Fig 1.
A subset of P3HR1 strain EBV-infected CBH mice develop tumors.
Kaplan-Meier analysis was performed comparing tumor development over time (tumor incidence curve) in CBH mice infected with P3HR1 (EBNA2-deleted) virus versus B95.8 (WT, EBNA2-sufficient) virus. B95.8 data was previously published [44]. P3HR1 infected mice were infected with a 25- to 50-fold higher dose of virus compared to B95.8 infected mice as described in the methods. Log-Rank test was performed on tumor incidence curves.
Fig 2.
P3HR1 virus induces invasive B cell lymphomas.
H&E staining was performed on P3HR1 virus, B95.8 virus or AG876 virus induced lymphomas (each invading the liver) as shown. In addition, IHC analysis was performed using antibodies against CD20 (B cell marker), IRF4 (marker of activated B cells), EBNA1 (EBV latency protein expressed in all EBV-infected cells) and EBNA2 (EBV latency protein expressed in cells with type III latency that is deleted in the P3HR1 virus). The P3HR1 tumor shown is from mouse #6 in S1 Table.
Table 1.
Summary of P3HR1 tumor morphologies.
Fig 3.
P3HR1 infected lymphomas contain foci with Wp-restricted latency as well as foci with type II latency.
IHC analysis using antibodies against EBNA-LP (expressed in type III latency and Wp-restricted latency) or LMP1 (expressed in type II and type III latency) was performed as indicated in tumors infected with either the P3HR1 virus, B95.8 virus or AG876 virus. P3HR1 “1” is from mouse #3 and P3HR1 “2” is from mouse #6 in S1 Table.
Fig 4.
LMP1 expression and EBNA-LP expression are mutually exclusive in P3HR1 infected lymphomas in CBH mice, and the type 1/II latency EBNA1 Qp promoter is used in P3HR1 tumors.
A. IHC co-staining was performed using an antibody against EBNA-LP (dark blue staining) and an antibody against LMP1 (brown staining) in lymphomas infected with P3HR1 or B95.8 viruses. Examples of cells that express only EBNA-LP are indicated by blue arrows, and examples of cells expressing only LMP1 by brown arrows. Co-staining cells are indicated by red arrows. The P3HR1 tumor shown in from mouse #2 in S1 Table. B. qPCR analysis of RNA isolated from P3HR1 and B95.8 EBV-induced lymphomas was performed using primers that recognize EBNA1 derived from the Qp promoter (type I/type II latency specific) (left panel); qPCR analysis of RNA isolated from BL control cell lines (Kem I and Kem III) with type I and type III latency, respectively, and EBV-negative Akata BL cells served as positive and negative controls in these assays (right panel).
Fig 5.
P3HR1-infected lymphoma cells with high level LMP1 expression also have high level CD30.
IHC analysis of P3HR1 infected, AG876 infected or B95.8 virus infected lymphomas were performed using antibodies that detect LMP1, EBNA-LP or CD30 as indicated. P3HR1 “1” and P3HR1 “2” are derived from geographically separated tumor foci in the kidney of mouse #2 in S1 Table.
Fig 6.
LMP1-high/EBNA-LP-low cells in P3HR1 virus infected lymphomas resemble RS cells of CHLs.
A. H & E staining was performed on P3HR1 virus infected lymphomas expressing high level LMP1/low level EBNA-LP (left panel: mouse #2; middle panel: mouse #7 in S1 Table), or low level LMP1/high level EBNA-LP (right panel: mouse #2 in S1 Table) as indicated at low power (20X) or high power (100X). B. LMP1 staining was also performed to show that RS-like cells in the LMP1 high/EBNA-LP-low tumor foci express LMP1 (mouse #6 in S1 Table). C. CD45 staining of LMP1-high/EBNA-LP-low tumor regions shows loss of CD45 expression on large RS cells (black arrows) but continued CD45 expression on smaller cells (mouse #2 in S1 Table).
Fig 7.
P3HR1-infected lymphoma cells express high level of the BZLF1 lytic viral protein in EBNA-LP-high/LMP1-low regions of tumors.
IHC analysis of P3HR1 infected lymphomas was performed using antibodies that detect BZLF1 (lytic EBV protein) and CD20 (B cell marker). Both P3HR1 sections were derived from different tumor foci in the same kidney of mouse #2 in S1 Table.
Fig 8.
P3HR1 infected lymphomas express both Wp-restricted and type II viral latency proteins and the immediate-early lytic protein BZLF1.
A. Immunoblot analysis of proteins isolated from P3HR1 infected, AG876 infected or B95.8 virus infected lymphomas was performed using antibodies against EBNA1, EBNA2, EBNA3A (EBV latency protein expressed in type III and Wp-restricted latency), LMP1 (EBV latency protein expressed in type II and III latency), LMP2A (EBV latency protein expressed in type II and type III latency), lytic protein BZLF1 or alpha tubulin (loading control). P3HR1 “1” protein is isolated from mouse #1 and P3HR1 “2” protein isolated from mouse #2 in S1 Table. EBNA-LP protein as expected can be expressed in multiple different sizes, and EBNA2 protein as expected is larger in type 1 EBV B95.8 infected tumors compared to type 2 EBV AG876 infected tumors. B. IHC co-staining studies were performed using antibodies that detect LMP1 or BZLF1 in a P3HR1 infected tumor (mouse #2 in S1 Table). No co-staining was observed. Blue arrow shows an example of an EBNA-LP staining cell, and brown arrow shows an example of an LMP1 positive cell. C. IHC co-staining studies were performed on a P3HR1 infected tumor using antibodies that detect BZLF1 or CD138. (mouse #2 in S1 Table). Blue arrow shows an example of a BZLF1 only staining cell, brown arrow shows an example of a CD138 only staining cell, and red arrows show examples of BZLF1/CD138 co-staining cells.
Fig 9.
P3HR1 virus-infected tumors express a complex mix of latent and lytic genes and are oligoclonal.
A. RNA-seq reads were mapped to the EBV genomes of 2 different tumors infected with P3HR1 virus (top panel), or two different tumors infected with B95.8 virus (middle panel). The locations of the Cp and the EBER, EBNA-LP, EBNA2, BHRF1, EBNA3, BZLF1 (Z), BRLF1 (R), EBNA1, LMP1 and LMP2A transcripts are indicated above the EBV genome map. P3HR1 “1” tumor is derived from mouse #6, and P3HR1 “2” tumor derived from mouse #7 in S1 Table. B. RNA-seq analysis was performed using RNA isolated from 2 different P3HR1 infected tumors shown in top panel in A. The relative frequency of the IGH transcripts containing various different IGHV genes is shown for each tumor. C. The frequency of the T cell receptor TRBV gene reads in the RNA-seq analysis of each P3HR1 tumor was determined by comparing the number of reads from each individual TRBV gene to the total number of TRBV gene reads.
Fig 10.
P3HR1 infected lymphomas express cellular genes upregulated in RS cells of Hodgkin lymphomas.
RNA was isolated from tumors infected with B95.8 or P3HR1 virus-induced 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. A. The relative levels of cellular gene transcripts in P3HR1-infected tumors, versus B95.8-infected tumors, are shown for known EBNA2 target genes (yellow shading), immunosuppressive factors which are highly expressed in RS cells and are known LMP1 targets (gray shading), markers for helper T cells and T cell exhaustion (blue shading), and markers for enhanced PDGF signaling (which is often increased in HL tumors)(green shading). The fold-change in cellular gene expression in P3HR1-infected tumors versus B95.8-infected tumors is indicated, as well as the p-value for each difference. B. qPCR was performed using cDNA isolated from two different P3HR1 virus-induced lymphomas versus two different B95.8 virus-infected lymphomas, using human specific primers to amplify genes. Results were normalized to the level of GAPDH transcript. Standard error is shown. C. IHC analysis using antibodies against PDGFRA or CD137 was performed in areas of tumors with low or high level LMP1 expression. Both P3HR1 tumor sections shown are derived from mouse #2 (S1 Table).
Fig 11.
P3HR1 virus infected tumors are heavily infiltrated with CD4 positive T cells and show a T reg signature.
A. Gene set enrichment analysis (GSEA) plot for the (“Go_T_cell receptor _complex”) and the “Gavin_foxp3-targets cluster _P4” gene set are shown in P3HR1 virus-induced lymphomas compared to B95.8 virus-infected lymphomas. B. IHC analysis using antibodies against CD20, CD3 or CD4 is shown in tumors infected with P3HR1 (mouse # 1 in S1 Table) or B95.8 viruses as indicated.
Fig 12.
P3HR1 virus infected tumors express more IgG in comparison to AG876 and B95.8 virus infected tumors.
A. The relative level of IGHM and various IGHG gene transcripts (as determined by RNAseq analysis) in P3HR1 virus-infected tumors, versus B95.8 virus-infected tumors is shown. Values are represented as PRKMs. B. Examples of IHC analysis using antibodies against IgM or IgG performed in tumors infected with P3HR1, AG876 or B95.8 viruses are shown. The P3HR1 tumor shown is derived from mouse #1 in S1 Table.
Fig 13.
P3HR1 virus infected tumors have a gene signature pattern suggestive of EMT and are infiltrated with collagen.
A. A gene set enrichment analysis (GSEA) plot for the “Hallmark_epithelial-mesenchymal_transition” and “Go extracellular matrix component” gene set are shown in P3HR1 virus-induced lymphomas compared to B95.8 virus-infected lymphomas. Normalized enrichment score (NES) and FDR q-value are shown to indicate strength of enrichment. A NES >1.6 and q-value < 0.05 were considered enriched. B. Expression levels of various human collagen genes and EMT markers (obtained from RNAseq data), along with the fold-upregulation in P3HR1 virus–infected versus B95.8 virus-infected cells (and the associated p-value) are shown. C. Masson’s trichrome staining (to detect collagen fibers in blue) and CD20 IHC staining (to detect lymphoma cells) was performed in a P3HR1 virus infected tumor derived from mouse #6 in S1 Table.
Table 2.
Antibodies used for immunohistochemistry (IHC) and western blot (WB).
Table 3.
Primers for qPCR analysis.