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1.
JCI Insight ; 7(1)2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-35014624

RESUMO

Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of neurodevelopmental disorders. However, the neuropathogenesis remains largely elusive due to a lack of informative animal models. In this study, we developed a congenital murine CMV (cMCMV) infection mouse model with high survival rate and long survival period that allowed long-term follow-up study of neurodevelopmental disorders. This model involves in utero intracranial injection and mimics many reported clinical manifestations of cCMV infection in infants, including growth restriction, hearing loss, and impaired cognitive and learning-memory abilities. We observed that abnormalities in MRI/CT neuroimaging were consistent with brain hemorrhage and loss of brain parenchyma, which was confirmed by pathological analysis. Neuropathological findings included ventriculomegaly and cortical atrophy associated with impaired proliferation and migration of neural progenitor cells in the developing brain at both embryonic and postnatal stages. Robust inflammatory responses during infection were shown by elevated inflammatory cytokine levels, leukocyte infiltration, and activation of microglia and astrocytes in the brain. Pathological analyses and CT neuroimaging revealed brain calcifications induced by cMCMV infection and cell death via pyroptosis. Furthermore, antiviral treatment with ganciclovir significantly improved neurological functions and mitigated brain damage as shown by CT neuroimaging. These results demonstrate that this model is suitable for investigation of mechanisms of infection-induced brain damage and long-term studies of neurodevelopmental disorders, including the development of interventions to limit CNS damage associated with cCMV infection.


Assuntos
Infecções por Citomegalovirus , Modelos Animais de Doenças , Neuroimagem , Animais , Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/diagnóstico por imagem , Infecções por Citomegalovirus/fisiopatologia , Infecções por Citomegalovirus/terapia , Feminino , Seguimentos , Camundongos , Camundongos Endogâmicos ICR , Gravidez
2.
Front Immunol ; 12: 713611, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34867944

RESUMO

Background: Methotrexate (MTX) is the first line treatment of rheumatoid arthritis (RA), and methylation changes in bulk T cells have been reported after treatment with MTX. We have investigated cell-type specific DNA methylation changes across the genome in naïve and memory CD4+ T cells before and after MTX treatment of RA patients. DNA methylation profiles of newly diagnosed RA patients (N=9) were assessed by reduced representation bisulfite sequencing. Results: We found that MTX treatment significantly influenced DNA methylation levels at multiple CpG sites in both cell populations. Interestingly, we identified differentially methylated sites annotated to two genes; TRIM15 and SORC2, previously reported to predict treatment outcome in RA patients when measured in bulk T cells. Furthermore, several of the genes, including STAT3, annotated to the significant CpG sites are relevant for RA susceptibility or the action of MTX. Conclusion: We detected CpG sites that were associated with MTX treatment in CD4+ naïve and memory T cells isolated from RA patients. Several of these sites overlap genetic regions previously associated with RA risk and MTX treatment outcome.


Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Metilação de DNA/efeitos dos fármacos , Metotrexato/uso terapêutico , Adulto , Idoso , Antirreumáticos/farmacologia , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Ilhas de CpG , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , /imunologia , Metotrexato/farmacologia , Pessoa de Meia-Idade , Receptores CCR6/genética , Receptores de Superfície Celular/genética , Fator de Transcrição STAT3/genética , Sinaptogirinas/genética
3.
Front Immunol ; 12: 663736, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33897713

RESUMO

Rheumatoid arthritis (RA) is a complex disease with a wide range of underlying susceptibility factors. Recently, dysregulation of microRNAs (miRNAs) in RA have been reported in several immune cell types from blood. However, B cells have not been studied in detail yet. Given the autoimmune nature of RA with the presence of autoantibodies, CD19+ B cells are a key cell type in RA pathogenesis and alterations in CD19+ B cell subpopulations have been observed in patient blood. Therefore, we aimed to reveal the global miRNA repertoire and to analyze miRNA expression profile differences in homogenous RA patient phenotypes in blood-derived CD19+ B cells. Small RNA sequencing was performed on CD19+ B cells of newly diagnosed untreated RA patients (n=10), successfully methotrexate (MTX) treated RA patients in remission (MTX treated RA patients, n=18) and healthy controls (n=9). The majority of miRNAs was detected across all phenotypes. However, significant expression differences between MTX treated RA patients and controls were observed for 27 miRNAs, while no significant differences were seen between the newly diagnosed patients and controls. Several of the differentially expressed miRNAs were previously found to be dysregulated in RA including miR-223-3p, miR-486-3p and miR-23a-3p. MiRNA target enrichment analysis, using the differentially expressed miRNAs and miRNA-target interactions from miRTarBase as input, revealed enriched target genes known to play important roles in B cell activation, differentiation and B cell receptor signaling, such as STAT3, PRDM1 and PTEN. Interestingly, many of those genes showed a high degree of correlated expression in CD19+ B cells in contrast to other immune cell types. Our results suggest important regulatory functions of miRNAs in blood-derived CD19+ B cells of MTX treated RA patients and motivate for future studies investigating the interactive mechanisms between miRNA and gene targets, as well as the possible predictive power of miRNAs for RA treatment response.


Assuntos
Artrite Reumatoide/etiologia , Artrite Reumatoide/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Metotrexato/farmacologia , MicroRNAs/genética , Antígenos CD19/metabolismo , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Biomarcadores , Biologia Computacional/métodos , Gerenciamento Clínico , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Metotrexato/uso terapêutico , Interferência de RNA
5.
RNA Biol ; 17(9): 1284-1292, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32436772

RESUMO

High-throughput sequencing has emerged as the favoured method to study microRNA (miRNA) expression, but biases introduced during library preparation have been reported. We recently compared the performance (sensitivity, reliability, titration response and differential expression) of six commercially-available kits on synthetic miRNAs and human RNA, where library preparation was performed by the vendors. We hereby supplement this study with data from two further commonly used kits (NEBNext, NEXTflex) whose manufacturers initially declined to participate. NEXTflex demonstrated the highest sensitivity, which may reflect its use of partially-randomized adapter sequences, but overall performance was lower than the QIAseq and TailorMix kits. NEBNext showed intermediate performance. We reaffirm that biases are kit specific, complicating the comparison of miRNA datasets generated using different kits.


Assuntos
Biblioteca Gênica , Engenharia Genética , MicroRNAs/genética , Engenharia Genética/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reagentes de Laboratório/normas , Reprodutibilidade dos Testes , Análise de Sequência de RNA/métodos
6.
Front Immunol ; 11: 194, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32117312

RESUMO

Background: Differences in DNA methylation have been reported in B and T lymphocyte populations, including CD4+ T cells, isolated from rheumatoid arthritis (RA) patients when compared to healthy controls. CD4+ T cells are a heterogeneous cell type with subpopulations displaying distinct DNA methylation patterns. In this study, we investigated DNA methylation using reduced representation bisulfite sequencing in two CD4+ T cell populations (CD4+ memory and naïve cells) in three groups: newly diagnosed, disease modifying antirheumatic drugs (DMARD) naïve RA patients (N = 11), methotrexate (MTX) treated RA patients (N = 18), and healthy controls (N = 9) matched for age, gender and smoking status. Results: Analyses of these data revealed significantly more differentially methylated positions (DMPs) in CD4+ memory than in CD4+ naïve T cells (904 vs. 19 DMPs) in RA patients compared to controls. The majority of DMPs (72%) identified in newly diagnosed and DMARD naïve RA patients with active disease showed increased DNA methylation (39 DMPs), whereas most DMPs (80%) identified in the MTX treated RA patients in remission displayed decreased DNA methylation (694 DMPs). Interestingly, we also found that about one third of the 101 known RA risk loci overlapped (±500 kb) with the DMPs. Notably, introns of the UBASH3A gene harbor both the lead RA risk SNP and two DMPs in CD4+ memory T cells. Conclusion: Our results suggest that RA associated DNA methylation differences vary between the two T cell subsets, but are also influenced by RA characteristics such as disease activity, disease duration and/or MTX treatment.


Assuntos
Artrite Reumatoide/imunologia , Linfócitos T CD4-Positivos/metabolismo , Metilação de DNA , Memória Imunológica , Metotrexato/uso terapêutico , Adulto , Idoso , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Ilhas de CpG , Feminino , Ontologia Genética , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único
8.
J Med Virol ; 92(5): 476-478, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32056235

RESUMO

The outbreak of a novel coronavirus (SARS-CoV-2) since December 2019 in Wuhan, the major transportation hub in central China, became an emergency of major international concern. While several etiological studies have begun to reveal the specific biological features of this virus, the epidemic characteristics need to be elucidated. Notably, a long incubation time was reported to be associated with SARS-CoV-2 infection, leading to adjustments in screening and control policies. To avoid the risk of virus spread, all potentially exposed subjects are required to be isolated for 14 days, which is the longest predicted incubation time. However, based on our analysis of a larger dataset available so far, we find there is no observable difference between the incubation time for SARS-CoV-2, severe acute respiratory syndrome coronavirus (SARS-CoV), and middle east respiratory syndrome coronavirus (MERS-CoV), highlighting the need for larger and well-annotated datasets.


Assuntos
Número Básico de Reprodução , Betacoronavirus/patogenicidade , Infecções por Coronavirus/patologia , Período de Incubação de Doenças Infecciosas , Pneumonia Viral/patologia , COVID-19 , China , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Conjuntos de Dados como Assunto , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , Vírus da SARS/patogenicidade , SARS-CoV-2 , Síndrome Respiratória Aguda Grave/patologia , Fatores de Tempo , Latência Viral
9.
RNA Biol ; 17(1): 75-86, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31559901

RESUMO

High-throughput sequencing is increasingly favoured to assay the presence and abundance of microRNAs (miRNAs) in biological samples, even from low RNA amounts, and a number of commercial vendors now offer kits that allow miRNA sequencing from sub-nanogram (ng) inputs. Although biases introduced during library preparation have been documented, the relative performance of current reagent kits has not been investigated in detail. Here, six commercial kits capable of handling <100ng total RNA input were used for library preparation, performed by kit manufactures, on synthetic miRNAs of known quantities and human total RNA samples. We compared the performance of miRNA detection sensitivity, reliability, titration response and the ability to detect differentially expressed miRNAs. In addition, we assessed the use of unique molecular identifiers (UMI) sequence tags in one kit. We observed differences in detection sensitivity and ability to identify differentially expressed miRNAs between the kits, but none were able to detect the full repertoire of synthetic miRNAs. The reliability within the replicates of all kits was good, while larger differences were observed between the kits, although none could accurately quantify the relative levels of the majority of miRNAs. UMI tags, at least within the input ranges tested, offered little advantage to improve data utility. In conclusion, biases in miRNA abundance are heavily influenced by the kit used for library preparation, suggesting that comparisons of datasets prepared by different procedures should be made with caution. This article is intended to assist researchers select the most appropriate kit for their experimental conditions.


Assuntos
Biblioteca Gênica , Engenharia Genética/métodos , MicroRNAs/genética , Engenharia Genética/normas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , MicroRNAs/síntese química , Reprodutibilidade dos Testes , Análise de Sequência de RNA/métodos
10.
Electrophoresis ; 41(1-2): 56-64, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31544246

RESUMO

In vitro derived simplified 3D representations of human organs or organ functionalities are predicted to play a major role in disease modeling, drug development, and personalized medicine, as they complement traditional cell line approaches and animal models. The cells for 3D organ representations may be derived from primary tissues, embryonic stem cells or induced pluripotent stem cells and come in a variety of formats from aggregates of individual or mixed cell types, self-organizing in vitro developed "organoids" and tissue mimicking chips. Microfluidic devices that allow long-term maintenance and combination with other tissues, cells or organoids are commonly referred to as "microphysiological" or "organ-on-a-chip" systems. Organ-on-a-chip technology allows a broad range of "on-chip" and "off-chip" analytical techniques, whereby "on-chip" techniques offer the possibility of real time tracking and analysis. In the rapidly expanding tool kit for real time analytical assays, mass spectrometry, combined with "on-chip" electrophoresis, and other separation approaches offer attractive emerging tools. In this review, we provide an overview of current 3D cell culture models, a compendium of current analytical strategies, and we make a case for new approaches for integrating separation science and mass spectrometry in this rapidly expanding research field.


Assuntos
Técnicas de Cultura de Células , Eletroforese , Dispositivos Lab-On-A-Chip , Espectrometria de Massas , Organoides , Animais , Cromatografia Líquida , Humanos , Modelos Biológicos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/fisiologia
11.
Bioinformatics ; 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31681943

RESUMO

MOTIVATION: The existence of complex subpopulations of miRNA isoforms, or isomiRs, is well established. While many tools exist for investigating isomiR populations, they differ in how they characterize an isomiR, making it difficult to compare results across different tools. Thus, there is a need for a more comprehensive and systematic standard for defining isomiRs. Such a standard would allow investigation of isomiR population structure in progressively more refined sub-populations, permitting the identification of more subtle changes between conditions and leading to an improved understanding of the processes that generate these differences. RESULTS: We developed Jasmine, a software tool that incorporates a hierarchal framework for characterizing isomiR populations. Jasmine is a Java application that can process raw read data in fastq/fasta format, or mapped reads in SAM format to produce a detailed characterization of isomiR populations. Thus, Jasmine can reveal structure not apparent in a standard miRNA-Seq analysis pipeline. AVAILABILITY: Jasmine is implemented in Java and R and freely available at bitbucket https://bitbucket.org/bipous/jasmine/src/master/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

12.
Noncoding RNA ; 5(4)2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31661777

RESUMO

A necessary pre-processing data analysis step is the removal of adapter sequences from the raw reads. While most adapter trimming tools require adapter sequence as an essential input, adapter information is often incomplete or missing. This can impact quantification of features, reproducibility of the study and might even lead to erroneous conclusions. Here, we provide examples to highlight the importance of specifying the adapter sequence by demonstrating the effect of using similar but different adapter sequences and identify additional potential sources of errors in the adapter trimming step. Finally, we propose solutions by which users can ensure their small RNA-seq data is fully annotated with adapter information.

13.
Front Physiol ; 10: 932, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31417412

RESUMO

It has been reported that several aspects of human health could be disturbed during a long-term isolated environment (for instance, the Mars-500 mission), including psychiatric disorders, circadian disruption, temporal dynamics of gut microbiota, immune responses, and physical-activity-related neuromuscular performance. Nevertheless, the mechanisms underlying these disturbances and the interactions among different aspects of human adaptation to extreme environments remain to be elucidated. Epigenetic features, like DNA methylation, might be a linking mechanism that explains the involvement of environmental factors between the human genome and the outcome of health. We conducted an exploration of personalized longitudinal DNA methylation patterns of the peripheral whole blood cells, profiling six subjects across six sampling points in the Mars-500 mission. Specifically, we developed a Personalized Epigenetic-Phenotype Synchronization Analysis (PeSa) algorithm to explore glucose- and mood-state-synchronized DNA methylation sites, focusing on finding the dynamic associations between epigenetic patterns and phenotypes in each individual, and exploring the underling epigenetic connections between glucose and mood-state disturbance. Results showed that DMPs (differentially methylated-probes) were significantly enriched in pathways related to glucose metabolism (Type II diabetes mellitus pathway), mood state (Long-term depression) and circadian rhythm (Circadian entrainment pathway) during the mission. Furthermore, our data revealed individualized glucose-synchronized and mood-state-synchronized DNA methylation sites, and PTPRN2 was found to be associated with both glucose and mood state disturbances across all six subjects. Our findings suggest that personalized phenotype-synchronized epigenetic features could reflect the effects on the human body, including the disturbances of glucose and mood-states. The association analysis of DNA methylation and phenotypes, like the PeSa analysis, could provide new possibilities in understanding the intrinsic relationship between phenotypic changes of the human body adapting to long-term isolation environmental factors.

14.
RNA Biol ; 16(11): 1534-1546, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31251108

RESUMO

microRNAs are small non-coding RNA molecules playing a central role in gene regulation. miRBase is the standard reference source for analysis and interpretation of experimental studies. However, the richness and complexity of the annotation is often underappreciated by users. Moreover, even for experienced users, the size of the resource can make it difficult to explore annotation to determine features such as species coverage, the impact of specific characteristics and changes between successive releases. A further consideration is that each new miRBase release contains entries that have had limited review and which may subsequently be removed in a future release to ensure the quality of annotation. To aid the miRBase user, we developed a software tool, miRBaseMiner, for investigating miRBase annotation and generating custom annotation sets. We apply the tool to characterize each release from v9.2 to v22 to examine how annotation has changed across releases and highlight some of the annotation features that users should keep in mind when using for miRBase for data analysis. These include: (1) entries with identical or very similar sequences; (2) entries with multiple annotated genome locations; (3) hairpin precursor entries with extremely low-estimated minimum free energy; (4) entries possessing reverse complementary; (5) entries with 3' poly(A) ends. As each of these factors can impact the identification of dysregulated features and subsequent clinical or biological conclusions, miRBaseMiner is a valuable resource for any user using miRBase as a reference source.


Assuntos
Biologia Computacional/métodos , MicroRNAs/genética , Anotação de Sequência Molecular/métodos , Animais , Humanos , Camundongos , Software , Terminologia como Assunto
15.
PLoS Negl Trop Dis ; 13(1): e0007036, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30640911

RESUMO

Coordinated surveillance, vaccination and public information efforts have brought the Chinese rabies epizootic under control, but significant numbers of fatalities are still reported annually with some cases occurring in previously rabies free regions. Tibet has remained virtually rabies free for 16 years, but since 2015 one human rabies case has been reported each year. To better understand the origins of these cases, we sequenced three human samples and an additional sample isolated from a dog in 2012. Three genomes were sequenced from brain samples: human case 1 (reported in 2015), human case 3 (2017), and the 2012 dog case. For human case 2 (2016), the rabies N gene was sequenced from a limited saliva sample. Phylogenetic analysis shows that Case 1 (CXZ1501H) and the dog case (CXZ1201D) belong to China IV lineage (equivalent to Arctic-like-2 in global rabies), suggesting an association with a wildlife spillover event. However, Case 2 (CXZ1601H) is placed within the dominant lineage China I, and was most similar with recent strains from neighboring Yunnan province, indicating the current epizootic has finally reached Tibet. Most surprisingly however, was the finding that Case 3 (CXZ1704H) is distinct from other Chinese isolates. This isolate is placed in the Indian Subcontinent clade, similar to recent Nepal strains, indicating that cross-border transmission is a new source for rabies infections. Thus, the complex mixture of the rabies epizootic in Tibet represents a major new challenge for Tibet and national rabies control.


Assuntos
Vírus da Raiva/classificação , Vírus da Raiva/genética , Raiva/epidemiologia , Raiva/veterinária , Adulto , Animais , Análise por Conglomerados , Cães , Feminino , Humanos , Masculino , Epidemiologia Molecular , Filogenia , Vírus da Raiva/isolamento & purificação , Análise de Sequência de DNA , Homologia de Sequência , Tibet/epidemiologia , Adulto Jovem
16.
PLoS Comput Biol ; 14(7): e1006185, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30005074

RESUMO

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by binding to partially complementary regions within the 3'UTR of their target genes. Computational methods play an important role in target prediction and assume that the miRNA "seed region" (nt 2 to 8) is required for functional targeting, but typically only identify ∼80% of known bindings. Recent studies have highlighted a role for the entire miRNA, suggesting that a more flexible methodology is needed. We present a novel approach for miRNA target prediction based on Deep Learning (DL) which, rather than incorporating any knowledge (such as seed regions), investigates the entire miRNA and 3'TR mRNA nucleotides to learn a uninhibited set of feature descriptors related to the targeting process. We collected more than 150,000 experimentally validated homo sapiens miRNA:gene targets and cross referenced them with different CLIP-Seq, CLASH and iPAR-CLIP datasets to obtain ∼20,000 validated miRNA:gene exact target sites. Using this data, we implemented and trained a deep neural network-composed of autoencoders and a feed-forward network-able to automatically learn features describing miRNA-mRNA interactions and assess functionality. Predictions were then refined using information such as site location or site accessibility energy. In a comparison using independent datasets, our DL approach consistently outperformed existing prediction methods, recognizing the seed region as a common feature in the targeting process, but also identifying the role of pairings outside this region. Thermodynamic analysis also suggests that site accessibility plays a role in targeting but that it cannot be used as a sole indicator for functionality. Data and source code available at: https://bitbucket.org/account/user/bipous/projects/MIRAW.


Assuntos
Simulação por Computador , Aprendizado Profundo , Marcação de Genes , MicroRNAs/genética , RNA Mensageiro/genética , Regiões 3' não Traduzidas/genética , Sítios de Ligação , Conjuntos de Dados como Assunto , Regulação da Expressão Gênica/genética , Humanos , MicroRNAs/metabolismo , Redes Neurais de Computação , Reprodutibilidade dos Testes , Termodinâmica
17.
Front Microbiol ; 9: 1067, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29922247

RESUMO

Zika virus (ZIKV) infection is associated with severe neurological defects in fetuses and newborns, such as microcephaly. However, the underlying mechanisms remain to be elucidated. In this study, proteomic analysis on ZIKV-infected primary human fetal neural progenitor cells (NPCs) revealed that virus infection altered levels of cellular proteins involved in NPC proliferation, differentiation and migration. The transcriptional levels of some of the altered targets were also confirmed by qRT-PCR. Among the altered proteins, doublecortin (DCX) plays an important role in NPC differentiation and migration. Results showed that ZIKV infection downregulated DCX, at both mRNA and protein levels, as early as 1 day post infection (1 dpi), and lasted throughout the virus replication cycle (4 days). The downregulation of DCX was also observed in a ZIKV-infected fetal mouse brain model, which displayed decreased body weight, brain size and weight, as well as defective cortex structure. By screening the ten viral proteins of ZIKV, we found that both the expression of NS4A and NS5 were correlated with the downregulation of both mRNA and protein levels of DCX in NPCs. These data suggest that DCX is modulated following infection of the brain by ZIKV. How these observed changes of DCX expression translate in the pathological consequences of ZIKV infection and if other cellular proteins are equally involved remains to be investigated.

18.
J Virol ; 92(9)2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29437978

RESUMO

WD repeat-containing protein 5 (WDR5) is essential for assembling the VISA-associated complex to induce a type I interferon antiviral response to Sendai virus infection. However, the roles of WDR5 in DNA virus infections are not well described. Here, we report that human cytomegalovirus exploits WDR5 to facilitate capsid nuclear egress. Overexpression of WDR5 in fibroblasts slightly enhanced the infectious virus yield. However, WDR5 knockdown dramatically reduced infectious virus titers with only a small decrease in viral genome replication or gene expression. Further investigation of late steps of viral replication found that WDR5 knockdown significantly impaired formation of the viral nuclear egress complex and induced substantially fewer infoldings of the inner nuclear membrane. In addition, fewer capsids were associated with these infoldings, and there were fewer capsids in the cytoplasm. Restoration of WDR5 partially reversed these effects. These results suggest that WDR5 knockdown impairs the nuclear egress of capsids, which in turn decreases virus titers. These findings reveal an important role for a host factor whose function(s) is usurped by a viral pathogen to promote efficient replication. Thus, WDR5 represents an interesting regulatory mechanism and a potential antiviral target.IMPORTANCE Human cytomegalovirus (HCMV) has a large (∼235-kb) genome with over 170 open reading frames and exploits numerous cellular factors to facilitate its replication. HCMV infection increases protein levels of WD repeat-containing protein 5 (WDR5) during infection, overexpression of WDR5 enhances viral replication, and knockdown of WDR5 dramatically attenuates viral replication. Our results indicate that WDR5 promotes the nuclear egress of viral capsids, the depletion of WDR5 resulting in a significant decrease in production of infectious virions. This is the first report that WDR5 favors HCMV, a DNA virus, replication and highlights a novel target for antiviral therapy.


Assuntos
Capsídeo/metabolismo , Citomegalovirus/fisiologia , Replicação do DNA/genética , DNA Viral/biossíntese , Histona-Lisina N-Metiltransferase/metabolismo , Replicação Viral/fisiologia , Linhagem Celular , Sobrevivência Celular , DNA Viral/genética , Genoma Viral/genética , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Pulmão/citologia , Pulmão/virologia , Transporte Proteico/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Regulação para Cima , Carga Viral/genética , Internalização do Vírus
19.
PLoS Pathog ; 13(7): e1006542, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28750047

RESUMO

Congenital human cytomegalovirus (HCMV) infection is the leading cause of neurological disabilities in children worldwide, but the mechanisms underlying these disorders are far from well-defined. HCMV infection has been shown to dysregulate the Notch signaling pathway in human neural progenitor cells (NPCs). As an important downstream effector of Notch signaling, the transcriptional regulator Hairy and Enhancer of Split 1 (Hes1) is essential for governing NPC fate and fetal brain development. In the present study, we report that HCMV infection downregulates Hes1 protein levels in infected NPCs. The HCMV 72-kDa immediate-early 1 protein (IE1) is involved in Hes1 degradation by assembling a ubiquitination complex and promoting Hes1 ubiquitination as a potential E3 ubiquitin ligase, followed by proteasomal degradation of Hes1. Sp100A, an important component of PML nuclear bodies, is identified to be another target of IE1-mediated ubiquitination. A C-terminal acidic region in IE1, spanning amino acids 451 to 475, is required for IE1/Hes1 physical interaction and IE1-mediated Hes1 ubiquitination, but is dispensable for IE1/Sp100A interaction and ubiquitination. Our study suggests a novel mechanism linking downregulation of Hes1 protein to neurodevelopmental disorders caused by HCMV infection. Our findings also complement the current knowledge of herpesviruses by identifying IE1 as the first potential HCMV-encoded E3 ubiquitin ligase.


Assuntos
Infecções por Citomegalovirus/enzimologia , Infecções por Citomegalovirus/virologia , Citomegalovirus/metabolismo , Células-Tronco Neurais/metabolismo , Fatores de Transcrição HES-1/genética , Ubiquitina-Proteína Ligases/metabolismo , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Regulação para Baixo , Interações Hospedeiro-Patógeno , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Células-Tronco Neurais/enzimologia , Células-Tronco Neurais/virologia , Ligação Proteica , Proteólise , Fatores de Transcrição HES-1/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
20.
Virology ; 510: 205-215, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28750324

RESUMO

T98G cells have been shown to support long-term human cytomegalovirus (HCMV) genome maintenance without infectious virus release. However, it remains unclear whether these viral genomes could be reactivated. To address this question, a recombinant HCMV (rHCMV) containing a GFP gene was used to infect T98G cells, and the infected cells absent of infectious virus production were designated T98G-LrV. Upon dibutyryl cAMP plus IBMX (cAMP/IBMX) treatment, a serial of phenomena were observed, including GFP signal increase, viral genome replication, lytic genes expression and infectious viruses release, indicating the reactivation of HCMV in T98G-LrV cells from a latent status. Mechanistically, HCMV reactivation in the T98G-LrV cells induced by cAMP/IBMX was associated with the PKA-CREB signaling pathway. These results demonstrate that HCMV was latent in T98G-LrV cells and could be reactivated. The T98G-LrV cells represent an effective model for investigating the mechanisms of HCMV reactivation from latency in the context of neural cells.


Assuntos
Citomegalovirus/fisiologia , Ativação Viral , Latência Viral , 1-Metil-3-Isobutilxantina/metabolismo , Bucladesina/metabolismo , Linhagem Celular Tumoral , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Humanos , Coloração e Rotulagem/métodos
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