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1.
PLoS Pathog ; 12(5): e1005655, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27218650

RESUMO

Herpesviruses persist indefinitely in their host through complex and poorly defined interactions that mediate latent, chronic or productive states of infection. Human cytomegalovirus (CMV or HCMV), a ubiquitous ß-herpesvirus, coordinates the expression of two viral genes, UL135 and UL138, which have opposing roles in regulating viral replication. UL135 promotes reactivation from latency and virus replication, in part, by overcoming replication-suppressive effects of UL138. The mechanism by which UL135 and UL138 oppose one another is not known. We identified viral and host proteins interacting with UL138 protein (pUL138) to begin to define the mechanisms by which pUL135 and pUL138 function. We show that pUL135 and pUL138 regulate the viral cycle by targeting that same receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR). EGFR is a major homeostatic regulator involved in cellular proliferation, differentiation, and survival, making it an ideal target for viral manipulation during infection. pUL135 promotes internalization and turnover of EGFR from the cell surface, whereas pUL138 preserves surface expression and activation of EGFR. We show that activated EGFR is sequestered within the infection-induced, juxtanuclear viral assembly compartment and is unresponsive to stress. Intriguingly, these findings suggest that CMV insulates active EGFR in the cell and that pUL135 and pUL138 function to fine-tune EGFR levels at the cell surface to allow the infected cell to respond to extracellular cues. Consistent with the role of pUL135 in promoting replication, inhibition of EGFR or the downstream phosphoinositide 3-kinase (PI3K) favors reactivation from latency and replication. We propose a model whereby pUL135 and pUL138 together with EGFR comprise a molecular switch that regulates states of latency and replication in HCMV infection by regulating EGFR trafficking to fine tune EGFR signaling.


Assuntos
Infecções por Citomegalovirus/metabolismo , Receptores ErbB/metabolismo , Latência Viral/fisiologia , Replicação Viral/fisiologia , Linhagem Celular , Citomegalovirus , Citometria de Fluxo , Imunofluorescência , Humanos , Immunoblotting , Imunoprecipitação , Espectrometria de Massas , Transporte Proteico
2.
J Virol ; 88(11): 5987-6002, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24623432

RESUMO

UNLABELLED: The mechanisms by which viruses persist and particularly those by which viruses actively contribute to their own latency have been elusive. Here we report the existence of opposing functions encoded by genes within a polycistronic locus of the human cytomegalovirus (HCMV) genome that regulate cell type-dependent viral fates: replication and latency. The locus, referred to as the UL133-UL138 (UL133/8) locus, encodes four proteins, pUL133, pUL135, pUL136, and pUL138. As part of the ULb' region of the genome, the UL133/8 locus is lost upon serial passage of clinical strains of HCMV in cultured fibroblasts and is therefore considered dispensable for replication in this context. Strikingly, we could not reconstitute infection in permissive fibroblasts from bacterial artificial chromosome clones of the HCMV genome where UL135 alone was disrupted. The loss of UL135 resulted in complex phenotypes and could ultimately be overcome by infection at high multiplicities. The requirement for UL135 but not the entire locus led us to hypothesize that another gene in this locus suppressed virus replication in the absence of UL135. The defect associated with the loss of UL135 was largely rescued by the additional disruption of the UL138 latency determinant, indicating a requirement for UL135 for virus replication when UL138 is expressed. In the CD34(+) hematopoietic progenitor model of latency, viruses lacking only UL135 were defective for viral genome amplification and reactivation. Taken together, these data indicate that UL135 and UL138 comprise a molecular switch whereby UL135 is required to overcome UL138-mediated suppression of virus replication to balance states of latency and reactivation. IMPORTANCE: Mechanisms by which viruses persist in their host remain one of the most poorly understood phenomena in virology. Herpesviruses, including HCMV, persist in an incurable, latent state that has profound implications for immunocompromised individuals, including transplant patients. Further, the latent coexistence of HCMV may increase the risk of age-related pathologies, including vascular disease. The key to controlling or eradicating HCMV lies in understanding the molecular basis for latency. In this work, we describe the complex interplay between two viral proteins, pUL135 and pUL138, which antagonize one another in infection to promote viral replication or latency, respectively. We previously described the role of pUL138 in suppressing virus replication for latency. Here we demonstrate a role of pUL135 in overcoming pUL138-mediated suppression for viral reactivation. From this work, we propose that pUL135 and pUL138 constitute a molecular switch balancing states of latency and reactivation.


Assuntos
Infecções por Citomegalovirus/fisiopatologia , Citomegalovirus/genética , Proteínas Virais/genética , Latência Viral/genética , Replicação Viral/genética , Cromossomos Artificiais Bacterianos , Clonagem Molecular , Infecções por Citomegalovirus/genética , Primers do DNA/genética , Fibroblastos , Loci Gênicos/genética , Vetores Genéticos/genética , Humanos , Immunoblotting , Microscopia Eletrônica de Transmissão
3.
J Virol ; 86(16): 8653-62, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22674978

RESUMO

We previously described a novel genetic locus within the ULb' region of the human cytomegalovirus (HCMV) genome that, while dispensable for replication in fibroblasts, suppresses replication in hematopoietic progenitors and augments replication in endothelial cells. This locus, referred to as the UL133-UL138 locus, encodes four proteins, pUL133, pUL135, pUL136, and pUL138. In this work, we have mapped the interactions among these proteins. An analysis of all pairwise interactions during transient expression revealed a robust interaction between pUL133 and pUL138. Potential interactions between pUL136 and both pUL133 and pUL138 were also revealed. In addition, each of the UL133-UL138 locus proteins self-associated, suggesting a potential to form higher-order homomeric complexes. As both pUL133 and pUL138 function in promoting viral latency in CD34(+) hematopoietic progenitor cells (HPCs) infected in vitro, we further focused on this interaction. pUL133 and pUL138 are the predominant complex detected when all proteins are expressed together and require no other proteins in the locus for their association. During infection, the interaction between pUL133 and pUL138 or pUL136 can be detected. A recombinant virus that fails to express both pUL133 and pUL138 exhibited a latency phenotype similar to that of viruses that fail to express either pUL133 or pUL138, indicating that these proteins function cooperatively in latency and do not have independent functions that additively contribute to HCMV latency. These studies identify protein interactions among proteins encoded by the UL133-UL138 locus and demonstrate an important interaction impacting the outcome of HCMV infection.


Assuntos
Citomegalovirus/fisiologia , Mapeamento de Interação de Proteínas , Proteínas Virais/metabolismo , Latência Viral , Linhagem Celular , Humanos , Ligação Proteica , Multimerização Proteica
4.
Cell Microbiol ; 14(5): 644-55, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22329758

RESUMO

Viral persistence is the rule following infection with all herpesviruses. The ß-herpesvirus, human cytomegalovirus (HCMV), persists through chronic and latent states of infection. Both of these states of infection contribute to HCMV persistence and to the high HCMV seroprevalence worldwide. The chronic infection is poorly defined molecularly, but clinically manifests as low-level virus shedding over extended periods of time and often in the absence of symptoms. Latency requires long-term maintenance of viral genomes in a reversibly quiescent state in the immunocompetent host. In this review, we focus on recent advances in the biology of HCMV persistence, particularly with respect to the latent mode of persistence. Latently infected individuals harbour HCMV genomes in haematopoietic cells and maintain large subsets of HCMV-specific T-cells. In the last few years, impressive advances have been made in understanding virus-host interactions important to HCMV infection, many of which will profoundly impact HCMV persistence. We discuss these advances and their known or potential impact on viral latency. As herpesviruses are met with similar challenges in achieving latency and often employ conserved strategies to persist, we discuss current and future directions of HCMV persistence in the context of the greater body of knowledge regarding α- and γ-herpesviruses persistence.


Assuntos
Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Citomegalovirus/imunologia , Citomegalovirus/patogenicidade , Interações Hospedeiro-Patógeno , Latência Viral , Doença Crônica , Humanos , Evasão da Resposta Imune , Modelos Biológicos
5.
Lipids Health Dis ; 10: 198, 2011 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-22051061

RESUMO

BACKGROUND: Obesity is characterized by increased cell death and inflammatory reactions in the adipose tissue. Here, we explored pathophysiological alterations taking place in the adipose tissue in long-standing obesity. In the epididymal fat of C57BL/6 mice fed a high-fat diet for 20 weeks, the prevalence and distribution of dead adipocytes (crown-like structures), mast cells (toluidine blue, mMCP6), macrophages (F4/80), and apoptotic cells (cleaved caspase-3) were measured. Moreover, gene and/or protein expression of several adipocytokines (leptin, adiponectin, TNF-α, IL-10, IL-6, MCP-1), F4/80, mMCP6, cleaved caspase-3 were determined. RESULTS: We observed that the epididymal fat mass was lower in obese than in lean mice. In obese mice, the epididymal fat mass correlated inversely with body weight and liver mass. Dead adipocytes, mast cells, macrophages, and apoptotic cells were abundant in the epididymal fat of obese mice, especially in the rostral vs. caudal zone. Accordingly, mMCP6, F4/80, and cleaved caspase-3 gene and/or protein expression was increased. Conversely, adiponectin, leptin, IL-6, and MCP-1 gene expression levels were lower in the epididymal fat of obese than lean mice. Although TNF-α and IL-10 gene expression was higher in the epididymal fat of obese mice, their expression relative to F4/80 and mMCP6 expression were lower in the heavily infiltrated rostral than caudal zone. CONCLUSIONS: This study demonstrates that in mice with long-standing obesity diminished gene expression of several adipocytokines accompany apoptosis and reduced mass of the epididymal fat. Our findings suggest that this is due to both increased prevalence of dead adipocytes and altered immune cell activity. Differential distribution of metabolically challenged adipocytes is indicative of the presence of biologically diverse zones within the epididymal fat.


Assuntos
Adiponectina/genética , Adiposidade , Leptina/genética , Obesidade/patologia , Adiponectina/metabolismo , Tecido Adiposo/patologia , Animais , Apoptose , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Dieta Hiperlipídica , Regulação para Baixo , Epididimo/enzimologia , Epididimo/metabolismo , Expressão Gênica , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Leptina/metabolismo , Fígado/patologia , Macrófagos/patologia , Masculino , Mastócitos/patologia , Mastocitose , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Tamanho do Órgão , Especificidade de Órgãos , Distribuição Aleatória , Triptases/genética , Triptases/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
6.
CBE Life Sci Educ ; 18(4): ar62, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31755820

RESUMO

College science instructors need continuous professional development (PD) to meet the call to evidence-based practice. New PD efforts need to focus on the nuanced blend of factors that influence instructors' teaching practices. We used persona methodology to describe the diversity among instructors who were participating in a long-term PD initiative. Persona methodology originates from ethnography. It takes data from product users and compiles those data in the form of fictional characters. Personas facilitate user-centered design. We identified four personas among our participants: Emma the Expert views herself as the subject-matter expert in the classroom and values her hard-earned excellence in lecturing. Ray the Relater relates to students and focuses on their points of view about innovative pedagogies. Carmen the Coach coaches her students by setting goals for them and helping them develop skill in scientific practices. Beth the Burdened owns the responsibility for her students' learning and feels overwhelmed that students still struggle despite her use of evidence-based practice. Each persona needs unique PD. We suggest ways that PD facilitators can use our personas as a reflection tool to determine how to approach the learners in their PD. We also suggest further avenues of research on learner-centered PD.


Assuntos
Educação Profissionalizante , Docentes , Aprendizagem , Feminino , Humanos , Masculino , Estudantes , Ensino
7.
CBE Life Sci Educ ; 15(2)2016.
Artigo em Inglês | MEDLINE | ID: mdl-27193288

RESUMO

We present our design for a cell biology course to integrate content with scientific practices, specifically data interpretation and model-based reasoning. A 2-yr research project within this course allowed us to understand how students interpret authentic biological data in this setting. Through analysis of written work, we measured the extent to which students' data interpretations were valid and/or generative. By analyzing small-group audio recordings during in-class activities, we demonstrated how students used instructor-provided models to build and refine data interpretations. Often, students used models to broaden the scope of data interpretations, tying conclusions to a biological significance. Coding analysis revealed several strategies and challenges that were common among students in this collaborative setting. Spontaneous argumentation was present in 82% of transcripts, suggesting that data interpretation using models may be a way to elicit this important disciplinary practice. Argumentation dialogue included frequent co-construction of claims backed by evidence from data. Other common strategies included collaborative decoding of data representations and noticing data patterns before making interpretive claims. Focusing on irrelevant data patterns was the most common challenge. Our findings provide evidence to support the feasibility of supporting students' data-interpretation skills within a large lecture course.


Assuntos
Biologia Celular/educação , Interpretação Estatística de Dados , Biologia do Desenvolvimento/educação , Modelos Educacionais , Estudantes , Comportamento Cooperativo , Avaliação Educacional , Humanos , Transdução de Sinais , Estudantes/psicologia
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