The gamma-herpesvirus Epstein Barr virus (EBV) is a master regulator of B-cell biology. EBV persistently infects >95% of adults, making it one of the most successful viruses worldwide. While EBV typically establishes a safe balance with the host, it is the cause of infectious mononucleosis, is associated with multiple sclerosis and causes ~200,000 cancers per year. Intriguingly, these include B cell, T cell, NK cell lymphomas and the epithelial cell cancers gastric and nasopharyngeal carcinoma. Studies of EBV/host interactions promise to reveal key aspects of EBV pathogenesis and of B lymphocyte biology. Indeed, EBV-infected B-cells were the first human lymphocytes that could be grown in culture and remain a key source of insights into B-cell biology, including NF-kB, MYC and Notch biology.
Establishment of latency in a long-lived cellular niche and subsequent lytic reactivation in response to environmental cues is a hallmark of herpesvirus infection, yet much remains to be learned about how EBV accomplishes these important roles. According to the “germinal center model” of EBV biology, EBV subverts normal B-cell biology pathways operative in lymphoid tissues in order to establish infection, expand the pool of latently infected B-cells, persist and spread. Despite encoding ~80 antigenic polypeptides, EBV navigates the B-cell compartment to colonize memory B-cells, the site of long-term persistence.
To drive B-cell growth transformation while also evading robust antiviral T and NK- responses, EBV uses multiple latency programs, in which combinations of viral nuclear and membrane oncoproteins and non-coding RNAs are expressed, but highly immunogenic lytic antigens remain silenced. Knowledge remains incomplete about how epigenetic mechanisms control EBV genome program selection. It is likely that the same mechanisms underlie the pathogenesis of multiple germinal center-derived EBV-driven B-cell malignancies, including Burkitt, Hodgkin and post-transplant lymphomas. Yet, the host/pathogen interactions that dictate key EBV B-cell latency states have remained incompletely characterized. Likewise, large questions remain to be answered, such as why EBV drives Burkitt lymphoma in regions of holoendemic malaria in sub-Saharan Africa versus nasopharyngeal carcinoma in other regions. Since primary human B-cells can be obtained in large numbers from discarded samples, we have the unique opportunity to study host/virus interactions in newly infected primary human cells.
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We are associated with the Harvard Department of Microbiology and Harvard Graduate Program in Virology
https://www.hms.harvard.edu/dms/virology/
https://micro.hms.harvard.edu/
We are associated with the Broad Institute Genetics Perturbation Platform and Infectious Disease Microbiome platforms: https://www.broadinstitute.org/genetic-perturbation-platform https://www.broadinstitute.org/infectious-disease-microbiome
Current research includes the following areas:
CRISPR/Cas9 screens for synthetic lethal targets in EBV-transformed B-cells
We performed the first genome-wide CRISPR/Cas9 loss-of-function systematic genetic analysis for host B-cell factors critical for EBV-transformed B-cell growth and survival. Our studies have identified multiple druggable lymphoblastoid B-cell and Burkitt lymphoma targets. We are using CRISPR genetics to further define newly-identified B-cell dependencies, including screens for host epigenetic factors that regulate the EBV lytic switch or that regulate viral genome latency program selection. CRISPR editing of the EBV genome is being used to screen for viral genes that subvert key innate and adaptive immune pathways. We are also using CRISPR/Cas9 knockout screens as well as CRISPR-activation genome-wide screens to characterize other key aspects of the EBV lifecycle and EBV pathogenesis, including epigenetic control of latency programs and sensitivity to immune recognition.
Host Dependency Factors. Human Genome-wide CRISPR Screen for host factors selectively important for EBV-transformed Burkitt versus lymphoblastoid B-cell growth and survival revealed mechanisms by which EBV subverts apoptosis pathways.
The EBV Lytic Switch
A human Genome-wide CRISPR/Cas9 screen in EBV-infected Burkitt lymphoma cells, a model for latent EBV infection, identified MYC as a suppressor of the lytic switch. EBV highly induces MYC in newly infected B-cells, where it establishes latent infection. We speculate that EBV senses decreases in MYC abundance as a readout of B-cell state, as MYC is suppressed in plasma cell development by Blimp1, a trigger for EBV lytic reactivation.
The LCL and primary human B-cell NF-κB Genomic Binding Landscape
The EBV oncoprotein LMP1 mimics CD40 signaling to constitutively activate the canonical and non-canonical NF-κB pathways, each of which are critical for EBV-mediated B-cell transformation. Yet, little is known about NF-kB pathway-specific roles in EBV-transformed B-cells, or in primary human B-cells. We used ChIP-seq to identify the LCL NF-κB genomic binding landscape, providing the first analysis of all NF-κB transcription factor subunits in a human cell. Our analysis identified a fascinating but complex NF-κB genomic binding landscape. We are using CRISPR to further identify NF-κB subunit-specific roles in EBV-infected and uninfected human B-cells. To enable comparison of LMP1 and CD40 B-cell effects, we are using ChIP-seq to identify NF-κB pathway roles in ex vivo CD40-stimulated primary human B-cells.
Quantitative proteomic analysis of EBV B-cell lytic replication
EBV B-cell replication enables lifelong B-cell colonization and seeding of oropharyngeal epithelial cells. Yet, systematic proteomic or genetic analysis of EBV lytic replication has not been performed. In collaboration with Michael Weekes (University of Cambridge) and Steve Gygi (Harvard Medical school), we are using multiplexed tandem-mass spectrometry to identify B-cell proteome-wide and cell-surface remodeling upon EBV lytic reactivation.
Proteomic Analysis of EBV of primary human B-cell transformation
EBV transformation of a resting, short-lived human B-cell into an immortalized lymphoblast is one of the most fascinating, yet incompletely understood aspects of EBV biology. We are using proteomic, CRISPR and chemical genetic approaches to study key metabolic pathways exploited by EBV at distinct stages of B-cell transformation.
Novel human immunodeficiencies manifest by susceptibility to EBV and EBV-associated malignancy
Rare primary immunodeficiencies highlight mechanisms that control EBV and result in markedly elevated EBV loads, hematological disorders and B-cell cancers. We are using immunologic and whole exome approaches to identify and characterize novel primary human immunodeficiency diseases that result in the inability to control EBV infection and EBV-driven cancers. We are also investigating the X-linked lymphoproliferative disease 2 syndrome to characterize key aspects of the disease, including why these individuals have high EBV viral loads but do not get EBV-associated lymphomas.
EBV and Gastric Carcinoma
There are ~80,000 cases/year of EBV-associated gastric carcinoma, which have a range of features that distinguish them from other types of gastric cancer, suggesting a key EBV role. These include high frequency of gain-of-function PI3K mutation and extreme DNA hyper-methylation, the highest degree of CpG methylation of any human cancer. We are performing CRISPR analysis of key EBV-associated gastric carcinoma features and have begun a program-project with Paul Lieberman, Italo Tempera and others at the Wistar Institute.
SARS-CoV-2 host/pathogen interactions
We are using metabolomic and CRISPR Genetic approaches to study SARS-COV-2 host/pathogen interactions. In collaboration with the Mootha laboratory of Massachusetts General Hospital, we are performing metabolomic analyses of newly infected cells. CRISPR is being used to study host factors important for SARS-CoV-2 replication
