Alternative EBNA1 expression in organ transplant patients

Abstract

In order to identify patients at risk for developing post-transplant lymphoproliferative disease (PTLD), a sensitive nested RT-PCR method for detection of EBNA1 gene expression in peripheral blood cells was used. EBNA1 expression in peripheral blood samples from 60 organ recipients was analyzed and compared with 24 healthy controls in a retrospective study. Overall, EBNA1-positive samples were detected at least once in 43% of the transplant patients with post-transplant lymphoproliferative disease, in 18% of the other transplant patients and in none of the healthy controls. The odds ratio for EBNA1 expression in patients with post-transplant lymphoproliferative disease was 3.42 (95% CI=1.02-11.54) compared to other transplant recipients. Together with normal EBV Q promoter initiated EBNA1 transcripts, an alternatively spliced form was expressed in peripheral blood cells in the above-mentioned transplant patients. This transcript lacks the U leader exon in the 5'-untranslated region (UTR). We have previously identified and characterized a functional internal ribosome entry site, the EBNA IRES, in the untranslated U leader exon of EBNA1. Transfection experiments with EBNA1 coding plasmids followed by Western blot showed that the EBNA IRES promotes cap-independent translation and increases the EBNA1 protein level. The alternative EBNA1 transcript lacking this function is expressed in the majority of the investigated EBNA1-positive patient samples as well as in some EBV-positive B-cell lines. Alternative splicing in this form gives EBV potential to regulate the translation of EBNA1 by modifying the 5' UTR. These findings indicate a new mechanism for EBNA1 expression in vivo.

Cell specific internal translation efficiency of Epstein–Barr virus present in solid organ transplant patients

“…Alternative splicing including long-range exon skipping in the 5 0 untranslated region upstream of the EBNA1-encoding K exon generates the EBNA1 mRNA. A functional alternative splice in the EBNA1 transcript that omits the U exon and consequently exclude the EBNA IRES element was recently reported [Berggren et al, 2005]. Transfection and Western blot experiments with an EBNA1 coding plasmid confirmed that the EBNA IRES promotes capindependent translation and increases the EBNA1 protein level.…”

“…Furthermore, analysis of the EBNA1 expression in peripheral blood samples from solid organ transplant patients revealed an alternatively spliced form of the EBNA1 transcript, which excludes the EBNA IRES element. The alternative transcript was expressed in the majority of the investigated EBNA1-positive organ transplant patient samples as well as in some EBVpositive cell lines [Berggren et al, 2005]. The presence of an IRES element within the EBV constitutes additional possibilities for the virus to regulate its gene expression.…”

“…The diagnosed disorders were all of B-cell origin, as shown by histopatholgy. All patients were under immunosuppressive treatments and are part of a clinical study published previously [Berggren et al, 2005].…”

“…In this study, experiments were undertaken to analyse the EBNA IRES sequence derived from immunosuppressed organ transplant patients. Starting with peripheral blood samples from seven EBNA1 expressing organ transplant patients, four of the patients were diagnosed with post transplant lymphoproliferative disorder and three patients without lymphoproliferative disorder [Berggren et al, 2005], DNA, and total RNA were prepared. The EBNA IRES cDNA and corresponding genomic EBV region were PCR amplified and sequence analysed.…”

“…RT-PCR, with total RNA prepared from patients or cell lines, was performed as previously described [Isaksson et al, 2003;Berggren et al, 2005]. From each patient sample 4-10 ml peripheral blood with EDTA was lysed with Erythrocytes Lysis buffer (QIAGEN, Hilden, Germany) and the nucleated cells were isolated by centrifugation and stored at À708C.…”