Browsing by Subject "Epstein-Barr virus"

The interaction of human tumor viruses with host growth suppressive pathways is a fine balance between controlled latent infection and virus-induced oncogenesis. This dissertation elucidates how Epstein-Barr virus interacts with the host growth suppressive DNA damage response signaling pathways (DDR) in order to transform infected human B lymphocytes.

Here I report that the activation of the ATM/Chk2 branch of the DDR in hyper-proliferating infected B cells results in G1/S cell cycle arrest and limits viral-mediated transformation. Similar growth arrest was found in mitogen-driven proliferating of B cells that sets the DDR as a default growth suppressive mechanism in human B cells. Hence, the viral protein EBNA3C functions to attenuate the host DDR and to promote immortalization of a small portion of infected B cells. Additionally, the pharmacological inhibition of the DDR in vitro increases viral immortalization of memory B cells that facilitates the isolation of broadly neutralizing antibodies to various infectious agents. Overall, this work defines early EBV-infected hyper-proliferating B cells as a new stage in viral infection that determines subsequent viral-mediated tumorigenesis.

Epstein-Barr virus (EBV) is a ubiquitous human gamma-herpesvirus which chronically infects >95% of the global population, and can give rise to a number of malignancies in B cells and epithelial cells. In EBV latently infected epithelial cells, such as nasopharyngeal carcinoma (NPC) and gastric carcinoma (GaCa) cells, viral protein expression is low. In contrast, a cluster of viral microRNAs (miRNAs) called miR-BARTs is highly expressed. MiRNAs are small non-coding RNAs which regulate gene expression by binding to complementary sequences in mRNAs. It is likely that miR-BARTs play a crucial role in EBV-infected epithelial cells, however a comprehensive understanding of miR-BARTs is currently lacking. Here, I present two studies utilizing the phenotypic and the target approaches, respectively, to demonstrate that miR-BARTs can inhibit apoptosis and activate the Wnt signaling pathway. To discover miR-BARTs that can inhibit apoptosis, I individually expressed miR-BARTs in the EBV- GaCa cell line AGS, and identified five miR-BARTs that conferred this phenotype. To identify pro-apoptotic genes targeted by the five anti-apoptotic miRNAs, I validated one previously published target and identified nine novel targets by performing photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) in the EBV+ NPC cell line C666. Next, I thoroughly demonstrated that the 10 candidate target genes were substantially suppressed by expression of the relevant miR-BARTs, as measured by 3’UTR-containing firefly luciferase (FLuc) expression, mRNA and protein levels, and knockdown of seven of the 10 candidate genes could suppress apoptosis, mimicking the effects of relevant miR-BARTs. On the other hand, in order to identify miR-BARTs that can activate the Wnt signaling pathway, I analyzed the PAR-CLIP data set of C666 cells and discovered nine anti-Wnt signaling targets of miR-BARTs, including seven novel genes and two pro-apoptotic genes identified above. Using FLuc 3’UTR indicator assays, I proved that the 3’UTRs of all seven newly identified anti-Wnt signaling genes were indeed targeted by the relevant miR-BARTs identified by PAR-CLIP. Utilizing a Wnt signaling FLuc reporter TOPflash which measures the Wnt signaling activation, I confirmed that expression of many miR-BARTs that target Wnt signaling inhibitors can indeed upregulate the Wnt signaling pathway. Together, my results identified and validated a substantial number of novel targets of miR-BARTs involved in apoptosis and the Wnt signaling pathway, indicating that EBV may employ miR-BARTs to heavily target these two pathways to facilitate chronic infection.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that infects over 95% of the adult population. While infection is typically asymptomatic, in some individuals, particularly the immunocompromised, EBV is the causative agent of several cancers including lymphomas and epithelial cancers. Specifically, gastric carcinoma, nasopharyngeal carcinoma, and lymphoepithelioma-like carcinoma which occurs across multiple sites in the body, most notably, in the lung. EBV-associated gastric cancer (EBVaGC) is a unique subset of gastric cancer that makes up 10% of all GCs worldwide. EBVaGCs display an 80% rate of activating PIK3CA mutation and are also the most hypermethylated of any tumor type, displaying what is known as a CpG island hypermethylator phenotype (CIMP). EBV infection of B cells can be easily modeled in vitro using primary B cells and lymphoblastoid cell lines (LCLs). However, epithelial cell infection models have proven much more difficult to develop. Given these difficulties, EBV infection and outgrowth in epithelial cells is comparatively understudied and the process of tumorigenesis in vivo is poorly understood. In this dissertation, I developed methods to generate EBV infected epithelial cell lines derived from both gastric cancer and lung adenocarcinoma using diverse strains of EBV. I used these models to complete a CRISPR/Cas9 whole genome knockout screen to identify cellular restriction factors of infection and outgrowth. Together, these data will provide novel insights into the process of EBV infection and the dynamic interplay between virus and host during tumorigenesis. Furthermore, I have used these models to explore unique therapeutic approaches for EBV+ epithelial cancers. Specifically, I have characterized the lytic reactivation potential to histone deacetylase (HDAC) inhibitors and generated preclinical data supporting the use of HDAC inhibitors and the anti-viral ganciclovir for treatment of EBV+ epithelial tumors. Lastly, I have identified modulators of the response to a PI3Ka inhibitor in PIK3CA mutant gastric cancers. I found that loss of NEDD9 or inhibition of BCL-XL rendered cells hyper-sensitive to the PI3Ka inhibitor BYL719. Additionally, I found that loss of CBFB conferred resistance to BYL719 through up- regulation of the protein kinase PIM1 and defined the clinical utility of our data in the context of PI3K inhibition more broadly. The work outlined in this dissertation contributes to the study of EBV infection and tumorigenesis in the stomach as well as provides mechanistic insights into novel therapeutic approaches for EBV+ epithelial cancers and PIK3CA mutant gastric cancers.

Epstein-Barr virus (EBV) is an oncogenic gamma-herpesvirus that infects over 90% of adults worldwide. Typically, EBV establishes a benign latent infection that is controlled by a strong cytotoxic T cell immune response. However, EBV infection in immunocompromised patients has been associated with development of several lymphoid and epithelial cell malignancies, including Burkitt’s lymphoma, Hodgkin’s lymphoma, post-transplant lymphoproliferative disease, and nasopharyngeal carcinoma. In primary human B cells, EBV infection has been shown to induce a transient period of hyper-proliferation, but many of these infected cells succumb to a DNA damage response (DDR)-mediated growth arrest. We hypothesize that EBV infection triggers replicative stress early after infection and facilitates persistent activation of the DDR establishing oncogene-induced senescence. To test this hypothesis, we infected primary human B cells with EBV and examined cellular proliferation and host DNA damage response pathways at early and late stages post infection. We found that early after EBV infection, rapidly proliferating B cells exhibited signs of replication stress and reduced levels of purine dNTP nucleotide pools that are necessary for sustained proliferation. These findings suggest that purine dNTP biosynthesis plays a critical role in the early stages of EBV-mediated B cell immortalization. Furthermore, we observed the formation of persistent DDR foci in arrested B cells and identified key regulators of long-term outgrowth of EBV-infected B cells. Ultimately, this work has shown that early after EBV infection, cells that experience aberrant proliferation establish oncogene-induced senescence by chronically activating the DDR in the context of reduced dNTP nucleotide pools.

Epstein-Barr virus (EBV) is a ubiquitous human pathogen that establishes a lifelong latent infection in over ninety percent of all adult humans worldwide. While typically benign, EBV has been causally associated with a number of human malignancies in the settings of immune suppression, genetic, and/or environmental factors. While a highly successful pathogen based on prevalence, the ability of the virus to immortalize human B cells (a stage of infection thought to be critical for the establishment of latency) is quite poor. We hypothesize that the interactions between the virus and the human host early after infection are ultimately important for the outcome of viral latency establishment. To answer this question we broadly profiled primary human B cells at both early and late times after EBV infection to assay both host mRNA expression and the host-driven response to apoptotic stimuli. We found that EBV infection induces host gene expression signatures early after infection that are functionally distinct from the gene expression program late after infection. These studies also led to the novel discovery that viral gene expression is controlled differently early after infection, including the delayed expression of a viral protein that is critical for the establishment of latency. Furthermore, we have also shown that EBV can use a single viral protein to alter and repress host apoptotic sensitivity in the face of an anti-viral apoptotic response.

Epstein-Barr Virus (EBV) is a ubiquitous gamma-herpesvirus in the human population and highly associated with lymphomas of the immune-suppressed. EBV maintains itself within the human host via temporal regulation of viral gene expression before establishing latency in resting memory B cells. Experimentally, we can model these immune-compromised lymphomas via in vitro infection of B cells isolated from peripheral blood. This infection model is biphasic where the initial phase is characterized by hyper-proliferation and expression of the EBV nuclear antigens (EBNAs) in the absence of the latent membrane proteins (LMPs) called latency IIb before transitioning to the NFkB-dependent latency III lymphoblastoid cell line (LCL) stage characterized by full expression of the LMPs in the presence of the EBNAs. This temporal regulation manipulates host cell proliferation rates and metabolic profiles of infected B lymphocytes. Our laboratory and my studies, in particular, are focused on the latency IIb to latency III transition as these states are often observed in patient biopsies of EBV-associated malignancies.

Latent membrane protein 1 (LMP1) expression defines latency III and has been used in lymphoma samples to identify EBV latency III in vivo. While LMP1 expression is lower in latency IIb, the mechanism by which it is repressed is currently unknown. Additionally, while LMP1 expression can be used to distinguish latency IIb from latency III, LMP1 expression varies widely within latency III and a subset of LMP1lo latency III cells express LMP1 at levels similar to latency IIb making these cells hard to distinguish in vitro and in vivo. Lastly, while EBV manipulates host cell metabolism, the viral and host factors important for metabolic rewiring have not been fully elucidated.

Using RT-qPCR, ChIP-qPCR, RNA-sequencing, Western Blotting, flow cytometry, and bioinformatic approaches we have identified c-Myc as a repressor of LMP1 during latency IIb. We have also identified host mRNA biomarkers capable of distinguishing latency IIb from latency III. This discovery was additionally leveraged to develop a multiplex RNA-FISH platform capable of distinguishing latency IIb from latency III. Lastly, we have identified host and viral proteins necessary to rewire host cell metabolism and sustain EBV-mediated B cell proliferation. This work, taken together, adds new understanding to EBV latency establishment, heterogeneity, and host B cell biology to develop new, more targeted, therapeutics for EBV-associated lymphomas in the future.