Epstein-Barr virus infection is known to convert resting B lymphocytes into immortal cells that continuously multiply, which leads to posttransplant lymphoproliferative disorder and can evolve to lymphoma and other lymphoproliferative disorders. In a recent study, Japanese researchers discovered the molecular mechanisms of this growth transformation, demonstrating the Epstein-Barr virus induces nucleolar enlargement and increased proliferation in B cells by activating the cancer-related gene IMPDH2.
The research also revealed strong evidence supporting the efficacy of mycophenolate mofetil, an approved immunosuppressant, in inhibiting PTLD, highlighting its therapeutic significance.
The study, published inMicrobiology Spectrum, used primary B cells from healthy donors instead of cell lines.
“Insights from EBV research that uses cell lines has been limited, because cell lines are already in an immortalized state,” Takayuki Murata, the study’s lead author, said in a statement. “To overcome this roadblock, we used primary B cells from healthy donors and then infected them with EBV. This allowed us to monitor the step-by-step growth transformation of B cells and analyze the mechanisms involved.”
The researchers observed the primary B cells infected with wild-type EBV using electron microscopy and immunostaining. As early as two days after infection, the morphology of B cells showed significant alterations. An enlargement of the nucleolus—a region within the nucleus that produces ribosomes, the cell’s protein-producing machinery—was observed, along with an increase in the number of nucleoli. Interestingly, nucleolar enlargement was followed by an enlargement of both—the nuclei and the cells.
The researchers then performed RNA sequencing on the infected B cells to understand the transcriptional changes.
“Among the genes showing significantly altered expression levels, one named IMPDH2 stood out, as it had previously been linked to similar morphological changes in glioblastoma,” Atsuko Sugimoto from Fujita Health University School of Medicine, who was also a part of the research team, said in a statement. “Careful analysis showed that the levels of the IMPDH2 peaked two days after infection—coinciding with the timing of nucleolar enlargement. This suggested that we were on the right path.”
The inhibition of IMPDH2 using silencer RNAs and the drug mycophenolic acid prevented the growth transformation of primary B cells after EBV infection, producing smaller nucleoli, nuclei, and cells. This confirmed that IMPDH2 induction played a key role in the growth transformation of EBV-infected B cells.
The next step involved understanding how EBV activates IMPDH2 expression. Three key viral genes—EBNA2 and LMP1—were tested because of their known role in EBV-induced B cell transformation. Interestingly, when EBV lacking EBNA2 was used for infection, IMPDH2 induction following primary EBV infection was blocked. This effect was not observed with LMP1 knockout.
“This very clearly demonstrated that EBV induces IMPDH2 expression EBNA2-dependent mechanisms. In addition, cellular transcription factor MYC was also involved in the IMPDH2 induction,” Sugimoto said.
With several key pieces of evidence on their plate, the researchers finally set out to find the final piece of the puzzle. To highlight the clinical significance of their findings, they examined whether the drug mycophenolate mofetil could prevent B cell transformation and PTLD.
“Like MPA, which we tested in the earlier part of our study, MMF is an IMPDH2 inhibitor. More importantly, MMF is already a clinically approved immunosuppressant. That is why it was useful to test if it could be applied for the clinical prevention of PTLD,” Murata said.
As expected, the administration of MMF in a pre-clinical mouse xenograft model led to improved survival and reduced splenomegaly. These observations confirmed that the use of MMF can inhibit PTLD development.
This study is the first to demonstrate that IMPDH2 activation and nucleolar hypertrophy are essential for EBV-induced B cell transformation and that IMPDH2 inhibition can suppress PTLD. It could lead to the adoption of MMF as an agent for the prevention of EBV-positive PTLD, providing significant relief to transplant patients.
