Eleonora Forte

An ATM/Chk2-Mediated DNA Damage-Responsive Signaling Pathway Suppresses Epstein-Barr Virus Transformation of Primary Human B Cells

SUMMARY Epstein-Barr virus (EBV), an oncogenic herpesvirus that causes human malignancies, infects and immortalizes primary human B cells in vitro into indefinitely proliferating lymphoblastoid cell lines, which represent a model for EBV-induced tumorigenesis. The immortalization efficiency is very low suggesting that an innate tumor suppressor mechanism is operative. We identify the DNA damage response (DDR) as a major component of the underlying tumor suppressor mechanism. EBV-induced DDR activation was not due to lytic viral replication nor did the DDR marks co-localize with latent episomes. Rather, a transient period of EBV-induced hyper-proliferation correlated with DDR activation. Inhibition of the DDR kinases ATM and Chk2 markedly increased transformation efficiency of primary B cells. Further, the viral latent oncoproteins EBNA3C was required to attenuate the EBV-induced DNA damage response We propose that heightened oncogenic activity in early cell divisions activates a growth-suppressive DDR which is attenuated by viral latency products to induce cell immortalization.

The Epstein-Barr Virus (EBV)-Induced Tumor Suppressor MicroRNA MiR-34a Is Growth Promoting in EBV-Infected B Cells

ABSTRACT Epstein-Barr virus (EBV) infection of primary human B cells drives their indefinite proliferation into lymphoblastoid cell lines (LCLs). B cell immortalization depends on expression of viral latency genes, as well as the regulation of host genes. Given the important role of microRNAs (miRNAs) in regulating fundamental cellular processes, in this study, we assayed changes in host miRNA expression during primary B cell infection by EBV. We observed and validated dynamic changes in several miRNAs from early proliferation through immortalization; oncogenic miRNAs were induced, and tumor suppressor miRNAs were largely repressed. However, one miRNA described as a p53-targeted tumor suppressor, miR-34a, was strongly induced by EBV infection and expressed in many EBV and Kaposis sarcoma-associated herpesvirus (KSHV)-infected lymphoma cell lines. EBV latent membrane protein 1 (LMP1) was sufficient to induce miR-34a requiring downstream NF-κB activation but independent of functional p53. Furthermore, overexpression of miR-34a was not toxic in several B lymphoma cell lines, and inhibition of miR-34a impaired the growth of EBV-transformed cells. This study identifies a progrowth role for a tumor-suppressive miRNA in oncogenic-virus-mediated transformation, highlighting the importance of studying miRNA function in different cellular contexts.

Analysis of Epstein-Barr Virus-Regulated Host Gene Expression Changes through Primary B-Cell Outgrowth Reveals Delayed Kinetics of Latent Membrane Protein 1-Mediated NF-κB Activation

ABSTRACT Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that dramatically reorganizes host gene expression to immortalize primary B cells. In this study, we analyzed EBV-regulated host gene expression changes following primary B-cell infection, both during initial proliferation and through transformation into lymphoblastoid cell lines (LCLs). While most EBV-regulated mRNAs were changed during the transition from resting, uninfected B cells through initial B-cell proliferation, a substantial number of mRNAs changed uniquely from early proliferation through LCL outgrowth. We identified constitutively and dynamically EBV-regulated biological processes, protein classes, and targets of specific transcription factors. Early after infection, genes associated with proliferation, stress responses, and the p53 pathway were highly enriched. However, the transition from early to long-term outgrowth was characterized by genes involved in the inhibition of apoptosis, the actin cytoskeleton, and NF-κB activity. It was previously thought that the major viral protein responsible for NF-κB activation, latent membrane protein 1 (LMP1), is expressed within 2 days after infection. Our data indicate that while this is true, LCL-level LMP1 expression and NF-κB activity are not evident until 3 weeks after primary B-cell infection. Furthermore, heterologous NF-κB activation during the first week after infection increased the transformation efficiency, while early NF-κB inhibition had no effect on transformation. Rather, inhibition of NF-κB was not toxic to EBV-infected cells until LMP1 levels and NF-κB activity were high. These data collectively highlight the dynamic nature of EBV-regulated host gene expression and support the notion that early EBV-infected proliferating B cells have a fundamentally distinct growth and survival phenotype from that of LCLs.

MDM2-Dependent Inhibition of p53 Is Required for Epstein-Barr Virus B-Cell Growth Transformation and Infected-Cell Survival

ABSTRACT Epstein-Barr virus (EBV) growth transformation of primary B lymphocytes into indefinitely proliferating lymphoblastoid cell lines (LCLs) depends on the concerted activities of a subset of viral proteins expressed during latency. EBV drives quiescent B cells into S phase, and consequently, a host response is activated that includes expression of p53 and its target genes. Since LCLs retain wild-type p53, it was of interest to determine what contribution the p53 pathway may have in controlling established LCL growth and EBV-mediated transformation of primary B cells. We found that liberation of p53 through chemical antagonism of one of its major ubiquitin ligases, MDM2, using the small-molecule Nutlin-3 led to apoptosis of established LCLs and suppressed EBV-mediated transformation of primary B cells. The activation of latent p53 induced target genes associated with apoptosis. Furthermore, MDM2 antagonism synergized with NF-κB inhibition in killing LCLs. NF-κB was important to increase steady-state MDM2 protein levels rather than in affecting p53-dependent transcription, suggesting a unique mechanism by which LCLs survive in the presence of a primed p53 pathway. Nutlin sensitivity of EBV-infected cells provides a novel system for studying the pathways that dictate LCL survival and regulate EBV transformation. Finally, MDM2 antagonists may be considered for therapeutic intervention in EBV-associated malignancies expressing wild-type p53.

The role of microRNAs in Epstein-Barr virus latency and lytic reactivation

Oncogenic viruses reprogram host gene expression driving proliferation, ensuring survival, and evading the immune response. The recent appreciation of microRNAs (miRNAs) as small non-coding RNAs that broadly regulate gene expression has provided new insight into this complex scheme of host control. This review highlights the role of viral and cellular miRNAs during the latent and lytic phases of the EBV life cycle.

2‐D Difference in gel electrophoresis combined with Pro‐Q Diamond staining: A successful approach for the identification of kinase/phosphatase targets

The protein tyrosine phosphatase PRL‐3 is an appealing therapeutic cancer target for its well described involvement in the metastasis progression. Nevertheless, very little is known about PRL‐3 role in tumorigenesis. In the attempt to identify the protein target of this phosphatase we have devised a model system based on the use of highly invasive HCT116 colon cancer cells over‐expressing PRL‐3. We used 2‐D difference gel electrophoresis combined with the fluorescence staining Pro‐Q Diamond selective for phosphorylated proteins to monitor changes in the phosphorylation status of possible substrates. Proteins whose phosphorylation level was negatively affected by PRL‐3 over‐expression were identified by MS. Two proteins were found to be significantly dephosphorylated in this condition, the cytoskeletal protein ezrin and elongation factor 2. Ezrin has already been described as having a proactive role in cancer metastasis through control of its phosphorylation status, and the PRL‐3‐induced modulation of ezrin phosphorylation in HCT116 and human umblical vascular endothelial cells is the subject of a separate paper by Forte et al. [Biochim. Biophys. Acta 2008, 1783, 334–344]. The combination of 2‐D difference in gel electrophoresis and Pro‐Q Diamond was hence confirmed successful in analyzing changes of protein phosphorylation which enable the identification of kinase/phosphatase targets.

MicroRNA-Mediated Transformation by the Kaposi's Sarcoma-Associated Herpesvirus Kaposin Locus

ABSTRACT The human oncogenic Kaposis sarcoma-associated herpesvirus (KSHV) expresses a set of ∼20 viral microRNAs (miRNAs). miR-K10a stands out among these miRNAs because its entire stem-loop precursor overlaps the coding sequence for the Kaposin (Kap) A/C proteins. The ectopic expression of KapA has been reported to lead to transformation of rodent fibroblasts. However, these experiments inadvertently also introduced miR-K10a, which raises the question whether the transforming activity of the locus could in fact be due to miR-K10a expression. To answer this question, we have uncoupled miR-K10a and KapA expression. Our experiments revealed that miR-K10a alone transformed cells with an efficiency similar to that when it was coexpressed with KapA. Maintenance of the transformed phenotype was conditional upon continued miR-K10a but not KapA protein expression, consistent with its dependence on miRNA-mediated changes in gene expression. Importantly, miR-K10a taps into an evolutionarily conserved network of miR-142-3p targets, several of which are expressed in 3T3 cells and are also known inhibitors of cellular transformation. In summary, our studies of miR-K10a serve as an example of an unsuspected function of an mRNA whose precursor is embedded within a coding transcript. In addition, our identification of conserved miR-K10a targets that limit transformation will point the way to a better understanding of the role of this miRNA in KSHV-associated tumors. IMPORTANCE Kaposis sarcoma-associated herpesvirus (KSHV) is a human tumor virus. The viral Kaposin locus has known oncogenic potential, which has previously been attributed to the encoded KapA protein. Here we show that the virally encoded miR-K10a miRNA, whose precursor overlaps the KapA-coding region, may account for the oncogenic properties of this locus. Our data suggest that miR-K10a mimics the cellular miRNA miR-142-3p and thereby represses several known inhibitors of oncogenic transformation. Our work demonstrates that functional properties attributed to a coding region may in fact be carried out by an embedded noncoding element and sheds light on the functions of viral miR-K10a.

Epstein-Barr Virus Induces Global Changes in Cellular mRNA Isoform Usage That Are Important for the Maintenance of Latency

ABSTRACT Oncogenic viruses promote cell proliferation through the dramatic reorganization of host transcriptomes. In addition to regulating mRNA abundance, changes in mRNA isoform usage can have a profound impact on the protein output of the transcriptome. Using Epstein-Barr virus (EBV) transformation of primary B cells, we have studied the ability of an oncogenic virus to alter the mRNA isoform profile of its host. Using the algorithm called SplicerEX with two complementary Affymetrix microarray platforms, we uncovered 433 mRNA isoform changes regulated by EBV during B-cell transformation. These changes were largely orthogonal with the 2,163 mRNA abundance changes observed during transformation, such that less than one-third of mRNAs changing at the level of isoform also changed in overall abundance. While we observed no preference for a mechanistic class of mRNA isoform change, we detected a significant shortening of 3′ untranslated regions and exclusion of cassette exons in EBV-transformed cells relative to uninfected B cells. Gene ontology analysis of the mRNA isoform changes revealed significant enrichment in nucleic acid binding proteins. We validated several of these isoform changes and were intrigued by those in two mRNAs encoding the proteins XBP1 and TCF4, which have both been shown to bind and activate the promoter of the major EBV lytic trans-activator BZLF1. Our studies indicate that EBV latent infection promotes the usage of mRNA isoforms of XBP1 and TCF4 that restrict BZLF1 activation. Therefore, characterization of global changes in mRNA isoform usage during EBV infection identifies a new mechanism for the maintenance of latent infection.

Use of Viral Systems to Study miRNA-Mediated Regulation of Gene Expression in Human Cells

MicroRNAs (miRNAs) are a class of small ∼22 nt regulatory RNAs that modulate mRNA expression in all multicellular eukaryotic organisms. Interestingly, viruses also encode miRNAs and these viral miRNAs target cellular and viral mRNAs to regulate virus replication and latent infection. In particular, herpesviruses encode a large number of miRNAs. Herpesvirus infection also changes the normal expression profile of cellular miRNAs. New genetic tools have recently been generated to study the function of viral and cellular miRNAs in virus-infected cells. The creation of these reagents and use in Epstein-Barr virus-infected lymphoblastoid cell lines are discussed as a model viral system for the investigation of miRNA function.

TNF-α induces reactivation of human cytomegalovirus independently of myeloid cell differentiation following post-transcriptional establishment of latency

We used the Kasumi-3 model to study HCMV latency and reactivation in myeloid progenitor cells. Kasumi-3 cells were infected with HCMV strain TB40/Ewt-GFP, flow sorted for GFP+ cells, and cultured for various times to monitor establishment of latency, as judged by repression of viral gene expression (RNA/DNA ratio) and loss of virus production. We found that latency was established post-transcriptionally: transcription was initially turned on, and then turned off. We did not find evidence to support a latency model in which viral gene expression is repressed at the outset of infection. The pattern of expression of lytic genes that have been associated with latency, including UL138, US28, and RNA2.7, was the same as that of other lytic genes, indicating that there was no preferential expression of these genes in latency. By analyzing expression of the progenitor cell marker CD34 as well as myeloid cell differentiation markers in IE+ cells after treatment with TNF-α, we showed that TNF-α induced reactivation independently of differentiation. In contrast, LPS did not induce reactivation in Kasumi-3 cells, despite activation of ERK and NF-κB, and did not induce transcription of inflammatory cytokines. We speculate that reactivation occurs in two steps: 1. activation of signal-specific transcription factors that bind to the MIEP; 2. reconfiguration of viral chromatin to allow access to the MIEP. We propose that differentiation is required to render cellular chromatin competent to express inflammatory cytokines that drive reactivation in response to LPS, but it is not required for reactivation in response to TNF-α itself. IMPORTANCE HCMV is an important human pathogen that establishes lifelong latent infection in myeloid progenitor cells, and reactivates frequently to cause significant disease in immunocompromised people. The epigenetic mechanisms that allow the virus to establish latency specifically in myeloid cells, and to reactivate in response to inflammatory mediators are not well understood. Our observation that viral gene expression is first turned on, and then turned off to establish latency suggests that there is a host defense, which may be myeloid-specific, responsible for transcriptional silencing of viral gene expression. Our observation that TNF-α induces reactivation independently of differentiation, but LPS does not, provides insight into molecular mechanisms that control reactivation.