Epstein Barr virus genomes reveal population structure and type 1 association with endemic Burkitt lymphoma [preprint]

Endemic Burkitt lymphoma (eBL), the most prevalent pediatric cancer in sub-Saharan Africa, is associated with malaria and Epstein Barr virus (EBV). In order to better understand the role of EBV in eBL, we improved viral DNA enrichment methods and generated a total of 98 new EBV genomes from both eBL cases (N=58) and healthy controls (N=40) residing in the same geographic region in Kenya. Comparing cases and controls, we found that EBV type 1 was significantly associated with eBL with 74.5% of patients (41/55) versus 47.5% of healthy children (19/40) carrying type 1 (OR=3.24, 95% CI=1.36 - 7.71, P=0.007). Controlling for EBV type, we also performed a genome-wide association study identifying 6 nonsynonymous variants in the genes EBNA1, EBNA2, BcLF1, and BARF1 that were enriched in eBL patients. Additionally, we observed that viruses isolated from plasma of eBL patients were identical to their tumor counterpart consistent with circulating viral DNA originating from the tumor. We also detected three intertypic recombinants carrying type 1 EBNA2 and type 2 EBNA3 regions as well as one novel genome with a 20 kb deletion resulting in the loss of multiple lytic and virion genes. Comparing EBV types, genes show differential variation rates as type 1 appears to be more divergent. Besides, type 2 demonstrates novel substructures. Overall, our findings address the complexities of EBV population structure and provide new insight into viral variation, which has the potential to influence eBL oncogenesis

Characterization of Epstein Barr Virus Latency Pattern in Argentine Breast Carcinoma

INTRODUCTION: Epstein-Barr virus (EBV)-associated tumors show different expression patterns of latency genes. Since in breast carcinoma this pattern is not yet fully described, our aim was to characterize EBV latency pattern in our EBV positive breast carcinoma series. METHODS: The study was conducted on 71 biopsies of breast carcinoma and in 48 non-neoplastic breast controls. EBNA1, LMP2A and LMP1 expression was assessed by immunohistochemistry with monoclonal antibodies, while viral genomic DNA and EBERs RNA transcripts expression was performed by in situ hybridization. EBV presence was confirmed by PCR. RESULTS: EBV genomic DNA and EBNA1 expression were detected in 31% (22/71) of patients specifically restricted to tumor epithelial cells in breast carcinoma while all breast control samples were negative for both viral DNA and EBNA1 protein. LMP2A was detected in 73% of EBNA1 positive cases, none of which expressed either LMP1 protein or EBERs transcripts. CONCLUSIONS: These findings suggest that EBV expression pattern in the studied biopsies could be different from those previously observed in breast carcinoma cell lines and lead us to suggest a new, EBNA1, LMP2A positive and LMP1 and EBERs negative latency profile in breast carcinoma in our population

Single-cell RNA-seq reveals transcriptomic heterogeneity mediated by host-pathogen dynamics in lymphoblastoid cell lines

Lymphoblastoid cell lines (LCLs) are generated by transforming primary B cells with Epstein-Barr virus (EBV) and are used extensively as model systems in viral oncology, immunology, and human genetics research. In this study, we characterized single-cell transcriptomic profiles of five LCLs and present a simple discrete-time simulation to explore the influence of stochasticity on LCL clonal evolution. Single-cell RNA sequencing (scRNA-seq) revealed substantial phenotypic heterogeneity within and across LCLs with respect to immunoglobulin isotype; virus-modulated host pathways involved in survival, activation, and differentiation; viral replication state; and oxidative stress. This heterogeneity is likely attributable to intrinsic variance in primary B cells and host-pathogen dynamics. Stochastic simulations demonstrate that initial primary cell heterogeneity, random sampling, time in culture, and even mild differences in phenotype-specific fitness can contribute substantially to dynamic diversity in populations of nominally clonal cells

Endemic Burkitt lymphoma avatar mouse models for exploring inter-patient tumor variation and testing targeted therapies

Endemic Burkitt lymphoma (BL) is a childhood cancer in sub-Saharan Africa characterized by Epstein-Barr virus and malaria-associated aberrant B-cell activation and MYC chromosomal translocation. Survival rates hover at 50% after conventional chemotherapies; therefore, clinically relevant models are necessary to test additional therapies. Hence, we established five patient-derived BL tumor cell lines and corresponding NSG-BL avatar mouse models. Transcriptomics confirmed that our BL lines maintained fidelity from patient tumors to NSG-BL tumors. However, we found significant variation in tumor growth and survival among NSG-BL avatars and in Epstein-Barr virus protein expression patterns. We tested rituximab responsiveness and found one NSG-BL model exhibiting direct sensitivity, characterized by apoptotic gene expression counterbalanced by unfolded protein response and mTOR pro-survival pathways. In rituximab-unresponsive tumors, we observed an IFN-α signature confirmed by the expression of IRF7 and ISG15. Our results demonstrate significant inter-patient tumor variation and heterogeneity, and that contemporary patient-derived BL cell lines and NSG-BL avatars are feasible tools to guide new therapeutic strategies and improve outcomes for these children

Epstein-Barr virus ensures B cell survival by uniquely modulating apoptosis at early and late times after infection

Abstract Latent Epstein-Barr virus (EBV) infection is causally linked to several human cancers. EBV expresses viral oncogenes that promote cell growth and inhibit the apoptotic response to uncontrolled proliferation. The EBV oncoprotein LMP1 constitutively activates NFkB and is critical for survival of EBV-immortalized B cells. However, during early infection EBV induces rapid B cell proliferation with low levels of LMP1 and little apoptosis. Therefore, we sought to define the mechanism of survival in the absence of LMP1/NFkB early after infection. We used BH3 profiling to query mitochondrial regulation of apoptosis and defined a transition from uninfected B cells (BCL-2) to early-infected (MCL-1/BCL-2) and immortalized cells (BFL-1). This dynamic change in B cell survival mechanisms is unique to virus-infected cells and relies on regulation of MCL-1 mitochondrial localization and BFL-1 transcription by the viral EBNA3A protein. This study defines a new role for EBNA3A in the suppression of apoptosis with implications for EBV lymphomagenesis

Upregulation of the cell-cycle regulator RGC-32 in Epstein-Barr virus-immortalized cells

Epstein-Barr virus (EBV) is implicated in the pathogenesis of multiple human tumours of lymphoid and epithelial origin. The virus infects and immortalizes B cells establishing a persistent latent infection characterized by varying patterns of EBV latent gene expression (latency 0, I, II and III). The CDK1 activator, Response Gene to Complement-32 (RGC-32, C13ORF15), is overexpressed in colon, breast and ovarian cancer tissues and we have detected selective high-level RGC-32 protein expression in EBV-immortalized latency III cells. Significantly, we show that overexpression of RGC-32 in B cells is sufficient to disrupt G2 cell-cycle arrest consistent with activation of CDK1, implicating RGC-32 in the EBV transformation process. Surprisingly, RGC-32 mRNA is expressed at high levels in latency I Burkitt's lymphoma (BL) cells and in some EBV-negative BL cell-lines, although RGC-32 protein expression is not detectable. We show that RGC-32 mRNA expression is elevated in latency I cells due to transcriptional activation by high levels of the differentially expressed RUNX1c transcription factor. We found that proteosomal degradation or blocked cytoplasmic export of the RGC-32 message were not responsible for the lack of RGC-32 protein expression in latency I cells. Significantly, analysis of the ribosomal association of the RGC-32 mRNA in latency I and latency III cells revealed that RGC-32 transcripts were associated with multiple ribosomes in both cell-types implicating post-initiation translational repression mechanisms in the block to RGC-32 protein production in latency I cells. In summary, our results are the first to demonstrate RGC-32 protein upregulation in cells transformed by a human tumour virus and to identify post-initiation translational mechanisms as an expression control point for this key cell-cycle regulator

The genetic landscape of immune-competent and HIV lymphoma

This journal supplement is Proceedings of the 13th International Conference on Malignancies in AIDS and Other Acquired Immunodeficiencies (ICMAOI)Open Access JournalBurkitt lymphoma (BL) and diffuse large B cell lymphoma (DLBCL) are aggressive forms of lymphoma in adults and demonstrate overlapping morphology, immunophenotype and clinical behavior. The risk of developing these tumors increases ten to hundred-fold in the setting of HIV infection. The genetic causes and the role of specific mutations, especially in the setting of HIV, are largely unknown. The decoding of the human genome and the advent of high-throughput sequencing have provided rich opportunities for the comprehensive identification of the genetic causes of cancer. In order to comprehensively identify genes that are recurrently mutated in immune-competent DLBCL and BL, we obtained a total of 92 cases of DLBCLs and 40 cases of BL. These cases were compared to a set of 5 DLBCLs and BL tumors derived from patients with HIV. The DLBCL cases were divided into a discovery set (N=34) and …link_to_OA_fulltextThe 13th International Conference on Malignancies in AIDS and Other Acquired Immunodeficiencies (ICAMAOI), Bethesda, MD., 7-8 November 2011. In Infectious Agents and Cancer, 2011, v. 7 suppl. 1, article no. O

Rapamycin Blocks Production of KSHV/HHV8: Insights into the Anti-Tumor Activity of an Immunosuppressant Drug

Infection with Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) often results in the development of fatal tumors in immunocompromised patients. Studies of renal transplant recipients show that use of the immunosuppressant drug rapamycin, an mTOR inhibitor, both prevents and can induce the regression of Kaposi's sarcoma (KS), an opportunistic tumor that arises within a subset of this infected population. In light of rapamycin's marked anti-KS activity, we tested whether the drug might directly inhibit the KSHV life cycle. We focused on the molecular switch that triggers this predominantly latent virus to enter the lytic (productive) replication phase, since earlier work links this transition to viral persistence and tumorigenesis.In latently infected human B cell lines, we found that rapamycin inhibited entry of the virus into the lytic replication cycle, marked by a loss of expression of the lytic switch protein, replication and transcription activator (RTA). To test for viral-specific effects of rapamycin, we focused our studies on a B cell line with resistance to rapamycin-mediated growth inhibition. Using this line, we found that the drug had minimal effect on cell cycle profiles, cellular proliferation, or the expression of other cellular or latent viral proteins, indicating that the RTA suppression was not a result of global cellular dysregulation. Finally, treatment with rapamycin blocked the production of progeny virions.These results indicate that mTOR plays a role in the regulation of RTA expression and, therefore, KSHV production, providing a potential molecular explanation for the marked clinical success of rapamycin in the treatment and prevention of post-transplant Kaposi's sarcoma. The striking inhibition of rapamycin on KSHV lytic replication, thus, helps explain the apparent paradox of an immunosuppressant drug suppressing the pathogenesis of an opportunistic viral infection