scholarly journals Cellular STAT3 Functions via PCBP2 To Restrain Epstein-Barr Virus Lytic Activation in B Lymphocytes

2015 ◽  
Vol 89 (9) ◽  
pp. 5002-5011 ◽  
Author(s):  
Siva Koganti ◽  
Carissa Clark ◽  
Jizu Zhi ◽  
Xiaofan Li ◽  
Emily I. Chen ◽  
...  
Keyword(s):  
Tumor Cells ◽  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
Lytic Cycle ◽  
Substantial Fraction ◽  
Data Set ◽  
Oncolytic Therapy ◽  
Barr Virus ◽  
Infected Cells ◽  
Epstein Barr

ABSTRACTA major hurdle to killing Epstein-Barr virus (EBV)-infected tumor cells using oncolytic therapy is the presence of a substantial fraction of EBV-infected cells that does not support the lytic phase of EBV despite exposure to lytic cycle-promoting agents. To determine the mechanism(s) underlying this refractory state, we developed a strategy to separate lytic from refractory EBV-positive (EBV+) cells. By examining the cellular transcriptome in separated cells, we previously discovered that high levels of host STAT3 (signal transducer and activator of transcription 3) curtail the susceptibility of latently infected cells to lytic cycle activation signals. The goals of the present study were 2-fold: (i) to determine the mechanism of STAT3-mediated resistance to lytic activation and (ii) to exploit our findings to enhance susceptibility to lytic activation. We therefore analyzed our microarray data set, cellular proteomes of separated lytic and refractory cells, and a publically available STAT3 chromatin immunoprecipitation sequencing (ChIP-Seq) data set to identify cellular PCBP2 [poly(C)-binding protein 2], an RNA-binding protein, as a transcriptional target of STAT3 in refractory cells. Using Burkitt lymphoma cells and EBV+cell lines from patients with hypomorphicSTAT3mutations, we demonstrate that single cells expressing high levels of PCBP2 are refractory to spontaneous and induced EBV lytic activation, STAT3 functions via cellular PCBP2 to regulate lytic susceptibility, and suppression of PCBP2 levels is sufficient to increase the number of EBV lytic cells. We expect that these findings and the genome-wide resources that they provide will accelerate our understanding of a longstanding mystery in EBV biology and guide efforts to improve oncolytic therapy for EBV-associated cancers.IMPORTANCEMost humans are infected with Epstein-Barr virus (EBV), a cancer-causing virus. While EBV generally persists silently in B lymphocytes, periodic lytic (re)activation of latent virus is central to its life cycle and to most EBV-related diseases. However, a substantial fraction of EBV-infected B cells and tumor cells in a population is refractory to lytic activation. This resistance to lytic activation directly and profoundly impacts viral persistence and the effectiveness of oncolytic therapy for EBV+cancers. To identify the mechanisms that underlie susceptibility to EBV lytic activation, we used host gene and protein expression profiling of separated lytic and refractory cells. We find that STAT3, a transcription factor overactive in many cancers, regulates PCBP2, a protein important in RNA biogenesis, to regulate susceptibility to lytic cycle activation signals. These findings advance our understanding of EBV persistence and provide important leads on devising methods to improve viral oncolytic therapies.

scholarly journals Nascent Transcriptomics Reveal Cellular Prolytic Factors Upregulated Upstream of the Latent-to-Lytic Switch Protein of Epstein-Barr Virus

2020 ◽  
Vol 94 (7) ◽  
Author(s):  
Tiffany R. Frey ◽  
Jozan Brathwaite ◽  
Xiaofan Li ◽  
Sandeepta Burgula ◽  
Ibukun A. Akinyemi ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Human Herpesvirus ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Viral Spread ◽  
Cellular Genes ◽  
Latently Infected ◽  
Infected Cells ◽  
Host Shutoff ◽  
Epstein Barr

ABSTRACT Lytic activation from latency is a key transition point in the life cycle of herpesviruses. Epstein-Barr virus (EBV) is a human herpesvirus that can cause lymphomas, epithelial cancers, and other diseases, most of which require the lytic cycle. While the lytic cycle of EBV can be triggered by chemicals and immunologic ligands, the lytic cascade is activated only when expression of the EBV latent-to-lytic switch protein ZEBRA is turned on. ZEBRA then transcriptionally activates other EBV genes and, together with some of those gene products, ensures completion of the lytic cycle. However, not every latently infected cell exposed to a lytic trigger turns on the expression of ZEBRA, resulting in responsive and refractory subpopulations. What governs this dichotomy? By examining the nascent transcriptome following exposure to a lytic trigger, we find that several cellular genes are transcriptionally upregulated temporally upstream of ZEBRA. These genes regulate lytic susceptibility to various degrees in latently infected cells that respond to mechanistically distinct lytic triggers. While increased expression of these cellular genes defines a prolytic state, such upregulation also runs counter to the well-known mechanism of viral-nuclease-mediated host shutoff that is activated downstream of ZEBRA. Furthermore, a subset of upregulated cellular genes is transcriptionally repressed temporally downstream of ZEBRA, indicating an additional mode of virus-mediated host shutoff through transcriptional repression. Thus, increased transcription of a set of host genes contributes to a prolytic state that allows a subpopulation of cells to support the EBV lytic cycle. IMPORTANCE Transition from latency to the lytic phase is necessary for herpesvirus-mediated pathology as well as viral spread and persistence in the population at large. Yet, viral genomes in only some cells in a population of latently infected cells respond to lytic triggers, resulting in subpopulations of responsive/lytic and refractory cells. Our investigations into this partially permissive phenotype of the herpesvirus Epstein-Barr virus (EBV) indicate that upon exposure to lytic triggers, certain cellular genes are transcriptionally upregulated, while viral latency genes are downregulated ahead of expression of the viral latent-to-lytic switch protein. These cellular genes contribute to lytic susceptibility to various degrees. Apart from indicating that there may be a cellular “prolytic” state, our findings indicate that (i) early transcriptional upregulation of cellular genes counters the well-known viral-nuclease-mediated host shutoff and (ii) subsequent transcriptional downregulation of a subset of early upregulated cellular genes is a previously undescribed mode of host shutoff.

scholarly journals CD8+ immunodominance among Epstein-Barr virus lytic cycle antigens directly reflects the efficiency of antigen presentation in lytically infected cells

2005 ◽  
Vol 201 (3) ◽  
pp. 349-360 ◽  
Author(s):  
Victoria A. Pudney ◽  
Alison M. Leese ◽  
Alan B. Rickinson ◽  
Andrew D. Hislop
Keyword(s):  
T Cell ◽  
Epstein Barr Virus ◽  
Cd8 T Cell ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
E Proteins ◽  
Barr Virus ◽  
Infected Cells ◽  
Epstein Barr ◽  
L Proteins

Antigen immunodominance is an unexplained feature of CD8+ T cell responses to herpesviruses, which are agents whose lytic replication involves the sequential expression of immediate early (IE), early (E), and late (L) proteins. Here, we analyze the primary CD8 response to Epstein-Barr virus (EBV) infection for reactivity to 2 IE proteins, 11 representative E proteins, and 10 representative L proteins, across a range of HLA backgrounds. Responses were consistently skewed toward epitopes in IE and a subset of E proteins, with only occasional responses to novel epitopes in L proteins. CD8+ T cell clones to representative IE, E, and L epitopes were assayed against EBV-transformed lymphoblastoid cell lines (LCLs) containing lytically infected cells. This showed direct recognition of lytically infected cells by all three sets of effectors but at markedly different levels, in the order IE > E ≫ L, indicating that the efficiency of epitope presentation falls dramatically with progress of the lytic cycle. Thus, EBV lytic cycle antigens display a hierarchy of immunodominance that directly reflects the efficiency of their presentation in lytically infected cells; the CD8+ T cell response thereby focuses on targets whose recognition leads to maximal biologic effect.

Functional Epstein-Barr virus reservoir in plasma cells derived from infected peripheral blood memory B cells

Blood ◽  
2009 ◽  
Vol 113 (3) ◽  
pp. 604-611 ◽  
Author(s):  
Yassine Al Tabaa ◽  
Edouard Tuaillon ◽  
Karine Bollore ◽  
Vincent Foulongne ◽  
Gael Petitjean ◽  
...  
Keyword(s):  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
Plasma Cells ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Ebv Dna ◽  
Epstein Barr ◽  
Immediate Early Antigen ◽  
Culture Supernatants ◽  
Healthy Carriers

AbstractThe Epstein-Barr virus (EBV) causes infectious mononucleosis, establishes latency in resting memory B lymphocytes, and is involved in oncogenesis through poorly understood mechanisms. The EBV lytic cycle is initiated during plasma cell differentiation by mRNAs transcripts encoded by BZLF1, which induce the synthesis of EBV proteins such as the immediate-early antigen ZEBRA and the late membrane antigen gp350. Therefore, we assessed the capacity of circulating EBV-infected B lymphocytes from healthy EBV-seropositive subjects to enter and complete the EBV lytic cycle. Purified B lymphocytes were polyclonally stimulated and BZLF1- or gp350-secreting cells (BZLF1-SCs or gp350-SCs) were enumerated by ELISpot assays. The number of BZLF1-SCs ranged from 50 to 480/107 lymphocytes (median, 80; 25th-75th percentiles, 70-150) and gp350-SCs from 10 to 40/107 lymphocytes (median, 17; 25th-75th percentiles, 10-20). gp350-SCs represented only 7.7% to 28.6% of BZLF1-SCs (median, 15%; 25th-75th percentiles, 10.5%-20%). This EBV functional reservoir was preferentially restricted to plasma cells derived from CD27+ IgD− memory B lymphocytes. In 9 of 13 subjects, EBV DNA quantification in B-cell culture supernatants gave evidence of completion of EBV lytic cycle. These results demonstrate that EBV proteins can be secreted by EBV-infected B lymphocytes from healthy carriers, a majority generating an abortive EBV lytic cycle and a minority completing the cycle.

scholarly journals Cleavage and Secretion of Epstein-Barr Virus Glycoprotein 42 Promote Membrane Fusion with B Lymphocytes

2009 ◽  
Vol 83 (13) ◽  
pp. 6664-6672 ◽  
Author(s):  
Jessica Sorem ◽  
Theodore S. Jardetzky ◽  
Richard Longnecker
Keyword(s):  
Membrane Fusion ◽  
B Lymphocytes ◽  
Viral Entry ◽  
Epstein Barr Virus ◽  
Class Ii ◽  
Hla Class Ii ◽  
Full Length ◽  
Barr Virus ◽  
Infected Cells ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) membrane glycoprotein 42 (gp42) is required for viral entry into B lymphocytes through binding to human leukocyte antigen (HLA) class II on the B-cell surface. EBV gp42 plays multiple roles during infection, including acting as a coreceptor for viral entry into B cells, binding to EBV glycoprotein H (gH) and gL during the process of membrane fusion, and blocking T-cell recognition of HLA class II-peptide complexes through steric hindrance. EBV gp42 occurs in two forms in infected cells, a full-length membrane-bound form and a soluble form generated by proteolytic cleavage that is secreted from infected cells due to loss of the N-terminal transmembrane domain. Both the full-length and the secreted gp42 forms bind to gH/gL and HLA class II, and the functional significance of gp42 cleavage is currently unclear. We found that in a virus-free cell-cell fusion assay, enhanced secretion of gp42 promoted fusion with B lymphocytes, and mutation of the site of gp42 cleavage inhibited membrane fusion activity. The site of gp42 cleavage was found to be physically distinct from the residues of gp42 necessary for binding to gH/gL. These results suggest that cleavage and secretion of gp42 are necessary for the process of membrane fusion with B lymphocytes, providing the first indicated functional difference between full-length and cleaved, secreted gp42.

scholarly journals Hyperphosphorylation of EBNA2 by Epstein-Barr Virus Protein Kinase Suppresses Transactivation of the LMP1 Promoter

2005 ◽  
Vol 79 (9) ◽  
pp. 5880-5885 ◽  
Author(s):  
Wei Yue ◽  
Edward Gershburg ◽  
Joseph S. Pagano
Keyword(s):  
Protein Kinase ◽  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
Lytic Cycle ◽  
Nuclear Antigen ◽  
Virus Protein ◽  
Protein Levels ◽  
Barr Virus ◽  
Latently Infected ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) BGLF4 gene encodes a serine/threonine protein kinase (PK) that is expressed in the cytolytic cycle. EBV nuclear antigen 2 (EBNA2) is a key latency gene essential for immortalization of B lymphocytes and transactivation of viral and cellular promoters. Here we report that EBV PK phosphorylates EBNA2 at Ser-243 and that these two proteins physically associate. PK suppresses EBNA2's ability to transactivate the LMP1 promoter, and Ser-243 of EBNA2 is involved in this suppression. Moreover, EBNA2 is hyperphosphorylated during EBV reactivation in latently infected B cells, which is associated with decreased LMP1 protein levels. This is the first report about the effect of EBV PK on the function of one of its target proteins and regulation of EBNA2 phosphorylation during the EBV lytic cycle.

scholarly journals An Epstein-Barr Virus That Expresses Only the First 231 LMP1 Amino Acids Efficiently Initiates Primary B-Lymphocyte Growth Transformation

1999 ◽  
Vol 73 (12) ◽  
pp. 10525-10530 ◽  
Author(s):  
Kenneth M. Kaye ◽  
Kenneth M. Izumi ◽  
Hong Li ◽  
Eric Johannsen ◽  
David Davidson ◽  
...  
Keyword(s):  
Amino Acids ◽  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
B Lymphocyte ◽  
Feeder Cells ◽  
Barr Virus ◽  
Infected Cells ◽  
Carboxyl Terminal ◽  
Epstein Barr

ABSTRACT An Epstein-Barr virus (EBV) recombinant (MS231) that expresses the first 231 amino acids (aa) of LMP1 and is truncated 155 aa before the carboxyl terminus transformed resting B lymphocytes into lymphoblastoid cell lines (LCLs) only when the infected cells were grown on fibroblast feeder cells (K. M. Kaye et al., J. Virol. 69:675–683, 1995). Higher-titer MS231 virus has now been compared to wild-type (WT) EBV recombinants for the ability to cause resting primary B-lymphocyte transformation. Unexpectedly, MS231 is as potent as WT EBV recombinants in causing infected B lymphocytes to proliferate in culture for up to 5 weeks. When more than one transforming event is initiated in a microwell, the MS231 recombinant supports efficient long-term LCL outgrowth and fibroblast feeder cells are not required. However, with limited virus input, MS231-infected cells differed in their growth from WT virus-infected cells as early as 6 weeks after infection. In contrast to WT virus-infected cells, most MS231-infected cells could not be grown into long-term LCLs. Thus, the LMP1 amino-terminal 231 aa are sufficient for initial growth transformation but the carboxyl-terminal 155 aa are necessary for efficient long-term outgrowth. Despite the absence of the carboxyl-terminal 155 aa, MS231- and WT-transformed LCLs are similar in latent EBV gene expression, in ICAM-1 and CD23 expression, and in NF-κB and c-jun N-terminal kinase activation. MS231 recombinant-infected LCLs, however, require 16- to 64-fold higher cell density than WT-infected LCLs for regrowth after limiting dilution. These data indicate that the LMP1 carboxyl-terminal 155 aa are important for growth at lower cell density and appear to reduce dependence on paracrine growth factors.

scholarly journals Negative Autoregulation of Epstein-Barr Virus (EBV) Replicative Gene Expression by EBV SM Protein

2009 ◽  
Vol 83 (16) ◽  
pp. 8041-8050 ◽  
Author(s):  
Dinesh Verma ◽  
Chen Ling ◽  
Eric Johannsen ◽  
Tirumuru Nagaraja ◽  
Sankar Swaminathan
Keyword(s):  
Epstein Barr Virus ◽  
Feedback Regulation ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Immediate Early ◽  
Mrna Levels ◽  
Barr Virus ◽  
Infected Cells ◽  
Epstein Barr ◽  
Sm Protein

ABSTRACT The Epstein-Barr virus (EBV) SM protein is essential for lytic EBV DNA replication and virion production. When EBV replication is induced in cells infected with an SM-deleted recombinant EBV, approximately 50% of EBV genes are expressed inefficiently. When EBV replication is rescued by transfection of SM, SM enhances expression of these genes by direct and indirect mechanisms. While expression of most EBV genes is either unaffected or enhanced by SM, expression of several genes is decreased in the presence of SM. Expression of BHRF1, a homolog of cellular bcl-2, is particularly decreased in the presence of SM. Investigation of the mechanism of BHRF1 downregulation revealed that SM downregulates expression of the immediate-early EBV transactivator R. In EBV-infected cells, R-responsive promoters, including the BHRF1 and SM promoters, were less active in the presence of SM, consistent with SM inhibition of R expression. SM decreased spliced R mRNA levels, supporting a posttranscriptional mechanism of R inhibition. R and BHRF1 expression were also found to decrease during later stages of EBV lytic replication in EBV-infected lymphoma cells. These data indicate that feedback regulation of immediate-early and early genes occurs during the lytic cycle of EBV regulation.

hTERT inhibits the Epstein-Barr virus lytic cycle and promotes the proliferation of primary B lymphocytes: Implications for EBV-driven lymphomagenesis

2007 ◽  
Vol 121 (3) ◽  
pp. 576-587 ◽  
Author(s):  
Liliana Terrin ◽  
Riccardo Dolcetti ◽  
Irene Corradini ◽  
Stefano Indraccolo ◽  
Jessica Dal Col ◽  
...  
Keyword(s):  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals Frequent latent Epstein-Barr virus infection of neoplastic T cells and bystander B cells in human immunodeficiency virus-negative European peripheral pleomorphic T-cell lymphomas

Blood ◽  
1993 ◽  
Vol 82 (1) ◽  
pp. 217-223 ◽  
Author(s):  
P Korbjuhn ◽  
I Anagnostopoulos ◽  
M Hummel ◽  
M Tiemann ◽  
F Dallenbach ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Tumor Cells ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Epstein Barr Virus Infection ◽  
Barr Virus ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Epstein Barr

We investigated 81 cases of peripheral pleomorphic T-cell lymphoma (PMTCL) occurring in human immunodeficiency virus-negative Europeans for the presence of Epstein-Barr virus (EBV)-DNA through polymerase chain reaction (PCR) for the presence of EBV-encoded small nuclear RNAs (EBER) and immediate early mRNAs (Bam H-fragment, lower strand frame [BHLF]) by in situ hybridization (ISH) and for EBV-encoded latent membrane protein (LMP) and nuclear antigen 2 (EBNA2) by immunohistology (IH). EBER-ISH, which could be applied on all cases, showed an overall incidence of EBV-infected cells in 38 of 81 cases (47%) of PMTCL. These data could be confirmed by PCR, which produced congruent results in the cases with amplifiable DNA. By EBER-ISH, the virus was located in the tumor cells in 30 of the 38 EBV-positive cases, with the proportion of the infected cells ranging from 1% to 100%. In 18 of these cases and in the 8 cases without EBV-infected tumor cells, the virus was, respectively, either additionally or exclusively detectable in occasional nonmalignant lymphoid bystander cells. An LMP expression was observed in several of the EBER-expressing tumor cells in 18 cases, whereas EBNA2 was detectable only in one case, which also displayed signs of viral replication. Some nonmalignant EBV-infected B immunoblasts also expressed LMP in several cases. Primary cutaneous and enteropathy-associated PMTCL displayed less frequent EBV infection when compared with other extranodal or nodal manifestations.

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