BioProject

Epstein-Barr virus (EBV) is an etiologic risk factor, and likely prerequisite, for the development of multiple sclerosis (MS). More...

Epstein-Barr virus (EBV) is an etiologic risk factor, and likely prerequisite, for the development of multiple sclerosis (MS). However, the role of EBV infected B cells in the immunopathology of MS is not well understood. Here, we characterized spontaneous lymphoblastoid cell lines (SLCLs) isolated from MS patients and healthy controls (HC) ex vivo to study EBV and host gene expression in the context of an individual’s endogenous EBV. SLCLs derived from MS patient B cells during active disease had higher EBV lytic gene expression than SLCLs from MS patients with stable disease or HCs. Host gene expression analysis revealed activation of pathways associated with hypercytokinemia and interferon signaling in MS SLCLs and differential expression of several genes, including upregulation of forkhead box protein 1 (FOXP1), which contributes to EBV lytic gene expression. In addition, we demonstrate that antiviral approaches targeting EBV replication decreased inflammatory cytokine production and diminished autologous CD4+ T cell responses. These data suggest that dysregulation of intrinsic B-cell control of EBV gene expression drives a pro-inflammatory, pathogenic B cell phenotype that can be attenuated by suppressing EBV lytic gene expression. Overall design: Genomic DNA was extracted from spontaneous lymphoblastoid cell lines derived from MS patients (n=7) and healthy controls (n=2). DNA was extracted from SLCLs using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). The Illumina DNA preparation kit was used for sequencing library preparation according to the manufacturer’s instructions. Sequencing was done with an Illumina NextSeq 500 system in high-output mode to generate a total of ∼4 × 108 paired-end 75-bp reads. Fastq reads were aligned against the EBV genome (accession NC_007605.1) using the BWA algorithm. Analysis of heterogeneous sites was performed following the procedure described by Čejková et al.. The consensus EBV genome for each sample was extracted using samtools59. Circus plots were generated using the R BioCircos package. Proteins were translated from nucleotide sequences using Geneious prime 2022.2 (https://www.geneious.com). Nucleotide and protein sequences were aligned using MAFFT v7.407 and visualized using Geneious prime 2022.2. Reference EBNA1 regions, in addition to the sequence from NC_007605.1, were obtained by querying each SLCL-derived sequence against nr using tblastn and taking differentially occurring sequences between matches for the three groups of samples (HC, AMS, SMS). One representative for each of three groups of identical sequences was kept. A phylogenetic tree of the 13 resulting sequences (9 samples, 4 reference) was constructed using PhyML with default parameters and visualized using iTOL. Less...