EBNA2-deleted Epstein-Barr virus (EBV) isolate, P3HR1, causes Hodgkin-like lymphomas and diffuse large B cell lymphomas with type II and Wp-restricted latency types in humanized mice

Abstract

EBV transforms B cells in vitro and causes 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 and is essential for EBV's ability to transform B cells in vitro. However, EBNA2 is not expressed in EBV-infected CHLs and BLs in humans. EBV-positive CHLs have type II latency and are largely driven by the EBV LMP1/LMP2A proteins, while EBV-positive BLs, which usually have type I latency are largely driven by c-Myc translocations, and only express the EBNA1 protein and viral noncoding RNAs. Approximately 15% of human BLs contain naturally occurring EBNA2-deleted viruses that support a form of viral latency known as Wp-restricted (expressing the EBNA-LP, EBNA3A/3B/3C, EBNA1 and BHRF1 proteins), but whether Wp-restricted latency and/ or EBNA2-deleted EBV can induce lymphomas in humanized mice, or in the absence of c-Myc translocations, is unknown. Here we show that a naturally occurring EBNA2-deleted EBV strain (P3HR1) isolated from a human BL induces EBV-positive B-cell lymphomas in a subset of infected cord blood-humanized (CBH) mice. Furthermore, we find that P3HR1infected lymphoma cells support two different viral latency types and phenotypes that are mutually exclusive: 1) Large (often multinucleated), CD30-positive, CD45-negative cells reminiscent of the Reed-Sternberg (RS) cells in CHL that express high levels of LMP1 but not EBNA-LP (consistent with type II viral latency); and 2) smaller monomorphic CD30-negative DLBCL-like cells that express EBNA-LP and EBNA3A but not LMP1 (consistent with Wp-restricted latency). These results reveal that EBNA2 is not absolutely required for EBV

Molecular Pathogenesis of Hodgkin Lymphoma: Past, Present, Future

“…In our experimental design, LMP1 induced TRF2 down-regulation was the key factor to lead to multi-nucleation [ 68 ]. Substantial experimental support of our findings was recently published by the group of Shannon C Kenney [ 69 ]. They demonstrated the formation of cHL-like tumors containing multi-nucleated RS-like cells with high LMP1 expression in a cord blood humanized mouse model infected with the EBNA2-deleted EBV strain P3HR1.…”

“…As the 3D cancer cell nucleus is a dynamic structure whose end-stage alterations are mostly identifiable [ 117 ] it is mandatory to detect and to analyze the very early changes on the road to malignancy with recently developed super-resolution microscopic techniques [ 118 ], humanized mouse model systems [ 69 ], and mass cytometry of nuclear structural proteins [ 106 ].…”

“…A “crippled” GC B-lymphocyte [ 24 ] or an EBV+ memory B-lymphocyte [ 25 ] re-entering GC reaction undergoes LMP1 oncoprotein induced transformation targeting the telomere-shelterin complex through a “pincer attack”. NF-kB driven mitosis [ 37 ] leading to telomere shortening and TRF2 down-regulation [ 60 , 68 ] resulting in telomere de-protection, both in concert generate short de-protected telomeres, prone to undergo sister chromatid fusion as the origin of repeated BBF-cycles, resulting first in H-cells [ 60 ], and, after several subsequent mitotic cycles, in the end-stage multi-faceted, often telomere poor RS-cells [ 60 , 61 , 62 , 67 , 68 , 69 ] The telomere-shelterin complex appears to emerge as a logical candidate for a common pathogenic denominator of both EBV-positive and EBV-negative cHL because LMP1 alone induces multi-nuclearity also in the 293 embryonic kidney cell line [ 38 ] and the myelomonocytic HD-MyZ cell line [ 30 ].…”

Modification of EBV Associated Lymphomagenesis and Its Immune Control by Co-Infections and Genetics in Humanized Mice

“…As a result, the in humans more prominent HL or BL with restricted latent gene expression cannot currently be modelled in humanized mice and therefore such models still have to be developed. Along these lines infection with EBNA2 deficient EBV has recently been reported to cause lymphomas with some HL characteristics and might be further explored to gain insights into this EBV associated malignancy ( 93 ). With further developments of humanized mice in the upcoming years we may be able to reveal even minor host and viral genetic as well as host immune factors that contribute to control of EBV infection.…”

Immune Escape by Non-coding RNAs of the Epstein Barr Virus

“…In germinal center B cells only EBNA1, LMP1 and 2, EBERs and BART miRNAs are expressed (latency IIa). This latency can, however, be also reached without prior EBNA2 dependent latency IIb and III in vivo ( Li et al, 2020 ). Latency IIa rescues infected cells from the germinal center reaction for persistence in memory B cells with only EBER and BART miRNA expression (latency 0) or additional EBNA1 expression during homeostatic proliferation (latency I) ( Babcock et al, 1998 ; Hochberg et al, 2004 ).…”