EBV persistence without its EBNA3A and 3C oncogenes in vivo

Abstract

The oncogenic Epstein Barr virus (EBV) infects the majority of the human population and usually persists within its host for life without symptoms. The EBV oncoproteins nuclear antigen 3A (EBNA3A) and 3C (EBNA3C) are required for B cell transformation in vitro and are expressed in EBV associated immunoblastic lymphomas in vivo. In order to address the necessity of EBNA3A and EBNA3C for persistent EBV infection in vivo, we infected NOD-scid γcnull mice with reconstituted human immune system components (huNSG mice) with recombinant EBV mutants devoid of EBNA3A or EBNA3C expression. These EBV mutants established latent infection in secondary lymphoid organs of infected huNSG mice for at least 3 months, but did not cause tumor formation. Low level viral persistence in the absence of EBNA3A or EBNA3C seemed to be supported primarily by proliferation with the expression of early latent EBV gene products transitioning into absent viral protein expression without elevated lytic replication. In vitro, EBNA3A and EBNA3C deficient EBV infected B cells could be rescued from apoptosis through CD40 stimulation, mimicking T cell help in secondary lymphoid tissues. Thus, even in the absence of the oncogenes EBNA3A and 3C, EBV can access a latent gene expression pattern that is reminiscent of EBV persistence in healthy virus carriers without prior expression of its whole growth transforming program.

In vivo analysis of γH2AX+ cells in skeletal muscle from aged and obese humans

“…Although many p16 antibodies used in previous studies of senescence are no longer available, 5,6,35 we utilized three different commercially available p16 antibodies. One, used by various labs for immunohistochemical analysis of p16, 36‐39 labeled > 60% of all nuclei in both young and old muscle (Figure S1), in spite of the fact that p16 mRNA expression is low in muscle, as is p21 mRNA (CT values 35‐36; data not shown). Consistent with mRNA results, two of the antibodies detected no p16 + cells in human skeletal muscle, regardless of age (data not shown).…”

The first days in the life of naïve human B-lymphocytes infected with Epstein-Barr virus

“…In mice reconstituted with functional components of a human immune system it is possible to study EBV's biology in vivo (Münz, 2015), but steps early after infection are difficult to investigate in this tractable model. To our knowledge, an EBNA2-deleted virus has not been studied in this animal model, yet, but, unexpectedly, EBV strains incapable of expressing EBNA3C were found to establish latency and cause B cell lymphomas albeit at a reduced frequency compared with wild-type EBV (Murer et al, 2018;Romero-Masters et al, 2018). EBNA3C is a transcriptional repressor and has been found to abrogate the expression of cell cycle inhibitors such as p16 INK4A encoded by CDKN2A, which is a prerequisite to establish lymphoblastoid cell lines with EBNA3C-deleted EBVs in vitro (Skalska et al, 2013).…”

Latency and lytic replication in Epstein–Barr virus-associated oncogenesis

“…As discussed above, these are essential latent EBV gene products that rescue EBV infected cells from cell death that is induced by EBNA2-driven proliferation 43 22 . This persistence was associated with EBNA2-driven proliferation during the first month of infection, which then, switched to EBV latency 0 persistence with only non-translated EBER expression after three months 22 . The observed absence of EBV latency III seems to be caused by a combination of EBNA3A or EBNA3C deficiency and immune control of rare completely virus transformed B cells, because in a HIS mouse model with less immunocompetence, LMPexpressing EBNA3C-negative lymphomas can be observed at lower frequency compared to wild-type EBV infection 59 .…”

“…This was recently queried using EBV deficient in EBNA3A and EBNA3C. As discussed above, these are essential latent EBV gene products that rescue EBV infected cells from cell death that is induced by EBNA2-driven proliferation 43 22 . This persistence was associated with EBNA2-driven proliferation during the first month of infection, which then, switched to EBV latency 0 persistence with only non-translated EBER expression after three months 22 .…”

“…Based on the detection of intermediate expression levels of only three latent EBV proteins during the B cell differentiation stage of germinal center B cells that could result from naïve B cells after their activation by the eight latent EBV proteins and precede memory B cell development 21 , it was suggested that the virus induces oncogenesis to drive infected B cells into differentiation in order to gain access to the memory B cell pool for persistence. However, recent evidence suggests that expression of all eight latent EBV proteins and B cell transformation by these proteins might not be required for EBV persistence and latency 22 . Furthermore, not only these eight latent EBV proteins, but also early lytic EBV proteins could enhance viral oncogenesis 23 .…”