Browsing by Author "Luftig, Micah A"

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 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.

The Epstein-Barr virus (EBV) is a ubiquitous human pathogen that infects more than >95% of the global adult population. In immunocompetent individuals, EBV infection is asymptomatic and takes place in the oral cavity, where EBV establishes a life-long latent infection in memory B cells by temporally regulating viral gene expression to mimic B cell maturation. In immunocompromised individuals, however, EBV infection can give rise to infectious mononucleosis, epithelial carcinomas, and lymphomas. To model EBV-mediated lymphomagenesis, infection of EBV in vitro generates growth-transformed and immortalized lymphoblastoid cell lines (LCLs), which allows for the characterization of dynamic viral and host gene expression. Our lab has found that the early phase after infection is transcriptionally distinct from the late phase when infected B cells are fully growth-transformed. We also found that apoptosis regulation in each phase of infection is uniquely regulated by a single viral nuclear protein that regulates host gene expression through epigenetic mechanisms. To determine if apoptosis regulation in EBV-infected B cells is virus-specific, I have characterized apoptosis regulation in uninfected maturing B cells and mitogen-stimulated B cells. For the upregulation of one anti-apoptotic protein, EBV infection promotes a chromatin structure resembling that in germinal center light zone B cells, indicating that EBV phenocopies germinal center chromatin regulation to promote apoptosis resistance. In addition to apoptosis regulation, EBV infection phenocopies various aspects of GC B cells and plasmablasts, where the inhibition of plasma cell differentiation increases the efficiency of immortalization and growth-transformation of B cells infected in vitro. The work outlined in this dissertation demonstrate that viral and host genes cooperate in mediating apoptosis regulation, differentiation, and ultimately fate-determination of EBV-infected B cells.

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.

Human cytomegalovirus (CMV) is the most common cause of congenital infection worldwide, affecting approximately 1 in 150 infants, and is a leading cause of morbidity and mortality among transplant recipients. Congenital CMV (cCMV) infection can lead to permanent hearing loss, brain damage, and neurodevelopmental delay, and cCMV alone is responsible for nearly 25% of all infant hearing loss. Chronic CMV infection has also been associated with a heightened inflammatory state and increased risk of aging-related diseases, including as cardiovascular disease and type 2 diabetes. Over the last fifty years, there have been many efforts to develop vaccines that can prevent CMV disease. However, vaccine development for CMV faces many challenges, including a limited understanding of the immune responses protective against infection.

Like other beta herpesviruses, CMV can establish a lifelong, persistent infection in hosts, marked by periods of latency and reactivation, and preexisting immunity does not protect against reinfection. Moreover, CMV is known to predominantly spread by direct cell-to-cell transmission, complicating efforts to design vaccines that not only prevent viral entry via antibody neutralization but also inhibit the spread of cell-associated virus. To identify targets for vaccine development, we investigated the antibody immune responses associated with protection from CMV in historical vaccine trials and the lineage maturation of neutralizing antibodies elicited in natural infection.

The most efficacious CMV vaccine to-date is the glycoprotein B (gB) subunit vaccine combined with the MF59 adjuvant (gB/MF59), which achieved 50% protection against primary CMV acquisition in multiple Phase 2 clinical trials. CMV gB is a viral envelope protein that mediates fusion with host cell membranes and is required for viral entry into all known cell types and for cell-cell spread. Previous studies had found that in cohorts of CMV-seronegative postpartum women and renal transplant recipients, gB/MF59 vaccination did not elicit broadly neutralizing antibodies but instead generated robust nonneutralizing antibody responses, namely antibody-dependent cellular phagocytosis (ADCP). These studies lacked the statistical power to determine whether ADCP responses contributed to the partial efficacy of the gB/MF59 vaccine. We aimed to define the immune responses correlated with protection from primary CMV acquisition in Phase 2 gB/MF59 clinical trials in cohorts of adolescent girls and postpartum women. We first evaluated the vaccine-elicited sera IgG binding, neutralizing, and nonneutralizing responses against CMV, and we observed distinct immunogenicity profiles in the adolescent and postpartum cohorts, wherein adolescent vaccinees but not postpartum vaccinees developed broadly neutralizing CMV antibodies. We then compared the sera antibody responses between vaccinees who acquired infection and those who remained uninfected during the course of the trial. By multiple logistic regression analysis, we found that protection against primary CMV acquisition was associated with the presence of sera IgG binding to cell-associated gB, but not IgG binding to soluble gB as used in the gB/MF59 vaccine. These results suggested that there may be conformational differences between cell-associated and soluble gB. Supporting this, we identified gB-specific monoclonal antibodies (mAbs) that differentially recognized these gB structures. Our findings indicated the importance of the native, cell-associated gB conformation in future CMV vaccine design.

In our immunogenicity studies of gB/MF59, we observed that the vaccine failed to elicit antibodies against the gB antigenic domain 2 site 1 region (AD-2S1), which is a highly conserved, linear epitope at the far N terminus of gB that is known to be a target for potently neutralizing antibodies in natural infection. The presence of sera antibodies against gB AD-2S1 in naturally infected individuals have been associated with decreased risk for cCMV transmission and less severe CMV disease in transplant recipients. Yet, only about half of naturally infected individuals develop anti-gB AD-2S1 antibodies, and gB-based vaccines to-date have failed to elicit these responses. Thus, it remained unclear how to generate neutralizing antibodies against this poorly immunogenic epitope by vaccination. With the goal to identify a gB AD-2S1 structure that could elicit these potently neutralizing antibodies from the germline, we employed a B cell lineage-targeted vaccine strategy. We mapped the phylogeny of a well-characterized, potently neutralizing anti-gB AD-2S1 mAb from its germline precursor, then by empiric testing, we identified the antibody mutations that conferred neutralizing function. We found that a single heavy chain mutation in the CDR1 region was both necessary and sufficient to confer neutralizing function to the otherwise nonneutralizing IGHV3-30/IGKV3-11 germline ancestor mAb. This study identified a critical, early B cell receptor mutation that can serve a target for mutation-guided CMV vaccine design.

Only a limited number of gB AD-2S1 mAb sequences have been published to-date, with few clonally related members. To identify additional antibodies for lineage analysis and for evaluation as potential passive therapeutics, we developed a pipeline to sequence anti-CMV gB AD-2S1 mAbs and estimate their antigen binding in high-throughput. This study leveraged modern advances in B cell sequencing technologies to expedite the discovery of potently neutralizing CMV antibodies.

In sum, this work contributes to our understanding of protective immunity to CMV. We found that the immune correlate of protection for the gB/MF59 vaccine was sera IgG binding to cell-associated gB, suggesting that the next generation of gB-based vaccines should be designed to adopt a cell-associated or native conformation. Then, we investigated the lineage maturation of potently neutralizing antibodies against CMV gB AD-2S1 and identified a critical mAb mutation from the germline associated with the development of neutralization function, which will may serve as target for immunogen design. Additionally, we developed a pipeline for the high-throughput isolation of anti-gB AD-2S1 mAbs. These findings provide insight into antibody-mediated protection from CMV and illuminate paths forward for rational vaccine design.

The Flavivirus genus contains several arthropod-borne viruses that pose global health threats, including dengue virus (DENV). We identified two ribosomal proteins, RPLP1 and RPLP2 (RPLP1/2), that are crucial host factors required for translation of flaviviruses and efficient flavivirus infection of human cell lines and Aedes aegypti mosquitoes, which are natural vectors for these viruses. We hypothesized that RPLP1/2 are accessory ribosomal proteins that function to promote translation of specific cellular mRNAs sharing undefined features with the DENV genome. We found that these proteins are not broadly required for cellular translation and but are necessary for efficient accumulation of DENV proteins early in infection and ectopically expressed DENV structural proteins. The ribosome profiling technique allowed us to quantitative map ribosomes across the transcriptome during early DENV infection in human cell lines depleted for RPLP1/2. We observed that local ribosome occupancy is altered in the viral open reading frame with RPLP1/2 knockdown, consistent with a role for RPLP1/2 in promoting translation elongation. The most prominent ribosome pausing site in the DENV RNA was in the 5’ end of the E protein coding sequence which is located 210 nts downstream of two adjacent TMs. We also observed that RPLP1/2 depletion resulted in altered ribosome density in mRNAs encoding two or more transmembrane domains. This work increases our knowledge on DENV translation regulation and sheds light on the function of RPLP1/2 in translation of specific cellular RNAs.

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.

Epstein Barr virus (EBV) is a gamma-herpesvirus that latently infects the majority of adults worldwide. EBV infection, and particularly lytic replication, is linked to various cancers and autoimmune disorders. For example, endemic Burkitt lymphoma (BL) is an EBV-associated B cell lymphoma that primarily affects children in malaria endemic regions of Sub-Saharan Africa. In this dissertation, I characterize new EBV strains derived from Kenyan BL patient tumors isolated by collaborators in Kisumu, Kenya. Like all herpesviruses, EBV prototypically maintains a tight latency in infected cells until reactivated by an external stimulus, such as B cell receptor crosslinking. However, Type 2 genetic variants of EBV are known to spontaneously enter the lytic cycle. Surprisingly, a Type 1 (T1) BL-derived EBV strain described in this study also spontaneously reactivates and produces virions in culture. We extensively characterized this T1 spontaneous lytic phenotype using molecular and bioinformatic techniques and identified viral and host factors that contribute to the phenotype. Specifically, I determined that a variant of the viral lytic transcription factor RTA increases lytic reactivation, that this lytic phenotype is further enhanced by the virus maintaining a latency III state, and that the T1 spontaneous lytic phenotype is favored in differentiated, plasma-like cells. Overall, the results of this research increase our appreciation of EBV phenotypic diversity and support the possibility that EBV lytic replication is an important factor in EBV associated diseases.

Epstein-Barr virus latently infects over 90% of the adult global population. While it generally results in a completely asymptomatic lifelong infection, EBV is associated with and the causative agent of several human malignancies, particularly in cases of immune suppression. There is extensive interplay between the virus and normal host factors and processes that are critical for maintenance of a successful latent infection. We sought to expand upon the current knowledge of this interplay in three areas: i) appropriation of the Notch signaling pathway via the host factor RBPJ, ii) global regulation of host gene expression and mRNA isoform choice, and iii) host factor regulation of the viral latent/lytic switch. Here we undertook biochemical and structural approaches to investigate the protein-protein interactions between viral latency-associated proteins and RBPJ. This revealed new information differentiating the surfaces of RBPJ bound by viral and Notch signaling proteins. Furthermore we utilized a novel algorithm with microarray data prior to infection and after establishment of latency in B cells to study host mRNA changes associated with latent infection. Here we identified transcription and splicing factors to be uniquely regulated by EBV at the level of mRNA isoform. Finally we identified two host factors that regulate the switch between latent and lytic replication based on their EBV-directed isoform usage.