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1.
PLoS Pathog ; 18(4): e1010453, 2022 04.
Article in English | MEDLINE | ID: mdl-35472072

ABSTRACT

Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV.


Subject(s)
B-Lymphocytes , Burkitt Lymphoma , Herpesvirus 4, Human , Interferon Regulatory Factors , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , B-Lymphocytes/virology , Burkitt Lymphoma/metabolism , Burkitt Lymphoma/pathology , Burkitt Lymphoma/virology , Herpesvirus 4, Human/metabolism , Humans , Interferon Regulatory Factors/metabolism , Phenotype , Viral Proteins/metabolism
2.
PLoS Pathog ; 16(6): e1008590, 2020 06.
Article in English | MEDLINE | ID: mdl-32542010

ABSTRACT

EBV transforms B cells in vitro and causes human B-cell lymphomas including classical Hodgkin lymphoma (CHL), Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). The EBV latency protein, EBNA2, transcriptionally activates the promoters of all latent viral protein-coding genes expressed in type III EBV latency and is essential for EBV's ability to transform B cells in vitro. However, EBNA2 is not expressed in EBV-infected CHLs and BLs in humans. EBV-positive CHLs have type II latency and are largely driven by the EBV LMP1/LMP2A proteins, while EBV-positive BLs, which usually have type I latency are largely driven by c-Myc translocations, and only express the EBNA1 protein and viral non-coding RNAs. Approximately 15% of human BLs contain naturally occurring EBNA2-deleted viruses that support a form of viral latency known as Wp-restricted (expressing the EBNA-LP, EBNA3A/3B/3C, EBNA1 and BHRF1 proteins), but whether Wp-restricted latency and/or EBNA2-deleted EBV can induce lymphomas in humanized mice, or in the absence of c-Myc translocations, is unknown. Here we show that a naturally occurring EBNA2-deleted EBV strain (P3HR1) isolated from a human BL induces EBV-positive B-cell lymphomas in a subset of infected cord blood-humanized (CBH) mice. Furthermore, we find that P3HR1-infected lymphoma cells support two different viral latency types and phenotypes that are mutually exclusive: 1) Large (often multinucleated), CD30-positive, CD45-negative cells reminiscent of the Reed-Sternberg (RS) cells in CHL that express high levels of LMP1 but not EBNA-LP (consistent with type II viral latency); and 2) smaller monomorphic CD30-negative DLBCL-like cells that express EBNA-LP and EBNA3A but not LMP1 (consistent with Wp-restricted latency). These results reveal that EBNA2 is not absolutely required for EBV to form tumors in CBH mice and suggest that P3HR1 virus can be used to model EBV positive lymphomas with both Wp-restricted and type II latency in vivo.


Subject(s)
Epstein-Barr Virus Infections , Epstein-Barr Virus Nuclear Antigens/genetics , Gene Deletion , Herpesvirus 4, Human/physiology , Hodgkin Disease , Lymphoma, Large B-Cell, Diffuse , Viral Proteins/genetics , Virus Latency , Animals , Cell Line , Epstein-Barr Virus Infections/genetics , Epstein-Barr Virus Infections/metabolism , Epstein-Barr Virus Infections/pathology , Epstein-Barr Virus Infections/virology , Epstein-Barr Virus Nuclear Antigens/metabolism , Herpesvirus 4, Human/pathogenicity , Hodgkin Disease/genetics , Hodgkin Disease/metabolism , Hodgkin Disease/pathology , Hodgkin Disease/virology , Humans , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , Lymphoma, Large B-Cell, Diffuse/virology , Mice , Viral Proteins/metabolism
3.
J Virol ; 94(10)2020 05 04.
Article in English | MEDLINE | ID: mdl-32132242

ABSTRACT

Epstein-Barr virus (EBV) causes B cell lymphomas and transforms B cells in vitro The EBV protein EBNA3A collaborates with EBNA3C to repress p16 expression and is required for efficient transformation in vitro An EBNA3A deletion mutant EBV strain was recently reported to establish latency in humanized mice but not cause tumors. Here, we compare the phenotypes of an EBNA3A mutant EBV (Δ3A) and wild-type (WT) EBV in a cord blood-humanized (CBH) mouse model. The hypomorphic Δ3A mutant, in which a stop codon is inserted downstream from the first ATG and the open reading frame is disrupted by a 1-bp insertion, expresses very small amounts of EBNA3A using an alternative ATG at residue 15. Δ3A caused B cell lymphomas at rates similar to their induction by WT EBV but with delayed onset. Δ3A and WT tumors expressed equivalent levels of EBNA2 and p16, but Δ3A tumors in some cases had reduced LMP1. Like the WT EBV tumors, Δ3A lymphomas were oligoclonal/monoclonal, with typically one dominant IGHV gene being expressed. Transcriptome sequencing (RNA-seq) analysis revealed small but consistent gene expression differences involving multiple cellular genes in the WT EBV- versus Δ3A-infected tumors and increased expression of genes associated with T cells, suggesting increased T cell infiltration of tumors. Consistent with an impact of EBNA3A on immune function, we found that the expression of CLEC2D, a receptor that has previously been shown to influence responses of T and NK cells, was markedly diminished in cells infected with EBNA3A mutant virus. Together, these studies suggest that EBNA3A contributes to efficient EBV-induced lymphomagenesis in CBH mice.IMPORTANCE The EBV protein EBNA3A is expressed in latently infected B cells and is important for efficient EBV-induced transformation of B cells in vitro In this study, we used a cord blood-humanized mouse model to compare the phenotypes of an EBNA3A hypomorph mutant virus (Δ3A) and wild-type EBV. The Δ3A virus caused lymphomas with delayed onset compared to the onset of those caused by WT EBV, although the tumors occurred at a similar rate. The WT EBV and EBNA3A mutant tumors expressed similar levels of the EBV protein EBNA2 and cellular protein p16, but in some cases, Δ3A tumors had less LMP1. Our analysis suggested that Δ3A-infected tumors have elevated T cell infiltrates and decreased expression of the CLEC2D receptor, which may point to potential novel roles of EBNA3A in T cell and NK cell responses to EBV-infected tumors.


Subject(s)
Epstein-Barr Virus Infections/virology , Epstein-Barr Virus Nuclear Antigens/genetics , Epstein-Barr Virus Nuclear Antigens/metabolism , Fetal Blood/metabolism , Herpesvirus 4, Human/genetics , Lymphoma/virology , Animals , B-Lymphocytes/virology , Cell Transformation, Viral , Disease Models, Animal , Gene Expression Regulation, Neoplastic , HEK293 Cells , Herpesvirus 4, Human/physiology , Humans , Killer Cells, Natural/immunology , Lymphoma/genetics , Lymphoma/pathology , Lymphoma, B-Cell , Mice , Mutagenesis, Site-Directed , Sequence Analysis, RNA , Sequence Deletion , T-Lymphocytes/immunology , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Latency/genetics
4.
mBio ; 10(4)2019 08 20.
Article in English | MEDLINE | ID: mdl-31431547

ABSTRACT

Epstein-Barr virus (EBV) is a human herpesvirus that is associated with lymphomas as well as nasopharyngeal and gastric carcinomas. Although carcinomas account for almost 90% of EBV-associated cancers, progress in examining EBV's role in their pathogenesis has been limited by difficulty in establishing latent infection in nontransformed epithelial cells. Recently, EBV infection of human telomerase reverse transcriptase (hTERT)-immortalized normal oral keratinocytes (NOKs) has emerged as a model that recapitulates aspects of EBV infection in vivo, such as differentiation-associated viral replication. Using uninfected NOKs and NOKs infected with the Akata strain of EBV (NOKs-Akata), we examined changes in gene expression due to EBV infection and differentiation. Latent EBV infection produced very few significant gene expression changes in undifferentiated NOKs but significantly reduced the extent of differentiation-induced gene expression changes. Gene set enrichment analysis revealed that differentiation-induced downregulation of the cell cycle and metabolism pathways was markedly attenuated in NOKs-Akata relative to that in uninfected NOKs. We also observed that pathways induced by differentiation were less upregulated in NOKs-Akata. We observed decreased differentiation markers and increased suprabasal MCM7 expression in NOKs-Akata versus NOKs when both were grown in raft cultures, consistent with our transcriptome sequencing (RNA-seq) results. These effects were also observed in NOKs infected with a replication-defective EBV mutant (AkataΔRZ), implicating mechanisms other than lytic-gene-induced host shutoff. Our results help to define the mechanisms by which EBV infection alters keratinocyte differentiation and provide a basis for understanding the role of EBV in epithelial cancers.IMPORTANCE Latent infection by Epstein-Barr virus (EBV) is an early event in the development of EBV-associated carcinomas. In oral epithelial tissues, EBV establishes a lytic infection of differentiated epithelial cells to facilitate the spread of the virus to new hosts. Because of limitations in existing model systems, the effects of latent EBV infection on undifferentiated and differentiating epithelial cells are poorly understood. Here, we characterize latent infection of an hTERT-immortalized oral epithelial cell line (NOKs). We find that although EBV expresses a latency pattern similar to that seen in EBV-associated carcinomas, infection of undifferentiated NOKs results in differential expression of a small number of host genes. In differentiating NOKs, however, EBV has a more substantial effect, reducing the extent of differentiation and delaying the exit from the cell cycle. This effect may synergize with preexisting cellular abnormalities to prevent exit from the cell cycle, representing a critical step in the development of cancer.


Subject(s)
Cell Cycle/physiology , Cell Differentiation , Epithelial Cells/metabolism , Epithelial Cells/virology , Epstein-Barr Virus Infections/metabolism , Herpesvirus 4, Human/genetics , Cell Division , Cell Line , Cell Proliferation , Epithelial Cells/pathology , Gene Expression Regulation, Neoplastic , Gene Expression Regulation, Viral , Genes, Viral/genetics , Herpesvirus 4, Human/pathogenicity , Humans , Keratinocytes/metabolism , Keratinocytes/pathology , Keratinocytes/virology , Minichromosome Maintenance Complex Component 7/metabolism , Stomach Neoplasms , Telomerase/metabolism , Transcriptome , Virus Activation , Virus Latency
5.
PLoS Pathog ; 14(8): e1007221, 2018 08.
Article in English | MEDLINE | ID: mdl-30125329

ABSTRACT

EBV causes human B-cell lymphomas and transforms B cells in vitro. EBNA3C, an EBV protein expressed in latently-infected cells, is required for EBV transformation of B cells in vitro. While EBNA3C undoubtedly plays a key role in allowing EBV to successfully infect B cells, many EBV+ lymphomas do not express this protein, suggesting that cellular mutations and/or signaling pathways may obviate the need for EBNA3C in vivo under certain conditions. EBNA3C collaborates with EBNA3A to repress expression of the CDKN2A-encoded tumor suppressors, p16 and p14, and EBNA3C-deleted EBV transforms B cells containing a p16 germline mutation in vitro. Here we have examined the phenotype of an EBNAC-deleted virus (Δ3C EBV) in a cord blood-humanized mouse model (CBH). We found that the Δ3C virus induced fewer lymphomas (occurring with a delayed onset) in comparison to the wild-type (WT) control virus, although a subset (10/26) of Δ3C-infected CBH mice eventually developed invasive diffuse large B cell lymphomas with type III latency. Both WT and Δ3C viruses induced B-cell lymphomas with restricted B-cell populations and heterogeneous T-cell infiltration. In comparison to WT-infected tumors, Δ3C-infected tumors had greatly increased p16 levels, and RNA-seq analysis revealed a decrease in E2F target gene expression. However, we found that Δ3C-infected tumors expressed c-Myc and cyclin E at similar levels compared to WT-infected tumors, allowing cells to at least partially bypass p16-mediated cell cycle inhibition. The anti-apoptotic proteins, BCL2 and IRF4, were expressed in Δ3C-infected tumors, likely helping cells avoid c-Myc-induced apoptosis. Unexpectedly, Δ3C-infected tumors had increased T-cell infiltration, increased expression of T-cell chemokines (CCL5, CCL20 and CCL22) and enhanced type I interferon response in comparison to WT tumors. Together, these results reveal that EBNA3C contributes to, but is not essential for, EBV-induced lymphomagenesis in CBH mice, and suggest potentially important immunologic roles of EBNA3C in vivo.


Subject(s)
Cell Transformation, Viral/genetics , Epstein-Barr Virus Infections/complications , Epstein-Barr Virus Nuclear Antigens/genetics , Herpesvirus 4, Human/physiology , Lymphoma, B-Cell/virology , Virus Latency/genetics , Animals , Cells, Cultured , Disease Models, Animal , Epstein-Barr Virus Infections/genetics , Fetal Blood/immunology , HEK293 Cells , Herpesvirus 4, Human/genetics , Humans , Lymphoma, B-Cell/immunology , Lymphoma, B-Cell/pathology , Mice , Mice, Inbred NOD , Mice, Transgenic
6.
PLoS Genet ; 9(6): e1003549, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23785296

ABSTRACT

Genome-wide association studies have revealed that many low-penetrance breast cancer susceptibility loci are located in non-protein coding genomic regions; however, few have been characterized. In a comparative genetics approach to model such loci in a rat breast cancer model, we previously identified the mammary carcinoma susceptibility locus Mcs1a. We now localize Mcs1a to a critical interval (277 Kb) within a gene desert. Mcs1a reduces mammary carcinoma multiplicity by 50% and acts in a mammary cell-autonomous manner. We developed a megadeletion mouse model, which lacks 535 Kb of sequence containing the Mcs1a ortholog. Global gene expression analysis by RNA-seq revealed that in the mouse mammary gland, the orphan nuclear receptor gene Nr2f1/Coup-tf1 is regulated by Mcs1a. In resistant Mcs1a congenic rats, as compared with susceptible congenic control rats, we found Nr2f1 transcript levels to be elevated in mammary gland, epithelial cells, and carcinoma samples. Chromatin looping over ∼820 Kb of sequence from the Nr2f1 promoter to a strongly conserved element within the Mcs1a critical interval was identified. This element contains a 14 bp indel polymorphism that affects a human-rat-mouse conserved COUP-TF binding motif and is a functional Mcs1a candidate. In both the rat and mouse models, higher Nr2f1 transcript levels are associated with higher abundance of luminal mammary epithelial cells. In both the mouse mammary gland and a human breast cancer global gene expression data set, we found Nr2f1 transcript levels to be strongly anti-correlated to a gene cluster enriched in cell cycle-related genes. We queried 12 large publicly available human breast cancer gene expression studies and found that the median NR2F1 transcript level is consistently lower in 'triple-negative' (ER-PR-HER2-) breast cancers as compared with 'receptor-positive' breast cancers. Our data suggest that the non-protein coding locus Mcs1a regulates Nr2f1, which is a candidate modifier of differentiation, proliferation, and mammary cancer risk.


Subject(s)
Breast Neoplasms/genetics , COUP Transcription Factor I/genetics , Cell Differentiation/genetics , Genetic Loci , Genetic Predisposition to Disease , Mammary Neoplasms, Animal/genetics , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , COUP Transcription Factor I/metabolism , Cell Proliferation , Chromosome Mapping , Epithelial Cells/metabolism , Epithelial Cells/pathology , Female , Gene Expression Regulation, Neoplastic , Genome-Wide Association Study , Humans , Mice , Rats
7.
J Cell Sci ; 125(Pt 23): 5702-10, 2012 Dec 01.
Article in English | MEDLINE | ID: mdl-22992461

ABSTRACT

Microtubules control cell motility by positively regulating polarization in many cell types. However, how microtubules regulate leukocyte migration is not well understood, particularly in living organisms. Here we exploited the zebrafish system to study the role of microtubules in neutrophil migration in vivo. The localization of microtubules was visualized in motile neutrophils using various bioprobes, revealing that, in contrast to what has been seen in studies in vitro, the microtubule organizing center is positioned in front of the nucleus (relative to the direction of migration) in motile neutrophils. Microtubule disassembly impaired attraction of neutrophils to wounds but enhanced the polarity of F-actin dynamics as measured by the distribution of stable and dynamic F-actin. Microtubule depolymerization inhibited polarized phosphoinositol 3-kinase (PI(3)K) activation at the leading edge and induced rapid PI(3)K independent motility. Finally, we show that microtubules exert their effects on neutrophil polarity and motility at least in part by the negative regulation of both Rho and Rac activity. These results provide new insight into the role of microtubules in neutrophil migration in a living vertebrate and show that the motility of these professional migratory cells are subject to distinctly different rules from those established for other cell types.


Subject(s)
Microtubules/metabolism , Neutrophils/cytology , Neutrophils/metabolism , Zebrafish/metabolism , Animals , Cell Movement/physiology , Cell Polarity/physiology , Chemotaxis/physiology
8.
Biotechniques ; 53(1): 57-60, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22780320

ABSTRACT

Here we introduce a modified antibody staining method that uses up to 80% less antibody for flow cytometry. We demonstrate this method for the detection of antigens expressed at high, moderate, or low levels in mouse and rat lymphocytes as well as mouse mammary epithelial cells. We obtained reproducibly accurate results for the detection of up to seven parameters for activation induced-proliferation, cell cycle analysis, and phenotyping of cell-surface and intracellular antigens.


Subject(s)
Antigens, CD/analysis , Epithelial Cells/immunology , Flow Cytometry/methods , Lymphocytes/immunology , Animals , Antigens, CD/immunology , Cells, Cultured , Epithelial Cells/cytology , Female , Flow Cytometry/economics , Lymphocytes/cytology , Mammary Glands, Animal/cytology , Mice , Rats , Sample Size , Staining and Labeling/economics , Staining and Labeling/methods
9.
PLoS One ; 6(10): e26145, 2011.
Article in English | MEDLINE | ID: mdl-22022542

ABSTRACT

Rat mammary carcinogenesis models have been used extensively to study breast cancer initiation, progression, prevention, and intervention. Nevertheless, quantitative molecular data on epithelial cell differentiation in mammary glands of untreated and carcinogen-exposed rats is limited. Here, we describe the characterization of rat mammary epithelial cells (RMECs) by multicolor flow cytometry using antibodies against cell surface proteins CD24, CD29, CD31, CD45, CD49f, CD61, Peanut Lectin, and Thy-1, intracellular proteins CK14, CK19, and FAK, along with phalloidin and Hoechst staining. We identified the luminal and basal/myoepithelial populations and actively dividing RMECs. In inbred rats susceptible to mammary carcinoma development, we quantified the changes in differentiation of the RMEC populations at 1, 2, and 4 weeks after exposure to mammary carcinogens DMBA and MNU. DMBA exposure did not alter the percentage of basal or luminal cells, but upregulated CD49f (Integrin α6) expression and increased cell cycle activity. MNU exposure resulted in a temporary disruption of the luminal/basal ratio and no CD49f upregulation. When comparing DMBA- or MNU-induced mammary carcinomas, the RMEC differentiation profiles are indistinguishable. The carcinomas compared with mammary glands from untreated rats, showed upregulation of CD29 (Integrin ß1) and CD49f expression, increased FAK (focal adhesion kinase) activation especially in the CD29hi population, and decreased CD61 (Integrin ß3) expression. This study provides quantitative insight into the protein expression phenotypes underlying RMEC differentiation. The results highlight distinct RMEC differentiation etiologies of DMBA and MNU exposure, while the resulting carcinomas have similar RMEC differentiation profiles. The methodology and data will enhance rat mammary carcinogenesis models in the study of the role of epithelial cell differentiation in breast cancer.


Subject(s)
Carcinoma/pathology , Cell Differentiation , Epithelial Cells/pathology , Mammary Glands, Animal/pathology , Mammary Neoplasms, Experimental/pathology , 9,10-Dimethyl-1,2-benzanthracene , Animals , Antigens, Surface/metabolism , Biomarkers/metabolism , Carcinoma/metabolism , Cell Division , Cell Membrane/metabolism , Cell Proliferation , Epithelial Cells/metabolism , Female , Mammary Glands, Animal/metabolism , Mammary Neoplasms, Experimental/metabolism , Methylnitrosourea , Models, Biological , Rats , Up-Regulation
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