Search

We have previously shown that the EBV ZEBRA protein (also denoted EB1, Z, or Zta) encoded by the BZLF open reading frame is expressed in primary human thymocytes and in human T lymphoblastoid cell lines infected by EBV. Expression of EBV-encoded gene products in T lymphocytes could contribute to viral pathogenesis during acute EBV infection as well as in individuals coinfected with EBV and HIV. HPB-ALL and Jurkat T lymphoblastoid cell lines transiently and stably expressing ZEBRA were characterized in this work. Expression of ZEBRA protein in human T lymphoblastoid cells was associated with decreased expression of an NF-kappaB reporter gene, altered expression of the NF-kappaB p50 protein subunit, and decreased DNA binding by components of NF-kappaB. These observations suggest that inactivation of NF-kappaB transcription by ZEBRA in EBV-infected T cells may be a novel mechanism of viral pathogenesis analogous in part to over-expression of the endogenous cytoplasmic inhibitor of NF-kappaB, IkappaBalpha.

The Epstein-Barr virus (EBV)-positive Burkitt's lymphoma cell line, Akata, was treated with dimethyl sulfoxide (DMSO) for 96 hr in order to reversibly arrest cell cycle progression in G1 phase. Stimulation of the cells with anti-IgG antibody induced a marked and synchronous replication of EBV DNA within 12 hr, before the cells entered into S-phase after release from DMSO-induced arrest. Furthermore, a reduced efficiency of productive replication was demonstrated if anti-IgG stimulation was delayed after release. The results indicate that entry into S-phase of host cells is not only unnecessary for, but also may have negative consequences for the productive phase of EBV infection. Also, it was shown that addition of acyclovir, an inhibitor of the EBV-encoded DNA polymerase, to anti-IgG-stimulated Akata cells inhibited the productive replication of EBV DNA, but had no effect on the expression of early genes of the virus, including BZLF1, BRLF1, BMRF1, and BHRF1.

The presence of Epstein-Barr virus in different T-cell malignancies is now widely reported. In an effort to ascertain whether T cells are susceptible to EBV infection, we and others have detected the EBV receptor, CD21 on a population of immature thymocytes. We showed that EBV is a cofactor in stimulating proliferation of thymocytes. This proliferation may be a relevant factor in EBV-associated T-cell malignancies as well as EBV causation of acute infectious mononucleosis (AIM). We have further identified a subset of thymocytes that is infectable by EBV in which the genome remains linear in the first weeks after infection. We documented the transcription of the switch protein ZEBRA, an alternatively spliced form, RAZ, and EBNA-1 transcription from the Fp promoter. We hypothesise that EBV may be a cofactor in oncogenesis in T cells through several different pathways.

Epstein-Barr virus (EBV) is known to infect B cells and epithelial cells. We and others have shown that EBV can also infect a subset of thymocytes. Infection of thymocytes was accompanied by the appearance of linear EBV genome within 8 hr of infection. Circularization of the EBV genome was not detected. This is in contrast to the infection in B cells where the genome can circularize within 24 hr of infection. The appearance of the BamHI ZLF-1 gene product, ZEBRA, by RT-PCR, was observed within 8 hr of infection. The appearance of a novel fusion transcript (RAZ), which comprised regions of the BZLF-1 locus and the adjacent BRLF-1 locus, was detected by RT-PCR. ZEBRA protein was also identified in infected thymocytes by immunoprecipitation. In addition, we demonstrated that the EBNA-1 gene in infected thymocytes was transcribed from the Fp promoter, rather than from the Cp/Wp promoter which is used in latently infected B cells. Transcripts encoding gp350/220, the major coat protein of EBV, were identified, but we did not find any evidence of transcription from the LMP-2A or EBER-1 loci in infected thymocytes. These observations suggest that de novo EBV infection of thymocytes differs from infection of B cells. The main difference is that with thymocytes, no evidence could be found that the virus ever circularizes. Rather, EBV remains in a linear configuration from which replicative genes are transcribed.

The discovery of EBV in certain T cell malignancies and the expression of the EBV receptor, CR2/CD21, on a population of immature thymocytes, T lymphoblastoid cell lines, and childhood acute T lymphoblastic leukemia cells suggested that EBV-receptor interactions on T cells may be of importance. We have shown that, within the thymus, a population of large, immature cells expresses CD21. EBV altered the activation responses of immature thymocytes in vitro. Triggering through CD2 is mitogenic for mature, but not immature, T cells. However, during infection by EBV, ligation of CD2 caused thymocytes to proliferate in the absence of exogenous cytokines. This function was a result of the interaction of EBV with its receptor, CD21, but was caused by infection rather than surface signaling, because neither specific mAb nor the P3HR-1 strain of virus mimicked the effect of B95-8. Immature thymocytes were infected by EBV, as determined by the internalization of the viral genome and its transcriptional activity. Consistent with the activity of B95-8, EBNA-2 transcripts were identified within infected thymocyte populations. In addition, components of the viral replicative pathway were expressed during infection of thymocytes. These components included transcription of BZLF-1, an early gene that characterizes EBV-infected B cells after disruption of latency. A second transcript was identified as encoding the recently characterized RAZ, which also is associated with replicative infection. The consequences of EBV infection of T cells at an early stage of differentiation may lead to failure of normal T cell repertoire development, autoimmunity, or malignancy.