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

Abstract

Epstein-Barr virus (EBV) infects and activates resting human B-lymphocytes, reprograms them, induces their proliferation, and establishes a latent infection in them. In established EBV-infected cell lines many viral latent genes are expressed. Their roles in supporting the continuous proliferation of EBV-infected B cells in vitro are known, but their functions in the early, pre-latent phase of infection have not been investigated systematically. In studies during the first eight days of infection using derivatives of EBV with mutations in single genes of EBVs we found only EBNA2 to be essential for activating naïve human B-lymphocytes, inducing their growth in cell volume, driving them into rapid cell divisions, and preventing cell death in a subset of infected cells. EBNA-LP, LMP2A and the viral microRNAs have supportive, auxiliary functions, but mutants of LMP1, EBNA3A, EBNA3C, and the noncoding EBER RNAs had no discernable phenotype compared with wild-type EBV. B cells infected with a double mutant of EBNA3A and 3C had an unexpected proliferative advantage and did not regulate the DNA damage response (DDR) of the infected host cell in the pre-latent phase. Even EBNA1 which has very critical longterm functions in maintaining and replicating the viral genomic DNA in established cell lines, was dispensable for the early activation of infected cells. Our findings document that the virus dose is a critical parameter and indicate that EBNA2 governs the infected cells initially and implements a strictly controlled temporal program independent of other viral latent genes. It thus appears that EBNA2 is sufficient to control all requirements for clonal cellular expansion and to reprogram human B-lymphocytes from energetically quiescent to activated cells. Author summaryThe preferred target of Epstein-Barr virus (EBV) are human resting B-lymphocytes. We found that their infection induces a well-coordinated, time-driven program that starts with a substantial 3 increase in cell volume followed by cellular DNA synthesis after three days and subsequent rapid rounds of cell divisions on the next day accompanied by some DNA replication stress (DRS). Two to three days later the cells decelerate and turn into stably proliferating lymphoblast cell lines. With the aid of 16 different recombinant EBV strains we investigated the individual contributions of EBV's multiple latent genes during early B-cell infection and found that many do not exert a detectable phenotype or contribute little to EBV's pre-latent phase. The exception is EBNA2 that is essential in governing all aspects of B-cell reprogramming. EBV relies on EBNA2 to turn the infected B-lymphocytes into proliferating lymphoblasts preparing the infected host cell for the ensuing stable, latent phase of viral infection. In the early steps of B-cell reprogramming viral latent genes other than EBNA2 are dispensable but some, EBNA-LP for example, support the viral program and presumably stabilize the infected cells once viral latency is established.

Histone Loaders CAF1 and HIRA Restrict Epstein-Barr Virus B-Cell Lytic Reactivation

“…Notably, the EBV tegument protein BNRF1 targets ATRX and DAXX for sequestration in promyelocytic leukemia (PML) bodies at this time point ( 24 ), suggesting that HIRA and newly induced CAF1 may be responsible. H3.1 and H3.3 levels remained stable at day 4 postinfection, a time point at which cells divide every 8 to 12 h ( 11 – 13 ). Interestingly, after the period of Burkitt-like hyperproliferation that extended roughly from day 3 to day 7 postinfection, H3.1 and H3.3 levels nearly doubled, even controlling for increases in EBV genome copy numbers over this interval.…”

“…Incoming EBV genomes are organized into nucleosomes, which must then be maintained or remodeled on newly synthesized, damaged, or transcribed regions of EBV genomes. Burkitt lymphoma cells are among the fastest growing human tumor cells, and newly EBV-infected B-cells undergo Burkitt-like hyperproliferation between day 3 and day 7 postinfection in cell culture ( 11 – 13 ). Host machinery must therefore propagate chromatin-encoded epigenetic information with each cell cycle, a process which begins with histone loading.…”

“…It is plausible that other histone chaperones, in particular, HIRA, may play key roles in H3/H4 loading onto incoming EBV genomes prior to mitosis in newly infected cells. CAF1 may then carry out DNA replication-dependent roles, beginning with entry of the newly infected cell into S-phase at approximately 72 h postinfection ( 11 – 13 ), and HIRA plays ongoing roles that remain to be defined. Notably, EBV tegument protein BNRF1 subverts DAXX/ATRX-mediated H3.3 loading on viral chromatin for the first several days postinfection and presumably also with lytic reactivation ( 23 , 24 ) ( Fig.…”

“…Multiple layers of epigenetic regulation enable EBV to establish latency in newly infected B-cells, in which a small number of virus-encoded proteins and viral noncoding RNAs (ncRNAs) reprogram the metabolism, growth, and survival pathways of infected cells ( 7 – 9 ). Within 3 days of infection, quiescent B-cells are reprogramed to become rapidly growing lymphoblasts that divide as frequently as every 8 h ( 10 – 13 ).…”

Epigenetic and epitranscriptomic regulation of viral replication

“…Latency establishment is an intricately regulated process that allows viruses to temporarily adopt an alternative viral state. Taking EBV as an example, upon infection of B lymphocytes, the virus first activates all eight latency genes to aid in the activation of naive B cells (type III latency), and then certain genes are switched off (type I/II latency), to minimize the number of viral proteins that are detectable by the immune system, with different latency types utilizing distinct viral promoters that are associated with active chromatin marks only when needed [72][73][74] . Meanwhile, lytic viral genes remain transcriptionally silent and are associated with heterochromatic markers during latency 70,75 .…”