RNA-seq libraries were prepared using the TruSeq Stranded mRNA Prep kit (illumina)

RNA-seq libraries were prepared using the TruSeq Stranded mRNA Prep kit (illumina). detect EBNA1 or CD3 (mouse #7 in S1 Table). Blue arrow shows an example of a cell staining for CD3, and brown arrow shows an example of an EBNA1 staining cell.(TIF) ppat.1008590.s002.tif (3.8M) GUID:?21D9AB64-A4D9-4FC4-B6E1-910BB9C2FA30 S3 Fig: P3HR1 infected tumors contain RS-like cells. H & E staining of P3HR1 infected tumor (derived from mouse #7 in S1 Table) is shown. A RS-like cell is indicated BMS-582949 by black arrow.(TIF) ppat.1008590.s003.tif (1.9M) GUID:?E0CAC74E-CE56-481A-990D-7AACF9236179 S4 Fig: P3HR1 infected RS-like cells have variable expression of CD20. IHC staining of a P3HR1 infected tumor derived from mouse # 2# 2 in S1 Table was performed using a CD20 antibody. Examples of RS-like cells BMS-582949 with high level CD20 staining (red arrows), medium staining (yellow arrows) and negative CD20 staining (white arrows) are shown.(TIF) ppat.1008590.s004.tif (2.4M) GUID:?D1892AC9-D9B3-46BD-8BB2-7D09F862A804 S5 Fig: P3HR1 and B95.8 infected lymphomas contain differentially expressed cellular genes. RNA was isolated from tumors infected with B95.8 or P3HR1 virus infected lymphomas, and RNA-seq performed. Mouse cell transcripts were removed from further analysis, and the levels of human genes in each tumor type was compared as described in the methods. The top 100 differentially expressed cellular genes in the RNA-seq analysis are shown above. The B95.8 and P3HR1 virus-induced lymphomas cluster together in Ebf1 a distinct pattern.(PDF) ppat.1008590.s005.pdf (81K) GUID:?3920F5C6-ECF6-44E2-BF75-BFFDC4F7470D S6 Fig: P3HR1 infected lymphomas express variable levels of c-Myc. Immunoblot analysis of proteins isolated from P3HR1 infected, AG876 infected or B95.8 virus infected lymphomas were performed using antibodies against c-Myc or tubulin as indicated. P3HR1 1 protein is isolated from mouse #1 and P3HR1 2 protein isolated from mouse #2 in S1 Table.(TIF) ppat.1008590.s006.tif (280K) GUID:?36BC9876-AEF0-47B2-9B65-290DDD4D6711 S1 Table: P3HR1 virus source and dose in each infected mouse. (PDF) ppat.1008590.s007.pdf (11K) GUID:?5DAC5B29-E489-4489-865B-65A727CE0FBA S2 Table: Gene profiles in listed GSEA plots. A. Go_T_cell receptor _complex. B. Go_T_cell receptor _complex. C. Hallmark_epithelial-mesenchymal_transition. D. Go extracellular matrix BMS-582949 component.(XLSX) ppat.1008590.s008.xlsx (59K) GUID:?0D23CCA5-C199-4B0B-A893-26C1E7A83D9E Data Availability StatementThe P3HR1 RNA-seq data reported in this manuscript has been deposited to the SRA database with the BioProject accession PRJNA622980. The WT B95.8 RNAseq data was previously published and can be found in the GEO database under the accession number GSE113070 (EBNA3C reference). All other data is contained within this manuscript and the supplemental information. Abstract EBV transforms B cells and causes BMS-582949 human B-cell lymphomas including classical Hodgkin lymphoma (CHL), Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). The EBV latency protein, EBNA2, transcriptionally activates the promoters of all latent viral protein-coding genes expressed in type III EBV latency BMS-582949 and is essential for EBVs ability to transform B cells into immortalized lymphoblastoid cell lines (LCLs). EBV-associated B-cell lymphomas include diffuse large B-cell lymphomas (DLBCLs), Burkitt lymphomas (BLs), classical Hodgkin lymphomas (CHLs), plasmablastic lymphomas, primary effusion lymphomas, primary CNS lymphomas, and a spectrum of post-transplant lymphoproliferative disorders including marginal zone lymphoma [1C5]. EBV can infect cells in either lytic or latent forms of infection. The lytic form of infection is required for production of infectious viral particles and horizontal transmission of the virus from cell to cell (and host to host), while the latent forms of infection allow the virus to persist long-term in memory B cells and evade the host immune response. However, there are several different gene expression patterns observed in latent EBV infection (commonly referred to as type I, type II and type III) that differ in regard to the number of viral proteins expressed, whether the virus can transform B cells or model systems for deriving stably EBV-transformed B cells that have type II latency in the context of the intact viral genome, although combined transgenic expression of both LMP1 and LMP2A in mouse germinal center cells induces massive plasmablast proliferation when T cells and NK cells are depleted [17]. All human EBV-positive BLs have either type I or Wp-restricted EBV latency. Type I latency (in which EBNA1 is the only protein expressed, along with virally-encoded miRNAs and EBERs) occurs when the two potential EBNA2 promoters (Cp and Wp) become methylated through poorly understood mechanisms, and EBNA1 expression continues using the Qp [6,7]. In Wp-restricted latency, which occurs in approximately 15% of human Burkitt lymphomas, the EBNA2 gene is deleted in a stereotypical manner that allows the latent W promoter (Wp) to drive expression of the EBNA1, EBNA-LP, EBNA3A/B/C and BHRF1 genes, resulting in a greater number of EBV proteins expressed relative to type I latency [18C22]. Both type I and Wp-restricted latency are completely unable to transform B cells compared to BL cells line with type I latency [20], suggesting that viral proteins specifically expressed in cells with Wp-restricted latency.