(-)-Epigallocatechin-3-gallate inhibits EBV lytic replication via targeting LMP1-mediated MAPK signal axes

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) plays an important oncogenic role in the viral latent infection. Recently, increasing evidence indicate that the high expression of LMP1 during EBV lytic cycle is related to the viral lytic replication. However, the mechanism by which LMP1 regulates EBV lytic replication remains unclear. (-)-Epigallocatechin-3-gallate (EGCG) prevents carcinogenesis by directly targeting numerous membrane proteins and effectively inhibits EBV lytic cascade. Here we demonstrated that LMP1 promotes EBV lytic replication through the downstream signal molecules MAPKs, including ERKs, p38, and JNKs. LMP1 induces the phosphorylation of p53 through MAPKs to enhance the ability of wild-type p53 (wt-p53) to activate expression of BZLF1 gene. While the JNKs/c-Jun signal axis appears to be involved in EBV lytic replication induced by LMP1 in p53 mutant manner. And we provided the first evidence that EGCG directly targets the viral membrane LMP1 (Kd =0.36 μM, n=1) using fluorescence quenching, isothermal titration calorimetry (ITC) assay, and CNBR-activated Sepharose 4B pull-down affinity chromatography. Furthermore, we revealed that EGCG inhibits EBV lytic replication via suppressing LMP1 and thus blocking the downstream MAPKs/wt-p53 signal axis in AGS-EBV cells and JNKs/c-Jun signal axis in p53 mutant B95.8 cells. Our study, for the first time, reports the binding and inhibitory efficacy of EGCG to the LMP1 which is a key oncoprotein encoded by EBV. These findings suggest the novel function of LMP1 in the regulation of EBV lytic cycle and reveal the new role of EGCG in EBV-associated malignancies through destroying viral reactivation.

1778. Epstein–Barr Virus Genetic Diversity: Evaluation of BZLF1 Variants among Bone Marrow Transplant Patients and Individuals with Infectious Mononucleosis

Background Epstein–Barr virus (EBV) is associated with several diseases, including infectious mononucleosis (IM) and malignant disorders, including post-transplant lymphoproliferative disorder (PTLD). The relationship between strains of the virus and disease manifestations or illness severity is of interest. Such strains have been defined by genetic variations in the major viral genes. Data involving the patterns of genetic diversity of the virus in different populations are required. We examined the genetic diversity of the BZLF1 gene, which is a major lytic gene of the virus. Methods We sequenced the BZLF1 gene of EBV following amplification from DNA that was extracted from blood obtained from pediatric bone marrow transplant (BMT) patients and children and young adults with IM. Sequencing was done by Sanger methodology (dideoxy DNA sequencing) and the sequences were aligned with a reference strain of EBV using Geneious software. The variant burden and types of single nucleotide variants (SNV) were compared across the 3 exons of the BZLF1 gene. Results We sequenced the BZLF1 gene using 21 patients with IM (median age 14, age range 2–19 years) and 11 who underwent bone marrow transplantation (median age 6, range 3–13 years). Three of 11 BMT patients developed post-transplant lymphoproliferative disorder (PTLD). Among the 3 exons, exon 1 had the greatest diversity across both study groups. There was a tendency for less diversity among PTLD samples, with no sample containing >1 single nucleotide variant (SNV) in contrast to the other samples. In samples that contained SNVs, there was a non-statistically significant trend for more SNVs to occur among the IM samples compared with PTLD samples (median 4.5 and 0, respectively; P > 0.05). Additionally, 2/11 (18.2%) BMT sequences contained more than 1 SNV compared with 7/21 (33.3%) IM sequences (P > 0.05). Conclusion There was a tendency for more genetic diversity among samples from patients with IM compared with bone marrow transplant patients, notably those with PTLD. Further studies will determine if this tendency is due to selective pressures in the transplant setting, including but not limited to the use of antiviral agents directed at the lytic phase of EBV. Disclosures All authors: No reported disclosures.