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1.
PLoS Pathog ; 11(1): e1004561, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25590614

RESUMO

The human herpes virus Epstein-Barr virus (EBV) latently infects and drives the proliferation of B lymphocytes in vitro and is associated with several forms of lymphoma and carcinoma in vivo. The virus encodes ~30 miRNAs in the BART region, the function of most of which remains elusive. Here we have used a new mouse xenograft model of EBV driven carcinomagenesis to demonstrate that the BART miRNAs potentiate tumor growth and development in vivo. No effect was seen on invasion or metastasis, and the growth promoting activity was not seen in vitro. In vivo tumor growth was not associated with the expression of specific BART miRNAs but with up regulation of all the BART miRNAs, consistent with previous observations that all the BART miRNAs are highly expressed in all of the EBV associated cancers. Based on these observations, we suggest that deregulated expression of the BART miRNAs potentiates tumor growth and represents a general mechanism behind EBV associated oncogenesis.


Assuntos
Transformação Celular Viral/genética , Herpesvirus Humano 4/genética , MicroRNAs/genética , Latência Viral/genética , Animais , Células Cultivadas , Feminino , Regulação Viral da Expressão Gênica , Genes Virais/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , MicroRNAs/fisiologia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia
2.
Proc Natl Acad Sci U S A ; 111(30): 11157-62, 2014 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-25012295

RESUMO

EBV is an oncogenic human herpesvirus that has the ability to infect and transform B cells latently in vitro. However, the virus also establishes a lifetime, benign, persistent latent infection in resting memory B cells in vivo, where the virus is quiescent (i.e., expresses none of the known latent proteins). The virus encodes ∼40 micro-RNAs (miRNAs), most of which are transcribed from the BamH1 fragment A rightward transcript (BART) region of the virus. We have shown previously that a subset of these miRNAs is present at high copy numbers in latently infected memory B cells in vivo, suggesting a role in maintaining latency. Here, we describe the role of one of these miRNAs, BART 18-5p. We show that it targets the 3'UTR of the mRNA, encoding the important cellular signaling molecule MAP kinase kinase kinase 2 (MAP3K2), at exactly the same site as the oncogenic cellular miRNA mir-26a-5p. To our knowledge, this is the first report of a virus encoding a miRNA that suppresses a target in the MAP kinase signaling cascade, a central signal transduction pathway that governs a broad spectrum of biological processes. We further show that MAP3K2 is an intermediary in the signaling pathways that initiate lytic viral replication. Thus, 18-5p expression in latently infected B cells has the effect of blocking viral replication. We propose that the role of 18-5p is to maintain latency by reducing the risk of fortuitous reactivation of the virus in latently infected memory B cells.


Assuntos
Herpesvirus Humano 4/fisiologia , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases , MicroRNAs/metabolismo , RNA Viral/metabolismo , Latência Viral/fisiologia , Replicação Viral/fisiologia , Regiões 3' não Traduzidas , Linhagem Celular Transformada , Humanos , Memória Imunológica , MAP Quinase Quinase Quinase 2 , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/imunologia , MicroRNAs/genética , MicroRNAs/imunologia , RNA Viral/genética , RNA Viral/imunologia
3.
PLoS Pathog ; 10(5): e1004170, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24874410

RESUMO

Endemic Burkitt's lymphoma (eBL) arises from the germinal center (GC). It is a common tumor of young children in tropical Africa and its occurrence is closely linked geographically with the incidence of P. falciparum malaria. This association was noted more than 50 years ago. Since then we have learned that eBL contains the oncogenic herpes virus Epstein-Barr virus (EBV) and a defining translocation that activates the c-myc oncogene. However the link to malaria has never been explained. Here we provide evidence for a mechanism arising in the GC to explain this association. Accumulated evidence suggests that eBL arises in the GC when deregulated expression of AID (Activation-induced cytidine deaminase) causes a c-myc translocation in a cell latently infected with Epstein-Barr virus (EBV). Here we show that P. falciparum targets GC B cells via multiple pathways to increase the risk of eBL. 1. It causes deregulated expression of AID, thereby increasing the risk of a c-myc translocation. 2. It increases the number of B cells transiting the GC. 3. It dramatically increases the frequency of these cells that are infected with EBV and therefore protected from c-myc induced apoptosis. We propose that these activities combine synergistically to dramatically increase the incidence of eBL in individuals infected with malaria.


Assuntos
Linfoma de Burkitt/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4 , Malária Falciparum/imunologia , Plasmodium falciparum , Animais , Linfoma de Burkitt/parasitologia , Linfoma de Burkitt/virologia , Linhagem Celular , Infecções por Vírus Epstein-Barr/genética , Humanos , Malária Falciparum/genética , Translocação Genética/genética , Translocação Genética/fisiologia
4.
Curr Top Microbiol Immunol ; 390(Pt 1): 151-209, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26424647

RESUMO

Persistent infection by EBV is explained by the germinal center model (GCM) which provides a satisfying and currently the only explanation for EBVs disparate biology. Since the GCM touches on every aspect of the virus, this chapter will serve as an introduction to the subsequent chapters. EBV is B lymphotropic, and its biology closely follows that of normal mature B lymphocytes. The virus persists quiescently in resting memory B cells for the lifetime of the host in a non-pathogenic state that is also invisible to the immune response. To access this compartment, the virus infects naïve B cells in the lymphoepithelium of the tonsils and activates these cells using the growth transcription program. These cells migrate to the GC where they switch to a more limited transcription program, the default program, which helps rescue them into the memory compartment where the virus persists. For egress, the infected memory cells return to the lymphoepithelium where they occasionally differentiate into plasma cells activating viral replication. The released virus can either infect more naïve B cells or be amplified in the epithelium for shedding. This cycle of infection and the quiescent state in memory B cells allow for lifetime persistence at a very low level that is remarkably stable over time. Mathematically, this is a stable fixed point where the mechanisms regulating persistence drive the state back to equilibrium when perturbed. This is the GCM of EBV persistence. Other possible sites and mechanisms of persistence will also be discussed.


Assuntos
Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/fisiologia , Animais , Linfócitos B/virologia , Herpesvirus Humano 4/genética , Humanos , Replicação Viral
5.
PLoS Pathog ; 9(10): e1003685, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24146621

RESUMO

Previous analysis of Epstein-Barr virus (EBV) persistent infection has involved biological and immunological studies to identify and quantify infected cell populations and the immune response to them. This led to a biological model whereby EBV infects and activates naive B-cells, which then transit through the germinal center to become resting memory B-cells where the virus resides quiescently. Occasionally the virus reactivates from these memory cells to produce infectious virions. Some of this virus infects new naive B-cells, completing a cycle of infection. What has been lacking is an understanding of the dynamic interactions between these components and how their regulation by the immune response produces the observed pattern of viral persistence. We have recently provided a mathematical analysis of a pathogen which, like EBV, has a cycle of infected stages. In this paper we have developed biologically credible values for all of the parameters governing this model and show that with these values, it successfully recapitulates persistent EBV infection with remarkable accuracy. This includes correctly predicting the observed patterns of cytotoxic T-cell regulation (which and by how much each infected population is regulated by the immune response) and the size of the infected germinal center and memory populations. Furthermore, we find that viral quiescence in the memory compartment dictates the pattern of regulation but is not required for persistence; it is the cycle of infection that explains persistence and provides the stability that allows EBV to persist at extremely low levels. This shifts the focus away from a single infected stage, the memory B-cell, to the whole cycle of infection. We conclude that the mathematical description of the biological model of EBV persistence provides a sound basis for quantitative analysis of viral persistence and provides testable predictions about the nature of EBV-associated diseases and how to curb or prevent them.


Assuntos
Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Imunidade Celular , Modelos Imunológicos , Linfócitos B/patologia , Linfócitos T CD8-Positivos/patologia , Infecções por Vírus Epstein-Barr/patologia , Centro Germinativo/imunologia , Centro Germinativo/patologia , Humanos
6.
J Virol ; 86(22): 12330-40, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22951828

RESUMO

Epstein-Barr virus infection has been epidemiologically associated with the development of multiple autoimmune diseases, particularly systemic lupus erythematosus and multiple sclerosis. Currently, there is no known mechanism that can account for these associations. The germinal-center (GC) model of EBV infection and persistence proposes that EBV gains access to the memory B cell compartment via GC reactions by driving infected cells to differentiate using the virus-encoded LMP1 and LMP2a proteins, which act as functional homologues of CD40 and the B cell receptor, respectively. The ability of LMP2a, when expressed in mice, to allow escape of autoreactive B cells suggests that it could perform a similar role in infected GC B cells, permitting the survival of potentially pathogenic autoreactive B cells. To test this hypothesis, we cloned and expressed antibodies from EBV(+) and EBV(-) memory B cells present during acute infection and profiled their self- and polyreactivity. We find that EBV does persist within self- and polyreactive B cells but find no evidence that it favors the survival of pathogenic autoreactive B cells. On the contrary, EBV(+) memory B cells express lower levels of self-reactive and especially polyreactive antibodies than their uninfected counterparts do. Our work suggests that EBV has only a modest effect on the GC process, which allows it to access and persist within a subtly unique niche of the memory compartment characterized by relatively low levels of self- and polyreactivity. We suggest that this might reflect an active process where EBV and its human host have coevolved so as to minimize the virus's potential to contribute to autoimmune disease.


Assuntos
Linfócitos B/virologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/metabolismo , Memória Imunológica , Anticorpos/química , Doenças Autoimunes/imunologia , Doenças Autoimunes/virologia , DNA Complementar/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Vetores Genéticos , Células HEK293 , Humanos , Leucócitos Mononucleares/virologia , Risco , Análise de Sequência de DNA , Proteínas da Matriz Viral/metabolismo
7.
PLoS Pathog ; 7(8): e1002193, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21901094

RESUMO

We have performed the first extensive profiling of Epstein-Barr virus (EBV) miRNAs on in vivo derived normal and neoplastic infected tissues. We describe a unique pattern of viral miRNA expression by normal infected cells in vivo expressing restricted viral latency programs (germinal center: Latency II and memory B: Latency I/0). This includes the complete absence of 15 of the 34 miRNAs profiled. These consist of 12 BART miRNAs (including approximately half of Cluster 2) and 3 of the 4 BHRF1 miRNAs. All but 2 of these absent miRNAs become expressed during EBV driven growth (Latency III). Furthermore, EBV driven growth is accompanied by a 5-10 fold down regulation in the level of the BART miRNAs expressed in germinal center and memory B cells. Therefore, Latency III also expresses a unique pattern of viral miRNAs. We refer to the miRNAs that are specifically expressed in EBV driven growth as the Latency III associated miRNAs. In EBV associated tumors that employ Latency I or II (Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma and gastric carcinoma), the Latency III associated BART but not BHRF1 miRNAs are up regulated. Thus BART miRNA expression is deregulated in the EBV associated tumors. This is the first demonstration that Latency III specific genes (the Latency III associated BARTs) can be expressed in these tumors. The EBV associated tumors demonstrate very similar patterns of miRNA expression yet were readily distinguished when the expression data were analyzed either by heat-map/clustering or principal component analysis. Systematic analysis revealed that the information distinguishing the tumor types was redundant and distributed across all the miRNAs. This resembles "secret sharing" algorithms where information can be distributed among a large number of recipients in such a way that any combination of a small number of recipients is able to understand the message. Biologically, this may be a consequence of functional redundancy between the miRNAs.


Assuntos
Herpesvirus Humano 4/genética , MicroRNAs/genética , Neoplasias/virologia , Animais , Linfócitos B/metabolismo , Linfócitos B/virologia , Linhagem Celular Tumoral , Regulação para Baixo , Perfilação da Expressão Gênica/métodos , Regulação Viral da Expressão Gênica , Humanos , Camundongos , MicroRNAs/metabolismo , Família Multigênica , Regulação para Cima , Proteínas Virais/genética , Proteínas Virais/metabolismo , Latência Viral/genética
8.
Proc Natl Acad Sci U S A ; 107(14): 6328-33, 2010 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-20304794

RESUMO

Noncoding regulatory microRNAs (miRNAs) of cellular and viral origin control gene expression by repressing the translation of mRNAs into protein. Interestingly, miRNAs are secreted actively through small vesicles called "exosomes" that protect them from degradation by RNases, suggesting that these miRNAs may function outside the cell in which they were produced. Here we demonstrate that miRNAs secreted by EBV-infected cells are transferred to and act in uninfected recipient cells. Using a quantitative RT-PCR approach, we demonstrate that mature EBV-encoded miRNAs are secreted by EBV-infected B cells through exosomes. These EBV-miRNAs are functional because internalization of exosomes by MoDC results in a dose-dependent, miRNA-mediated repression of confirmed EBV target genes, including CXCL11/ITAC, an immunoregulatory gene down-regulated in primary EBV-associated lymphomas. We demonstrate that throughout coculture of EBV-infected B cells EBV-miRNAs accumulate in noninfected neighboring MoDC and show that this accumulation is mediated by transfer of exosomes. Thus, the exogenous EBV-miRNAs transferred through exosomes are delivered to subcellular sites of gene repression in recipient cells. Finally, we show in peripheral blood mononuclear cells from patients with increased EBV load that, although EBV DNA is restricted to the circulating B-cell population, EBV BART miRNAs are present in both B-cell and non-B-cell fractions, suggestive of miRNA transfer. Taken together our findings are consistent with miRNA-mediated gene silencing as a potential mechanism of intercellular communication between cells of the immune system that may be exploited by the persistent human gamma-herpesvirus EBV.


Assuntos
Linfócitos B/metabolismo , Exossomos/metabolismo , Herpesvirus Humano 4/genética , MicroRNAs/metabolismo , RNA Viral/metabolismo , Linfócitos B/imunologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Exossomos/ultraestrutura , Herpesvirus Humano 4/ultraestrutura , Humanos , MicroRNAs/genética , Microscopia Eletrônica , RNA Viral/genética , Internalização do Vírus
9.
J Immunol ; 185(11): 6753-64, 2010 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-21048112

RESUMO

Memory T cells cross-reactive with epitopes encoded by related or even unrelated viruses may alter the immune response and pathogenesis of infection by a process known as heterologous immunity. Because a challenge virus epitope may react with only a subset of the T cell repertoire in a cross-reactive epitope-specific memory pool, the vigorous cross-reactive response may be narrowly focused, or oligoclonal. We show in this article, by examining human T cell cross-reactivity between the HLA-A2-restricted influenza A virus-encoded M1(58-66) epitope (GILGFVFTL) and the dissimilar Epstein-Barr virus-encoded BMLF1(280-288) epitope (GLCTLVAML), that, under some conditions, heterologous immunity can lead to a significant broadening, rather than a narrowing, of the TCR repertoire. We suggest that dissimilar cross-reactive epitopes might generate a broad, rather than a narrow, T cell repertoire if there is a lack of dominant high-affinity clones; this hypothesis is supported by computer simulation.


Assuntos
Epitopos de Linfócito T/metabolismo , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/metabolismo , Vírus da Influenza A/imunologia , Vírus da Influenza A/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Adolescente , Adulto , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Células Clonais , Reações Cruzadas , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Humanos , Epitopos Imunodominantes/metabolismo , Pessoa de Meia-Idade , Oligopeptídeos/imunologia , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Proteínas da Matriz Viral/imunologia , Proteínas da Matriz Viral/metabolismo , Adulto Jovem
10.
J Virol ; 84(2): 1158-68, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19889783

RESUMO

In this study we show that in long-term persistent infection, Epstein-Barr virus (EBV)-infected cells undergoing a germinal center (GC) reaction in the tonsils are limited to the follicles and proliferate extensively. Despite this, the absolute number of infected cells per GC remains small (average of 3 to 4 cells per germinal center; range, 1 to 9 cells), and only about 38 to 55% (average, 45%) of all GCs carry infected cells. The data fit a model where, on average, cells in the GC divide approximately three times; however, only one progeny cell survives to undergo a further three divisions. Thus, a fraction of cells undergo multiple rounds of division without increasing in numbers; i.e., they die at the same rate that they are dividing. We conclude that EBV-infected cells in the GC undergo the extensive proliferation characteristic of GC cells but that the absolute number is limited either by the immune response or by the availability of an essential survival factor. We suggest that this behavior is a relic of the mechanism by which EBV establishes persistence during acute infection. Lastly, the expression of the viral latent protein LMP1 in GC B cells, unlike in vitro, does not correlate directly with the expression of bcl-2 or bcl-6. This emphasizes our claim that observations made regarding the functions of EBV proteins in cell lines or in transgenic mice should be treated with skepticism unless verified in vivo.


Assuntos
Linfócitos B/virologia , Proliferação de Células , Centro Germinativo/citologia , Herpesvirus Humano 4/patogenicidade , Proteínas da Matriz Viral/metabolismo , Latência Viral , Animais , Linfócitos B/citologia , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Memória Imunológica , Contagem de Linfócitos , Camundongos , Tonsila Palatina/citologia
11.
PLoS Pathog ; 5(7): e1000496, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19578433

RESUMO

To develop more detailed models of EBV persistence we have studied the dynamics of virus shedding in healthy carriers. We demonstrate that EBV shedding into saliva is continuous and rapid such that the virus level is replaced in < or =2 minutes, the average time that a normal individual swallows. Thus, the mouth is not a reservoir of virus but a conduit through which a continuous flow stream of virus passes in saliva. Consequently, virus is being shed at a much higher rate than previously thought, a level too high to be accounted for by replication in B cells in Waldeyer's ring alone. Virus shedding is relatively stable over short periods (hours-days) but varies through 3.5 to 5.5 logs over longer periods, a degree of variation that also cannot be accounted for solely by replication in B cells. This variation means, contrary to what is generally believed, that the definition of high and low shedder is not so much a function of variation between individuals but within individuals over time. The dynamics of shedding describe a process governing virus production that is occurring independently < or =3 times at any moment. This process grows exponentially and is then randomly terminated. We propose that these dynamics are best explained by a model where single B cells sporadically release virus that infects anywhere from 1 to 5 epithelial cells. This infection spreads at a constant exponential rate and is terminated randomly, resulting in infected plaques of epithelial cells ranging in size from 1 to 10(5) cells. At any one time there are a very small number (< or =3) of plaques. We suggest that the final size of these plaques is a function of the rate of infectious spread within the lymphoepithelium which may be governed by the structural complexity of the tissue but is ultimately limited by the immune response.


Assuntos
Portador Sadio/virologia , Células Epiteliais/virologia , Herpesvirus Humano 4/fisiologia , Modelos Biológicos , Linfócitos B/virologia , Linhagem Celular , Desoxirribonucleases/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/crescimento & desenvolvimento , Humanos , Modelos Lineares , Método de Monte Carlo , Reação em Cadeia da Polimerase , Saliva/virologia , Vírion/crescimento & desenvolvimento , Vírion/metabolismo , Replicação Viral , Eliminação de Partículas Virais
12.
J Virol ; 83(8): 3968-76, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19193789

RESUMO

The current model of Epstein-Barr virus (EBV) infection and persistence in vivo proposes that EBV uses the germinal center (the GC model) to establish a quiescent latent infection in otherwise-normal memory B cells. However, the evidence linking EBV-infected cells and the GC is only indirect and limited. Therefore, a key portion of the model, that EBV-infected cells physically reside and participate in GCs, has yet to be verified. Furthermore, recent experiments suggested that upon infection of GC cells the viral growth latency transcription program is dominant and GC functionality and phenotype are ablated, i.e., EBV infection is not consistent with GC function. In this study we show that in vivo, EBV-infected B cells in the tonsils retain expression of functional and phenotypic markers of GC cells, including bcl-6 and AID. Furthermore, these cells are physically located in the GC and express a restricted form of latency, the default latency program. Thus, the EBV default latency transcription program, unlike the growth latency program, is consistent with the retention of GC functionality in vivo. This work verifies key components of the GC model of EBV persistence and suggests that EBV and the GC can interact to produce the latently infected memory cells found in the periphery. Furthermore, it identifies latently infected GC B cells as a potential pathogenic nexus for the development of the EBV-positive, GC-associated lymphomas Hodgkin's disease and Burkitt's lymphoma.


Assuntos
Linfócitos B/virologia , Centro Germinativo/virologia , Herpesvirus Humano 4/crescimento & desenvolvimento , Tonsila Palatina/virologia , Adolescente , Linfócitos B/química , Linfócitos B/imunologia , Citidina Desaminase/análise , Proteínas de Ligação a DNA/análise , Centro Germinativo/imunologia , Herpesvirus Humano 4/fisiologia , Humanos , Tonsila Palatina/imunologia , Proteínas Proto-Oncogênicas c-bcl-6 , Latência Viral
13.
J Virol ; 83(5): 2357-67, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19091858

RESUMO

Epstein-Barr Virus (EBV) establishes a long-term latent infection and is associated with a number of human malignancies that are thought to arise from deregulation of different stages of the viral life cycle. Recently, a large number of microRNAs (miRNAs) have been described for EBV, and it has been suggested that their expression may vary between the different latency states found in normal and malignant tissue. To date, however, no technique has been utilized to comprehensively and quantitatively test this idea by profiling expression of the EBV miRNAs in primary infected tissues. We describe here a multiplex reverse transcription-PCR assay that allows the profiling of 39 of the 40 known mature EBV miRNAs from as little as 250 ng of RNA. With this approach, we present a comprehensive profile of EBV miRNAs in primary nasopharyngeal carcinoma (NPC) tumors including estimates of miRNA copy number per tumor cell. This is the first comprehensive profiling of EBV miRNAs in any EBV-associated tumor. In contrast to previous suggestions, we show that the BART-derived miRNAs are present in a wide range of copy numbers from < or =10(3) per cell in both primary tumors and the widely used NPC-derived C666-1 cell line. However, we confirm the hypothesis that the BHRF1 miRNAs are not expressed in NPC. Lastly, we demonstrate that EBV miRNA expression in the widely used NPC line C666-1 is, with some caveats, broadly representative of primary NPC tumors.


Assuntos
Carcinoma/virologia , Perfilação da Expressão Gênica , Herpesvirus Humano 4/genética , MicroRNAs/genética , Neoplasias Nasofaríngeas/virologia , Linhagem Celular Tumoral , Infecções por Vírus Epstein-Barr/genética , Humanos , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade
14.
PLoS Pathog ; 3(10): 1388-400, 2007 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-17953479

RESUMO

The possibility of using computer simulation and mathematical modeling to gain insight into biological and other complex systems is receiving increased attention. However, it is as yet unclear to what extent these techniques will provide useful biological insights or even what the best approach is. Epstein-Barr virus (EBV) provides a good candidate to address these issues. It persistently infects most humans and is associated with several important diseases. In addition, a detailed biological model has been developed that provides an intricate understanding of EBV infection in the naturally infected human host and accounts for most of the virus' diverse and peculiar properties. We have developed an agent-based computer model/simulation (PathSim, Pathogen Simulation) of this biological model. The simulation is performed on a virtual grid that represents the anatomy of the tonsils of the nasopharyngeal cavity (Waldeyer ring) and the peripheral circulation--the sites of EBV infection and persistence. The simulation is presented via a user friendly visual interface and reproduces quantitative and qualitative aspects of acute and persistent EBV infection. The simulation also had predictive power in validation experiments involving certain aspects of viral infection dynamics. Moreover, it allows us to identify switch points in the infection process that direct the disease course towards the end points of persistence, clearance, or death. Lastly, we were able to identify parameter sets that reproduced aspects of EBV-associated diseases. These investigations indicate that such simulations, combined with laboratory and clinical studies and animal models, will provide a powerful approach to investigating and controlling EBV infection, including the design of targeted anti-viral therapies.


Assuntos
Simulação por Computador , Herpesvirus Humano 4/fisiologia , Mononucleose Infecciosa/imunologia , Mononucleose Infecciosa/virologia , Modelos Imunológicos , Adolescente , Adulto , Linfócitos B/imunologia , Linfócitos B/patologia , Linfócitos B/virologia , Herpesvirus Humano 4/isolamento & purificação , Herpesvirus Humano 4/patogenicidade , Humanos , Mononucleose Infecciosa/patologia , Tonsila Palatina/imunologia , Tonsila Palatina/patologia , Software , Processos Estocásticos , Fatores de Tempo , Ativação Viral/imunologia , Latência Viral , Fenômenos Fisiológicos Virais
16.
Cancer Res ; 67(2): 474-81, 2007 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-17234754

RESUMO

Nonkeratinizing nasopharyngeal carcinoma (NPC) is 100% associated with Epstein-Barr Virus (EBV) and divided into two subtypes (WHO types II and III) based on histology. We tested whether these subtypes can be distinguished at the molecular genetic level using an algorithm that analyzes sets of related genes (gene set enrichment analysis). We found that a class of IFN-stimulated genes (ISG), frequently associated with the antiviral response, was significantly activated in type III versus type II NPC. Consistent with this, replication of the endogenous EBV was suppressed in type III. A strong association was also seen with a subset of ISGs previously identified in systemic lupus erythematosus, another disease in which 'normal' EBV biology is deregulated, suggesting that this pattern of ISG expression may be linked to the increased EBV activity in both diseases. In contrast, unsupervised hierarchical clustering of the complete expression profiles failed to distinguish the two subsets. These results suggest that type II and III NPC have not originated from obviously distinct epithelial precursors; rather, the histologic differences may be a consequence of a differential antiviral response, involving IFNs, to chronic EBV infection.


Assuntos
Herpesvirus Humano 4/genética , Interferon-alfa/genética , Neoplasias Nasofaríngeas/classificação , Neoplasias Nasofaríngeas/genética , Algoritmos , Antígenos Virais/biossíntese , Antígenos Virais/genética , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/virologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Humanos , Interferon-alfa/biossíntese , Neoplasias Nasofaríngeas/patologia , Neoplasias Nasofaríngeas/virologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
17.
J Virol ; 81(24): 13566-77, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17898050

RESUMO

Epstein-Barr virus (EBV) in vivo is known to establish persistent infection in resting, circulating memory B cells and to productively replicate in plasma cells. Until now, the molecular mechanism of how EBV switches from latency to lytic replication in vivo was not known. Here, we report that the plasma cell differentiation factor, XBP-1s, activates the expression of the master regulator of EBV lytic activation, BZLF1. Using reporter assays, we observed that XBP-1s was able to transactivate the BZLF1 promoter, Zp, in a plasma cell line and other lymphoid cell lines but, interestingly, not in epithelial cell lines. We have identified an XBP-1s binding site on the ZID/ZII region of Zp, which when abolished by site-directed mutagenesis led to abrogation of XBP-1s binding and promoter activation. Using the chromatin immunoprecipitation assay, we observed direct binding of XBP-1s to endogenous Zp in an EBV-infected plasma cell line. Finally, in the same cell line, we observed that overexpression of XBP-1s resulted in increased expression of BZLF1, while knockdown of XBP-1s with short hairpin RNA drastically reduces BZLF1 expression. We suggest that EBV harnesses the B-cell terminal differentiation pathway via XBP-1s as a physiological signal to reactivate and begin viral replication. We are currently investigating other signals, such as the endoplasmic reticulum stress response proteins, which act upstream of XBP-1s, to identify other interacting factors that initiate and/or amplify the lytic switch.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 4/fisiologia , Proteínas Nucleares/metabolismo , Plasmócitos/virologia , Regiões Promotoras Genéticas/genética , Transativadores/metabolismo , Ativação Transcricional , Proteínas Virais/metabolismo , Animais , Células COS , Diferenciação Celular , Linhagem Celular , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Regulação Viral da Expressão Gênica , Células HeLa , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , Células Jurkat , Proteínas Nucleares/genética , Fatores de Transcrição de Fator Regulador X , Transativadores/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Virais/genética , Ativação Viral , Replicação Viral , Proteína 1 de Ligação a X-Box
18.
Methods Mol Biol ; 292: 39-56, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15507700

RESUMO

The method described in this chapter uses limiting dilution analysis in conjunction with RT-PCR to determine quantitatively what percentage of EBV-infected cells within a given population are expressing the viral genes EBNA-1 Q-K, EBNA-2, LMP-1, LMP-2, BZLF-1, and the EBERs. Because this technique involves limiting dilution analysis, it is possible to define which viral transcription programs are being used at the single-cell level. This assay takes 3-4 d to complete and involves the following steps: (1) sample preparation and isolation of the cell population of interest; (2) DNA-PCR limiting dilution analysis to determine the frequency of infected cells within the cell population; (3) RNA isolation; (4) cDNA synthesis; (5) PCR; (6) visualization of PCR products by Southern blotting; and (7) calculations. As an example, we have used PBMCs from the blood of an acute infectious mononucleosis patient. However, this technique can be applied to other cell populations, such as B cells, and other patient groups, such as healthy long-term virus carriers and immunosuppressed organ transplant recipients.


Assuntos
Infecções por Vírus Epstein-Barr/metabolismo , Perfilação da Expressão Gênica/métodos , Expressão Gênica/fisiologia , Herpesvirus Humano 4/genética , RNA/análise , Infecções por Vírus Epstein-Barr/genética , Humanos
19.
Curr Opin Virol ; 3(3): 227-32, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23683686

RESUMO

Epstein-Barr virus (EBV) maintains a lifelong infection. According to the germinal center model (GCM), latently infected B cells transit the germinal center (GC) to become resting memory cells. Here, the virus resides quiescently, occasionally reactivating to infect new B cells, completing the cycle of infection. The GCM remains the only model that explains EBV biology and the pathogenesis of lymphoma. Recent work suggests modifications to the model notably that the virus contributes only modestly to the GC process and predictions from mathematical models that quiescence within memory B cells shapes the overall structure of viral infection but is not essential for persistence. Rather, it is the cycle of infection which allows viral persistence at the very low levels observed.


Assuntos
Infecções por Vírus Epstein-Barr/patologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/fisiologia , Latência Viral , Linfócitos B/virologia , Herpesvirus Humano 4/patogenicidade , Humanos
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