Binding of Epstein Barr Virus EBNA2 Unifies Multiple Sclerosis Genetic Mechanisms

多发性硬化症（MS）是一种中枢神经系统的毁灭性疾病，影响着超过230万人

Multiple sclerosis (MS) is a devastating disease of the central nervous system that affects over 2.3 million people worldwide. Current therapies for MS are at best only partially effective, and there is no cure. Improved understanding of MS disease mechanisms would lead to better diagnosis, treatment, and prevention. MS is caused by both genetic and environmental risk factors. Epstein-Barr virus (EBV) is the most consistently replicated environmental factor. Mounting evidence indicates that EBV-infected B cells, and their downstream immunological effects, are key drivers of MS disease processes. Recent work by our group and others implicates the EBV-encoded EBNA2 gene regulatory protein in mechanisms at almost half of the established MS genetic risk loci. In this proposal, we will test the hypothesis that EBNA2-driven allele-dependent alterations to human gene expression lead to distinct B and T cell phenotypes directly contributing to disease processes in MS. Aim 1. Quantification of EBV-specific human gene expression in multiple MS patient demographics. We will expand our cohort to include males and females of European, African, and Asian ancestries. We will measure MS- and EBV-specific human gene expression in these cohorts. Aim 2. Global discovery of EBV- and MS genotype-dependent gene regulatory mechanisms. We will use Massively Parallel Reporter Assays (MPRAs) to systematically identify EBV- and risk allele-dependent gene expression at all MS risk variants in primary B cells with and without EBV infection. We will identify human transcriptional regulators acting with EBNA2, EBNA3C, and/or EBNA-LP at these loci and confirm their allele- dependent actions in MS-derived B cells using cutting-edge functional genomics technologies. We will validate these EBV- and genotype-dependent gene regulatory mechanisms using CRISPR-based genome editing of patient-derived primary B cells at CD37, CD58, ZMIZ1, and other MS risk loci. Aim 3. Discovery of allelic EBV-based mechanisms altering cellular behavior in MS. We will gauge the necessity and sufficiency of EBV and specific MS variants (e.g: CD37, CD58, ZMIZ1) on B cell function using the CRISPR-edited B cells. We will measure impact on B cell receptor signaling, cytokine production, proliferation, and apoptosis. We will use an inducible pluripotent stem cell-derived blood brain barrier (BBB) endothelium model to measure the impact of EBV and MS risk alleles on B cell - BBB interactions. The concept that disease processes might be influenced by virus-controlled, allelic regulatory protein complexes is highly innovative and has never before been demonstrated. Our work for the first time provides mechanistic insight into the established role of EBV in MS through a unified gene by environment model. Comprehensive cataloging, dissection, and understanding of the downstream effects of genetic mechanisms impacted by EBV will be significant because it will provide strong rationale to develop therapies that interfere with these mechanisms, or even vaccines that prevent EBV infection to cure MS and other EBV-related diseases.