Epstein-Barr virus with the latent infection nuclear antigen 3B completely deleted is still competent for B-cell growth transformation in vitro

The Epstein-Barr virus (EBV) nuclear antigen 3B (EBNA-3B) is considered nonessential for EBV-mediated B-cell growth transformation in vitro based on three virus isolates with EBNA-3B mutations. Two of these isolates could potentially express truncated EBNA-3B products, and, similarly, we now show that the third isolate, IB4, has a point mutation and in-frame deletion of 263 amino acids. In order to test whether a virus with EBNA-3B completely deleted can immortalize B-cell growth, we first cloned the EBV genome as a bacterial artificial chromosome (BAC) and showed that the BAC-derived virus was B-cell immortalization competent. Deletion of the entire EBNA-3B open reading frame from the EBV BAC had no adverse impact on growth of EBV-immortalized B cells, providing formal proof that EBNA-3B is not essential for EBV-mediated B-cell growth transformation in vitro.     

The residues between the two transformation effector sites of Epstein-Barr virus latent membrane protein 1 are not critical for B-lymphocyte growth transformation

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for EBV-mediated transformation of primary B lymphocytes. LMP1 spontaneously aggregates in the plasma membrane and enables two transformation effector sites (TES1 and TES2) within the 200-amino-acid cytoplasmic carboxyl terminus to constitutively engage the tumor necrosis factor receptor (TNFR)-associated factors TRAF1, TRAF2, TRAF3, and TRAF5 and the TNFR-associated death domain proteins TRADD and RIP, thereby activating NF-kappaB and c-Jun N-terminal kinase (JNK). To investigate the importance of the 60% of the LMP1 carboxyl terminus that lies between the TES1-TRAF and TES2-TRADD and -RIP binding sites, an EBV recombinant was made that contains a specific deletion of LMP1 codons 232 to 351. Surprisingly, the deletion mutant was similar to wild-type (wt) LMP1 EBV recombinants in its efficiency in transforming primary B lymphocytes into lymphoblastoid cell lines (LCLs). Mutant and wt EBV-transformed LCLs were similarly efficient in long-term outgrowth and in regrowth after endpoint dilution. Mutant and wt LMP1 proteins were also similar in their constitutive association with TRAF1, TRAF2, TRAF3, TRADD, and RIP. Mutant and wt EBV-transformed LCLs were similar in steady-state levels of Bcl2, JNK, and activated JNK proteins. The wt phenotype of recombinants with LMP1 codons 232 to 351 deleted further demarcates TES1 and TES2, underscores their central importance in B-lymphocyte growth transformation, and provides a new perspective on LMP1 sequence variation between TES1 and TES2.     

T-cell-mediated regression of "spontaneous" and of Epstein-Barr virus-induced B-cell transformation in vitro: studies with cyclosporin A

The regression of Epstein-Barr (EB) virus-transformed B-cell outgrowth which is seen in experimentally-infected cultures of blood mononuclear (UM) cells from healthy seropositive donors can be abolished in medium containing the T-cell-suppressive agent cyclosporin A (CSA) at concentrations of 0.05 microgram/ml and above. CSA mediates its effect within the first 4 days post-infection of the UM cells and this prevents subsequent in vitro generation of the EB virus-specific cytotoxic-T-cell response which normally brings about regression. Regression can be fully restored by supplementing the CSA-treated culture with interleukin 2 (IL-2)-containing culture supernatants or indeed with purified IL-2 itself, suggesting that CSA mediates its effect in this system through inhibiting the endogenous production of IL-2 which is required to amplify the virus-specific cytotoxic response. "Spontaneous transformation" to EB virus genome-positive lymphoblastoid cell lines in noninfected cultures of UM cells from healthy seropositive donors, though rare in normal medium, is enhanced to such a degree in the presence of CSA that, for many donors, the phenomenon becomes titratable against input cell dose across the 2.0 X 10(6)-2.5 X 10(5) cells/culture range. Cell mixing experiments suggest that the spontaneously transformed cell lines which arise with such efficiency under these conditions do so not by direct in vitro outgrowth of progenitor cells transformed by the virus in vivo, but by a two-step mechanism involving virus release and secondary infection in vitro.     

Establishment of latent Epstein-Barr virus infection and stable episomal maintenance in murine B-cell lines

Epstein-Barr virus (EBV) is a strict human pathogen for which no small animal models exist. Plasmids that contain the EBV plasmid origin of replication, oriP, and express EBV nuclear antigen 1 (EBNA1) are stably maintained extrachromosomally in human cells, whereas these plasmids replicate poorly in rodent cells. However, the ability of oriP and EBNA1 to maintain the entire EBV episome in proliferating rodent cells has not been determined. Expression of the two human B-cell receptors for EBV on the surfaces of murine B cells allows efficient viral entry that leads to the establishment of latent EBV infection and long-term persistence of the viral genome. Latent gene expression in these cells resembles the latency II profile in that EBNA1 and LMP1 can be detected whereas EBNA2 and the EBNA3s are not expressed.     

Plasma cell-specific transcription factor XBP-1s binds to and transactivates the Epstein-Barr virus BZLF1 promoter

Epstein-Barr virus (EBV) in vivo is known to establish persistent infection in resting, circulating memory B cells and to productively replicate in plasma cells. Until now, the molecular mechanism of how EBV switches from latency to lytic replication in vivo was not known. Here, we report that the plasma cell differentiation factor, XBP-1s, activates the expression of the master regulator of EBV lytic activation, BZLF1. Using reporter assays, we observed that XBP-1s was able to transactivate the BZLF1 promoter, Zp, in a plasma cell line and other lymphoid cell lines but, interestingly, not in epithelial cell lines. We have identified an XBP-1s binding site on the ZID/ZII region of Zp, which when abolished by site-directed mutagenesis led to abrogation of XBP-1s binding and promoter activation. Using the chromatin immunoprecipitation assay, we observed direct binding of XBP-1s to endogenous Zp in an EBV-infected plasma cell line. Finally, in the same cell line, we observed that overexpression of XBP-1s resulted in increased expression of BZLF1, while knockdown of XBP-1s with short hairpin RNA drastically reduces BZLF1 expression. We suggest that EBV harnesses the B-cell terminal differentiation pathway via XBP-1s as a physiological signal to reactivate and begin viral replication. We are currently investigating other signals, such as the endoplasmic reticulum stress response proteins, which act upstream of XBP-1s, to identify other interacting factors that initiate and/or amplify the lytic switch.     

Effects of S-adenosylhomocysteine and analogs on Epstein-Barr virus-induced transformation, expression of the Epstein-Barr virus capsid antigen, and methylation of Epstein-Barr virus DNA.

S-Adenosylhomocysteine was found to have no effect on Epstein-Barr virus-induced transformation of B-lymphocytes and to stimulate viral capsid antigen expression only slightly in the FF41-1 cell line. In contrast, the S-adenosylhomocysteine analogs sinefungin and S-isobutyladenosine inhibited Epstein-Barr virus transformation and induced a significant increase in the numbers of cells expressing the viral capsid antigen. An inverse relationship between levels of viral DNA methylation and gene expression was demonstrated.