First Days in the Life of Naive 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, prelatent phase of infection have not been investigated systematically. In studies during the first 8 days of infection using derivatives of EBV with mutations in single genes of EBVs, we found only Epstein-Barr nuclear antigen 2 (EBNA2) to be essential for activating naive 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, latent membrane protein 2A (LMP2A), and the viral microRNAs have supportive, auxiliary functions, but mutants of LMP1, EBNA3A, EBNA3C, and the noncoding Epstein-Barr virus with small RNA (EBERs) had no discernible 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 prelatent phase. Even EBNA1, which has very critical long-term 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 decisive 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. IMPORTANCE The preferred target of Epstein-Barr virus (EBV) is human resting B lymphocytes. We found that their infection induces a well-coordinated, time-driven program that starts with a substantial increase in cell volume, followed by cellular DNA synthesis after 3 days and subsequent rapid rounds of cell divisions on the next day accompanied by some DNA replication stress (DRS). Two to 3 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 prelatent 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.

Direct evidence of abortive lytic infection-mediated establishment of Epstein-Barr virus latency during B-cell infection

“…This observation is consistent with previous findings that rapid growth of EBV-infected cells is coupled with an increase in biomass for energy and production of biosynthetic intermediates [24]. Hammerschmidt and colleagues also showed that infected cells did not divide within the first three days of infection but rapidly re-commenced growth at 4 dpi, during EBV infection to naïve human B-cells [25]. Thus, EBV reprogrammed the transcriptome of infected cells during the initial stage of infection even without immortalization, similar to EBV infection of naïve or resting B-cells [15, 25, 26].…”

“…Hammerschmidt and colleagues also showed that infected cells did not divide within the first three days of infection but rapidly re-commenced growth at 4 dpi, during EBV infection to naïve human B-cells [25]. Thus, EBV reprogrammed the transcriptome of infected cells during the initial stage of infection even without immortalization, similar to EBV infection of naïve or resting B-cells [15, 25, 26].…”

Histone loaders CAF1 and HIRA restrict Epstein-Barr virus B-cell lytic reactivation

“…Notably, the EBV tegument protein BNLF1 targets ATRX and DAXX for sequestration in PML bodies at this timepoint (23), suggesting that HIRA and newly induced CAF1 may be responsible. H3.1 and H3.3 levels remained stable at Day 4 post-infection, a timepoint at which cells have entered Burkitt-like hyperproliferation and divide every 8-12 hours (11–13). Interestingly, after the period of Burkitt-like hyper-proliferation that extends roughly from days 3-7 post-infection, H3.1 and H3.3 levels nearly doubled, even when controlling for increases in EBV genome copy number 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 are amongst the fastest growing human tumor cells, and newly EBV-infected B-cells undergo Burkitt-like hyperproliferation between days 3-7 post-infection in cell culture (11–13). Host machinery must therefore propagate chromatin-encoded epigenetic information with each cell cycle, 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 approximately 72 hours post-infection (11–13), and HIRA plays ongoing roles that remain to be defined. Notably, the EBV tegument protein BNLF1 subverts DAXX/ATRX-mediated H3.3 loading on viral chromatin for the first several days post-infection (22, 23) (Fig.…”