos , Células Matadoras Naturais/metabolismo , Receptores KIR3DS1/genética , Receptores KIR3DS1/metabolismo , Regulação para CimaRESUMO
The anaphase-promoting complex, or cyclosome (APC/C), is a large E3 ubiquitin ligase composed of 14 subunits. The activity of APC/C oscillates during the cell cycle to ensure a timely transition through each phase by promoting the degradation of important cell cycle regulators. Of the human herpesviruses, cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) both impair the activity of APC/C during their lytic replication cycle through virus-encoded protein kinases. Here, we addressed whether the oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) deregulates the activity of APC/C during the lytic replication cycle. To this end, we used the well-characterized iSLK.219 cell model of KSHV infection and established a new infection model of primary lymphatic endothelial cells (LECs) infected with a lytically replicating KSHV BAC16 mutant. In contrast to those of EBV and HCMV, the KSHV lytic cycle occurs while the APC/C is active. Moreover, interfering with the activity of APC/C did not lead to major changes in the production of infectious virus. We further investigated whether rereplication stress induced by the unscheduled activation of the APC/C-CDH1 complex affects the number and integrity of KSHV viral episomes. Deep sequencing of the viral episomes and host chromosomes in iSLK.219 cells revealed that, while distinct regions in the cellular chromosomes were severely affected by rereplication stress, the integrity of the viral episomes remained unaltered.IMPORTANCE DNA viruses have evolved complex strategies to gain control over the cell cycle. Several of them target APC/C, a key cellular machinery that controls the timely progression of the cell cycle, by either blocking or enhancing its activity. Here, we investigated the activity of APC/C during the lytic replication cycle of KSHV and found that, in contrast to that of KSHV's close relatives EBV and HCMV, KSHV lytic replication occurs while the APC/C is active. Perturbing APC/C activity by depleting a core protein or the adaptor proteins of the catalytic domain, and hence interfering with normal cell-cycle progression, did not affect virus replication. This suggests that KSHV has evolved to replicate independently of the activity of APC/C and in various cell cycle conditions.
Assuntos
Herpesvirus Humano 8/metabolismo , Latência Viral/genética , Replicação Viral/fisiologia , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Linhagem Celular , Células Endoteliais/metabolismo , Regulação Viral da Expressão Gênica/genética , Herpesvirus Humano 8/patogenicidade , Humanos , Cultura Primária de Células , Sarcoma de Kaposi/virologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Virais/metabolismo , Ativação Viral/genética , Latência Viral/fisiologiaRESUMO
Merkel cell polyomavirus (MCPyV) is the major cause for Merkel cell carcinoma (MCC), a rare but highly aggressive skin cancer predominantly found in elderly and immunosuppressed patients. The early viral gene products large T-antigen (LT) and small T-antigen (sT) are important for efficient viral DNA replication, and both contribute to transformation processes. These functions are executed mainly through interactions with host factors. Here, we identify the cellular ubiquitin-specific processing protease 7 (Usp7) as a new interaction partner of the MCPyV LT. Using glutathione S-transferase pulldown experiments, we show that MCPyV LT directly binds to Usp7 and that N- as well as C-terminal regions of LT bind to the TRAF (tumor necrosis factor receptor-associated) domain of Usp7. We demonstrate that endogenous Usp7 coprecipitates with MCPyV T-antigens and relocalizes to viral DNA replication centers in cells actively replicating MCPyV genomes. We show that Usp7 does not alter ubiquitination levels of the T-antigens; however, Usp7 binding increases the binding affinity of LT to the origin of replication, thereby negatively regulating viral DNA replication. Together, these data identify Usp7 as a restriction factor of MCPyV replication. In contrast to other DNA viruses, Usp7 does not affect MCPyV gene expression via its ubiquitination activity but influences MCPyV DNA replication solely via a novel mechanism that modulates binding of LT to viral DNA.IMPORTANCE MCPyV is the only human polyomavirus that is associated with cancer; the majority of Merkel cell cancers have a viral etiology. While much emphasis was placed on investigations to understand the transformation process by MCPyV oncoproteins and cellular factors, we have only limited knowledge of cellular factors participating in the MCPyV life cycle. Here, we describe Usp7, a cellular deubiquitination enzyme, as a new factor involved in MCPyV replication. Usp7 is known in the context of large DNA tumor viruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus, to restrict viral replication. Similar to EBV, where Usp7 binding to EBNA1 increases EBNA1 binding affinity to viral DNA, we find MCPyV LT binding to the origin of replication to be increased in the presence of Usp7, resulting in restriction of viral DNA replication. However, Usp7-induced restriction of MCPyV replication is independent of its enzymatic activity, thereby constituting a novel mechanism of Usp7-induced restriction of viral replication.
Assuntos
Antígenos Virais de Tumores/metabolismo , DNA Viral/metabolismo , Poliomavírus das Células de Merkel/genética , Poliomavírus das Células de Merkel/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo , Replicação Viral/fisiologia , Carcinoma de Célula de Merkel/virologia , Linhagem Celular , Proliferação de Células , Células HEK293 , Humanos , Poliomavírus das Células de Merkel/crescimento & desenvolvimento , Infecções por Polyomavirus/virologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Infecções Tumorais por Vírus/virologiaRESUMO
Latent Kaposi sarcoma-associated herpesvirus (KSHV) genomes rapidly acquire distinct patterns of the activating histone modification H3K4-me3 as well as repressive H3K27-me3 marks, a modification linked to transcriptional silencing by polycomb repressive complexes (PRC). Interestingly, PRCs have recently been reported to restrict viral gene expression in a number of other viral systems, suggesting they may play a broader role in controlling viral chromatin. If so, it is an intriguing possibility that latency establishment may result from viral subversion of polycomb-mediated host responses to exogenous DNA. To investigate such scenarios we sought to establish whether rapid repression by PRC constitutes a general hallmark of herpesvirus latency. For this purpose, we performed a comparative epigenome analysis of KSHV and the related murine gammaherpesvirus 68 (MHV-68). We demonstrate that, while latently replicating MHV-68 genomes readily acquire distinct patterns of activation-associated histone modifications upon de novo infection, they fundamentally differ in their ability to efficiently attract H3K27-me3 marks. Statistical analyses of ChIP-seq data from in vitro infected cells as well as in vivo latency reservoirs furthermore suggest that, whereas KSHV rapidly attracts PRCs in a genome-wide manner, H3K27-me3 acquisition by MHV-68 genomes may require spreading from initial seed sites to which PRC are recruited as the result of an inefficient or stochastic recruitment, and that immune pressure may be needed to select for latency pools harboring PRC-silenced episomes in vivo. Using co-infection experiments and recombinant viruses, we also show that KSHV's ability to rapidly and efficiently acquire H3K27-me3 marks does not depend on the host cell environment or unique properties of the KSHV-encoded LANA protein, but rather requires specific cis-acting sequence features. We show that the non-canonical PRC1.1 component KDM2B, a factor which binds to unmethylated CpG motifs, is efficiently recruited to KSHV genomes, indicating that CpG island characteristics may constitute these features. In accord with the fact that, compared to MHV-68, KSHV genomes exhibit a fundamentally higher density of CpG motifs, we furthermore demonstrate efficient acquisition of H2AK119-ub by KSHV and H3K36-me2 by MHV-68 (but not vice versa), furthermore supporting the notion that KSHV genomes rapidly attract PRC1.1 complexes in a genome-wide fashion. Collectively, our results suggest that rapid PRC silencing is not a universal feature of viral latency, but that some viruses may rather have adopted distinct genomic features to specifically exploit default host pathways that repress epigenetically naive, CpG-rich DNA.
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Herpesvirus Humano 8/genética , Proteínas do Grupo Polycomb/metabolismo , Rhadinovirus/genética , Latência Viral/genética , Animais , Linhagem Celular Transformada , Ilhas de CpG/genética , Epigenoma/genética , Feminino , Regulação Viral da Expressão Gênica/genética , Genoma Viral/genética , Código das Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The RNA-binding protein TDP-43 is heavily implicated in neurodegenerative disease. Numerous patient mutations in TARDBP, the gene encoding TDP-43, combined with data from animal and cell-based models, imply that altered RNA regulation by TDP-43 causes Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. However, underlying mechanisms remain unresolved. Increased cytoplasmic TDP-43 levels in diseased neurons suggest a possible role in this cellular compartment. Here, we examined the impact on translation of overexpressing human TDP-43 and the TDP-43A315T patient mutant protein in motor neuron-like cells and primary cultures of cortical neurons. In motor-neuron like cells, TDP-43 associates with ribosomes without significantly affecting global translation. However, ribosome profiling and additional assays revealed enhanced translation and direct binding of Camta1, Mig12, and Dennd4a mRNAs. Overexpressing either wild-type TDP-43 or TDP-43A315T stimulated translation of Camta1 and Mig12 mRNAs via their 5'UTRs and increased CAMTA1 and MIG12 protein levels. In contrast, translational enhancement of Dennd4a mRNA required a specific 3'UTR region and was specifically observed with the TDP-43A315T patient mutant allele. Our data reveal that TDP-43 can function as an mRNA-specific translational enhancer. Moreover, since CAMTA1 and DENND4A are linked to neurodegeneration, they suggest that this function could contribute to disease.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação a DNA/genética , Doenças Neurodegenerativas/genética , Transativadores/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Citoplasma/genética , Citoplasma/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação , Doenças Neurodegenerativas/patologia , Cultura Primária de Células , RNA Mensageiro/genética , Ribossomos/genéticaRESUMO
BACKGROUND: The detection of known human papillomaviruses (PVs) from targeted wet-lab approaches has traditionally used PCR-based methods coupled with Sanger sequencing. With the introduction of next-generation sequencing (NGS), these approaches can be revisited to integrate the sequencing power of NGS. Although computational tools have been developed for metagenomic approaches to search for known or novel viruses in NGS data, no appropriate tool is available for the classification and identification of novel viral sequences from data produced by amplicon-based methods. RESULTS: We have developed PVAmpliconFinder, a data analysis workflow designed to rapidly identify and classify known and potentially new Papillomaviridae sequences from NGS amplicon sequencing with degenerate PV primers. Here, we describe the features of PVAmpliconFinder and its implementation using biological data obtained from amplicon sequencing of human skin swab specimens and oral rinses from healthy individuals. CONCLUSIONS: PVAmpliconFinder identified putative new HPV sequences, including one that was validated by wet-lab experiments. PVAmpliconFinder can be easily modified and applied to other viral families. PVAmpliconFinder addresses a gap by providing a solution for the analysis of NGS amplicon sequencing, increasingly used in clinical research. The PVAmpliconFinder workflow, along with its source code, is freely available on the GitHub platform: https://github.com/IARCbioinfo/PVAmpliconFinder.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Papillomaviridae/isolamento & purificação , Interface Usuário-Computador , DNA Viral/química , DNA Viral/metabolismo , Humanos , Papillomaviridae/genética , Fluxo de TrabalhoRESUMO
MicroRNAs (miRNAs) are small RNAs that regulate diverse biological processes including multiple aspects of the host-pathogen interface. Consequently, miRNAs are commonly encoded by viruses that undergo long-term persistent infection. Papillomaviruses (PVs) are capable of undergoing persistent infection, but as yet, no widely-accepted PV-encoded miRNAs have been described. The incomplete understanding of PV-encoded miRNAs is due in part to lack of tractable laboratory models for most PV types. To overcome this, we have developed miRNA Discovery by forced Genome Expression (miDGE), a new wet bench approach to miRNA identification that screens numerous pathogen genomes in parallel. Using miDGE, we screened over 73 different PV genomes for the ability to code for miRNAs. Our results show that most PVs are unlikely to code for miRNAs and we conclusively demonstrate a lack of PV miRNA expression in cancers associated with infections of several high risk HPVs. However, we identified five different high-confidence or highly probable miRNAs encoded by four different PVs (Human PVs 17, 37, 41 and a Fringilla coelebs PV (FcPV1)). Extensive in vitro assays confirm the validity of these miRNAs in cell culture and two FcPV1 miRNAs are further confirmed to be expressed in vivo in a natural host. We show that miRNAs from two PVs (HPV41 & FcPV1) are able to regulate viral transcripts corresponding to the early region of the PV genome. Combined, these findings identify the first canonical PV miRNAs and support that miRNAs of either host or viral origin are important regulators of the PV life cycle.
Assuntos
Perfilação da Expressão Gênica/métodos , Regulação Viral da Expressão Gênica/genética , MicroRNAs/genética , Papillomaviridae/genética , RNA Viral/análise , Células HEK293 , Humanos , Infecções por Papillomavirus/genética , RNA Viral/genética , TranscriptomaRESUMO
Bromodomain-containing protein 4 (BRD4) is a member of the bromo- and extraterminal (BET) domain-containing family of epigenetic readers which is under intensive investigation as a target for anti-tumor therapy. BRD4 plays a central role in promoting the expression of select subsets of genes including many driven by oncogenic transcription factors and signaling pathways. However, the role of BRD4 and the effects of BET inhibitors in non-transformed cells remain mostly unclear. We demonstrate that BRD4 is required for the maintenance of a basal epithelial phenotype by regulating the expression of epithelial-specific genes including TP63 and Grainy Head-like transcription factor-3 (GRHL3) in non-transformed basal-like mammary epithelial cells. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription. Motif analyses of cell context-specific BRD4-enriched regions predicted the involvement of FOXO transcription factors. Consistently, activation of FOXO1 function via inhibition of EGFR-AKT signaling promoted the expression of TP63 and GRHL3. Moreover, activation of Src kinase signaling and FOXO1 inhibition decreased the expression of FOXO/BRD4 target genes. Together, our findings support a function for BRD4 in promoting basal mammary cell epithelial differentiation, at least in part, by regulating FOXO factor function on enhancers to activate TP63 and GRHL3 expression.
Assuntos
Mama/metabolismo , Proteínas de Ligação a DNA/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Mama/citologia , Proteínas de Ciclo Celular , Linhagem Celular , Proteínas de Ligação a DNA/biossíntese , Elementos Facilitadores Genéticos , Fatores de Transcrição Forkhead/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , RNA Polimerase II/metabolismo , Transdução de Sinais , Fatores de Transcrição/biossíntese , Transcrição Gênica , Proteínas Supressoras de Tumor/biossínteseRESUMO
Differences in T-cell phenotype, particularly the expression of markers of T-cell homeostasis, have been observed in fatal and nonfatal Ebola virus disease (EVD). However, the relationship between these markers with T-cell function and virus clearance during EVD is poorly understood. To gain biological insight into the role of T cells during EVD, combined transcriptomics and T-cell receptor sequencing was used to profile blood samples from fatal and nonfatal EVD patients from the recent West African EVD epidemic. Fatal EVD was characterized by strong T-cell activation and increased abundance of T-cell inhibitory molecules. However, the early T-cell response was oligoclonal and did not result in viral clearance. In contrast, survivors mounted highly diverse T-cell responses, maintained low levels of T-cell inhibitors, and cleared Ebola virus. Our findings highlight the importance of T-cell immunity in surviving EVD and strengthen the foundation for further research on targeting of the dendritic cell-T cell interface for postexposure immunotherapy.
Assuntos
Doença pelo Vírus Ebola/imunologia , Homeostase , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/imunologia , Biomarcadores , Estudos Transversais , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/mortalidade , Humanos , TranscriptomaRESUMO
Yersinia outer protein M (YopM) is a crucial immunosuppressive effector of the plaque agent Yersinia pestis and other pathogenic Yersinia species. YopM enters the nucleus of host cells but neither the mechanisms governing its nucleocytoplasmic shuttling nor its intranuclear activities are known. Here we identify the DEAD-box helicase 3 (DDX3) as a novel interaction partner of Y. enterocolitica YopM and present the three-dimensional structure of a YopM:DDX3 complex. Knockdown of DDX3 or inhibition of the exportin chromosomal maintenance 1 (CRM1) increased the nuclear level of YopM suggesting that YopM exploits DDX3 to exit the nucleus via the CRM1 export pathway. Increased nuclear YopM levels caused enhanced phosphorylation of Ribosomal S6 Kinase 1 (RSK1) in the nucleus. In Y. enterocolitica infected primary human macrophages YopM increased the level of Interleukin-10 (IL-10) mRNA and this effect required interaction of YopM with RSK and was enhanced by blocking YopM's nuclear export. We propose that the DDX3/CRM1 mediated nucleocytoplasmic shuttling of YopM determines the extent of phosphorylation of RSK in the nucleus to control transcription of immunosuppressive cytokines.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/biossíntese , Yersiniose/imunologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Cristalografia por Raios X , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/imunologia , Imunofluorescência , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Parasita/fisiologia , Humanos , Tolerância Imunológica/fisiologia , Imunoprecipitação , Macrófagos/microbiologia , Espectrometria de Massas , Microscopia Confocal , Reação em Cadeia da Polimerase , Transporte Proteico/fisiologia , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismo , Yersiniose/metabolismo , Yersinia enterocoliticaRESUMO
Multiple myeloma (MM) is a hematological cancer for which immune-based treatments are currently in development. Many of these rely on the identification of highly disease-specific, strongly and stably expressed antigens. Here, we profiled the myeloma B-cell immunome both to explore its predictive role in the context of autologous and allogeneic hematopoietic stem cell transplantation (HSCT) and to identify novel immunotherapeutic targets. We used random peptide phage display, reverse immunization, and next-generation sequencing-assisted antibody phage display to establish a highly myeloma-specific epitope fingerprint targeted by B-cell responses of 18 patients in clinical remission. We found that allogeneic HSCT more efficiently allowed production of myeloma-specific antibodies compared with autologous HSCT and that a highly reactive epitope recognition signature correlated with superior response to treatment. Next, we performed myeloma cell surface screenings of phage-displayed patient transplant immunomes. Although some of the screenings yielded clear-cut surface binders, the majority of screenings did not, suggesting that many of the targeted antigens may in fact not be accessible to the B-cell immune system in untreated myeloma cells. This fit well with the identification of heat-shock proteins as a class of antigens that showed overall the broadest reactivity with myeloma patient sera after allogeneic HSCT and that may be significantly translocated to the cell surface upon treatment as a result of immunogenic cell death. Our data reveal a disease-specific epitope signature of MM that is predictive for response to treatment. Mining of transplant immunomes for strong myeloma surface binders may open up avenues for myeloma immunotherapy.
Assuntos
Mapeamento de Epitopos/métodos , Epitopos/metabolismo , Terapia de Alvo Molecular/métodos , Mieloma Múltiplo/terapia , Proteoma/imunologia , Transplantes/imunologia , Transplantes/metabolismo , Adulto , Idoso , Linhagem Celular Tumoral , Feminino , Células HL-60 , Humanos , Imunoterapia/métodos , Células Jurkat , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/imunologia , Proteínas do Mieloma/análise , Proteínas do Mieloma/isolamento & purificação , Biblioteca de Peptídeos , Mapeamento de Peptídeos/métodos , Proteoma/análiseRESUMO
Atypical porcine pestivirus (APPV) was recently reported to be associated with neurologic disorders in newborn piglets. Investigations of 1,460 serum samples of apparently healthy pigs from different parts of Europe and Asia demonstrate a geographically wide distribution of genetically highly variable APPV and high APPV genome and antibody detection rates.
Assuntos
Anticorpos Antivirais/sangue , Variação Genética , Genoma Viral , Infecções por Pestivirus/veterinária , Pestivirus/genética , Doenças dos Suínos/epidemiologia , Animais , Animais Recém-Nascidos , Ásia/epidemiologia , Doenças Assintomáticas , Europa (Continente)/epidemiologia , Pestivirus/classificação , Pestivirus/isolamento & purificação , Infecções por Pestivirus/epidemiologia , Infecções por Pestivirus/transmissão , Infecções por Pestivirus/virologia , Filogenia , RNA Helicases/genética , RNA Viral/genética , Serina Endopeptidases/genética , Suínos , Doenças dos Suínos/transmissão , Doenças dos Suínos/virologia , Proteínas não Estruturais Virais/genéticaRESUMO
Merkel cell carcinoma (MCC) is an aggressive skin cancer entity that frequently leads to rapid death due to its high propensity to metastasize. The etiology of most MCC cases is linked to Merkel cell polyomavirus (MCPyV), a virus which is monoclonally integrated in up to 95% of tumors. While there are presently no animal models to study the role of authentic MCPyV infection on transformation, tumorigenesis or metastasis formation, xenograft mouse models employing engrafted MCC-derived cell lines (MCCL) represent a promising approach to study certain aspects of MCC pathogenesis. Here, the two MCPyV-positive MCC cell lines WaGa and MKL-1 were subcutaneously engrafted in scid mice. Engraftment of both MCC cell lines resulted in the appearance of circulating tumor cells and metastasis formation, with WaGa-engrafted mice showing a significantly shorter survival time as well as increased numbers of spontaneous lung metastases compared to MKL-1 mice. Interestingly, explanted tumors compared to parental cell lines exhibit an upregulation of MCPyV sT-Antigen expression in all tumors, with WaGa tumors showing significantly higher sT-Antigen expression than MKL-1 tumors. RNA-Seq analysis of explanted tumors and parental cell lines furthermore revealed that in the more aggressive WaGa tumors, genes involved in inflammatory response, growth factor activity and Wnt signalling pathway are significantly upregulated, suggesting that sT-Antigen is the driver of the observed differences in metastasis formation.