Epstein-Barr virus latency in vivo and in vitro

In this thesis, I studied 1) The latency situation of Epstein- Barr virus (EBV) in bone marrow transplanted (BMT) patients and healthy virus carriers. 2) The role of EBNA1 and cellular transcriptional factors Oct and Grg/TLE family in EBV latency switch.

B lymphocytes have been identified as the main reservoir of latent Epstein-Barr virus (EBV) in healthy virus carriers. We have established a semi-quantitative PCR method (SQ-PCR) to estimate the EBV genome load in the blood B-cell subpopulation in healthy individuals. EBV DNA was detected in subfractionated IgM-, IgG- and IgA-positive B cells. Between 80%. and 90% of the viral DNA was found in the lgA-positive compared with the lgA-negative fraction.

We used SQ-PCR to monitor the blood levels of EBV-DNA in 9 patients receiving allogeneic BMT. Four of 5 recipients of HLA-mismatched T-cell-depleted grafts showed a 4- to 5-log increase of EBV-DNA within 1 to 3 months after BMT. Administration of 2 to 4 infusions of 107 EBV-specific cytotoxic T-lymphocytes (CTLs)/M2 starting from the time of maximal virus load resulted in a 2- to 3-log decrease of virus titers in 3 patients. A moderate increase of virus titers was also detected in 3 of 4 patients receiving unmanipulated HLA-matched grafts, whereas one patient with Wiskott-Aldrich syndrome (WAS) reached a 5-log increase of EBVDNA load within 70 days after BMT. Our results suggest that a rapid increase of circulating EBV-DNA occurs in the absence of EBV-specific T-cell precursors or in the presence of congenital immune defects that prevent the reestablishment of virus-specific immunity. Prophylactic administration of EBV-CTI-s early after 13MT appears to provide the most effective protection against the development of EBV-associated lymphoproliferative disease.

We identified that Cp could also can be activated by octamer-binding factor (OCT) proteins. Physical binding to the FR by the cellular transcription factors OCT1 and OCT2 was demonstrated by using electrophoretic mobility shift assay (EMSA). Furthermore, OCT2 alone or OCTI in combination with co-regulator Bob-1could drive transcription of a heterologous thymidine kinase promoter liked to the FR both in B cells and epithelial cells. Cp controlled by the FR also activated by binding of OCT2 to FR. OCT proteins can recruit the Grg/TLE - farmly of transcription factor regulatory proteins (co-factors) and those proteins repress OCT2 promoter activity through FR. Although all Grg/TLE variants could repress the OCT2 induced activity from a FR-luciferase report vector, Grg-3 was most efficient. Grg/TLE proteins did not inhibit the EBNA1 activity on their own but when cotransfected with OCT2 also the EBNA I-induced FR enhancer effect was repressed. Addition of EBNA1 efficiently counteracted the transcriptional repression evoked by OCT-2 and Grg/TLE3. We also demonstrate that the levels of EBNA1 and OCT2 differ dramatically between latency III and I cells.

Using competition and base substitutions of oligo-probes in EMSA1 we mapped in detail the binding of OCT-proteins to all octamer sequence three bp away from the EBNA1 core binding site. Different FR repeats vary in their ability to form complexes with OCTI, OCT2 and their cofactors. Direct physical binding of OCT and Grg/TLE to FR repeats was demonstrated in vitro by affinity adsorption with FR specific DNA as bait. Both OCT2 and EBNA I were shown to bind FR in vivo in EBV-positive cells representing latency I or III utilizing Chromatin immunoprecipitation assay (ChIP). Based on our results, a model is suggested that the on-off switch of the EBV C promoter in latency is controlled by competition between EBNA1 and OCT-proteins together with their co regulators.

List of scientific papers

I. Ehlin-Henriksson B, Zou JZ, Klein G, Ernberg I (1999). Epstein-Barr virus genomes are found predominantly in IgA-positive B cells in the blood of healthy carriers. Int J Cancer. 83(1): 50-4.
https://pubmed.ncbi.nlm.nih.gov/10449607

II. Gustafsson A, Levitsky V, Zou JZ, Frisan T, Dalianis T, Ljungman P, Ringden O, Winiarski J, Ernberg I, Masucci MG (2000). Epstein-Barr virus (EBV) load in bone marrow transplant recipients at risk to develop posttransplant lymphoproliferative disease: prophylactic infusion of EBV-specific cytotoxic T cells. Blood. 95(3): 807-14.
https://pubmed.ncbi.nlm.nih.gov/10648390

III. Almqvist J, Zou JZ, Linderson Y, Borestrom C, Altiok E, Zetterberg H, Rymo L, Pettersson S, Ernberg I (2005). Functional interaction of Oct transcription factors with the family of repeats in Epstein-Barr virus oriP. J Gen Virol. 86(5): 1261-7.
https://pubmed.ncbi.nlm.nih.gov/15831936

IV. Almqvist J, Zou JZ, Borestrom C, Pettersson S, Rymo L, Ernberg I (2006). Repression of Epstein-Barr virus enhancer Family of Repeats mediated transcription by Oct and Grg/TLE transcriptional regulators, suggests an involvement in switching of latency programs. [Manuscript]

V. Zou JZ, Almqvist J, Ernberg I (2006). Characterization of the Oct-protein binding sites in the FR control element of the C promoter and in Q promoter in Epstein-Barr virus (EBV). [Manuscript]