Disease relevance of EBNA-1

• Extranodal, nasal NK/T-cell lymphomas are regularly Epstein-Barr virus (EBV)-positive, with a type II latency pattern, expressing thus EBNA-1 and LMP1 [1].
• The EBNA-1 gene was identified as another diverged locus, although its variation is believed not to correlate with EBV type [2].
• For example, endemic Burkitt lymphoma (BL) classically presents as a monoclonal, c-myc-translocation-positive tumor in which every cell carries EBV as an EBNA1-only (Latency I) infection; such homogeneity among EBV-positive cells, and the lack of EBV-negative comparators, hampers attempts to understand EBV's role in BL pathogenesis [3].
• The carboxyl-terminal one-third of the Epstein-Barr virus nuclear antigen (EBNA-1) encoded by the BamHI restriction fragment K was synthesized in Escherichia coli by use of a high-expression plasmid [4].
• EBNA-1 also regulates viral gene expression and is required for cell immortalization, suggesting that LANA performs similar functions in the context of HHV-8 infection [5].

High impact information on EBNA-1

• In addition, they contain multiple copies of the EBV genome, express the viral nuclear antigen (EBNA) and, most importantly, proceed to undergo transformation when placed back in culture [6].
• To examine the role of EBV nuclear antigen (EBNA) 3C in the proliferation of LCLs, we established LCLs infected with an EBV recombinant that expresses EBNA3C with a C-terminal fusion to a 4-hydroxytamoxifen (4HT)-dependent mutant estrogen receptor, E3C-HT [7].
• Both rabbit monospecific antibodies and mouse monoclonal antibodies against 28-kDa EBNA gave nuclear immunofluorescence staining on Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines and recognized the appropriate intact EBNA polypeptide bands on immunoblots [4].
• Despite continuous antigen presence due to persistent EBV infection, half of the proliferating EBNA1-specific CD4(+) T cells belonged to the central-memory compartment (T(CM)) [8].
• The remaining EBNA1-specific CD4(+) T cells displayed an effector-memory phenotype (T(EM)), of which a minority rapidly secreted IFN upon stimulation with EBNA1 [8].

Chemical compound and disease context of EBNA-1

• Survival of the EBV-positive cells is not consistently associated with EBV lytic gene expression or with the genes that are expressed in EBV latency I BL cells (i.e., EBNA1, EBERs, and BARTs) but correlates with reduced expression of the cellular proapoptotic BH3-only protein Bim [9].
• Regulation of the EBNA1 Epstein-Barr virus protein by serine phosphorylation and arginine methylation [10].

Biological context of EBNA-1

• DNA methylation does not control, however, Qp (a promoter for EBNA1 transcripts only) in lymphoblastoid cell lines (LCLs), although in vitro methylated Qp-reporter gene constructs are silenced [11].
• We hypothesized that transcription levels of EBNA1, the gene essential for EBV persistence within an infected cell, are similar in BL cell lines [12].
• Type I latency and type III latency can be distinguished by the expression of EBNA2, which has been shown to be regulated, in part, by the EBNA1-dependent enhancer activity of the origin of replication (OriP) [13].
• In the Epstein-Barr virus genome, a total of 26 EBNA-1-binding sites occur within three clustered loci referred to as the family of repeats and dyad symmetry locus of ori-P and the separate BamHI-Q locus [14].
• These reconstruction experiments demonstrated that a transactivation domain exists within the carboxy-terminal region of EBNA-1, that transactivation is more efficient when a nuclear localization signal is present, and that the natural karyophilic signal lies outside of the carboxy-terminal 191 amino acids [15].

Anatomical context of EBNA-1

• This cell line allowed us to examine expression from the endogenous latency gene promoters in the context of an actual latent infection and the presence of other EBNA proteins, in particular EBNA-2, which is presumed to coregulate transcription with EBNA-3C [16].
• CONCLUSION: Our results suggest uniform EBNA1 transcription levels in BL and that microarray profiling can reveal novel insights on quantitative EBV gene transcription and its impact on lymphocyte biology [12].
• Presumably, initiation of replication at the DS, mediated by EBNA-1, is important for the natural life cycle of EBV, perhaps in establishing latent infections of normal B cells [17].
• COS-1 cells will thus provide a valuable system in which to analyze functional domains of the EBNA gene [18].
• In mouse fibroblast cell lines expressing PyV large T antigen (LT) and either BPV1 E2 or EBV EBNA1, the long-term episomal replication of plasmids carrying the PyV minimal origin together with the MME or family of repeats (FR) element can be monitored easily for 1 month under nonselective conditions [19].

Associations of EBNA-1 with chemical compounds

• Similar results were obtained when a partially purified preparation of intact Raji EBNA-1 was substituted for the 28K EBNA-1, and the results were further supported by methylation interference studies which indicated contact points between EBNA-1 and the guanine residues at positions -8, -7, and +3 of the binding site [14].
• EBNA-1 was then purified by applying the whole cell extract soluble fraction to a Ni-NTA Superflow column and eluting with an imidazole gradient [20].
• EBNA-1 can subdue immune recognition by virtue of a long glycine and alanine-rich repeat, which interferes with the proteasomal degradation of EBNA-1 and in this way averts the presentation of antigenic peptides derived from it [21].
• Thereafter, an alternative EBNA promoter, Cp, becomes dominant, Wp activity declines dramatically, and bisulfite sequencing of EBV-transformed lymphoblastoid cell lines (LCLs) shows extensive Wp methylation [22].
• EBNA1 was also found to bind the arginine methyltransferases PRMT1 and PRMT5 [10].

Other interactions of EBNA-1

• EBV RNA positivity was even higher in fresh samples stored at -80 degrees C until RNA expression analyses (88% for both EBNA1 and BARF1) [23].
• When the new assays were used to screen a collection of endemic Burkitt's lymphoma tumours, abundant Qp-driven EBNA1 expression was found, whereas the other latent transcripts (with the exception of LMP2A) were either absent or detectable only at trace levels [24].

Analytical, diagnostic and therapeutic context of EBNA-1

• Three immobilization chemistries for the attachment of BSA-EBNA were investigated to optimize ligand density and minimize loss of EBNA-1 immunoreactivity [25].
• Epstein-Barr nuclear antigen 1 (EBNA1) and the carcinoma-specific BARF1 mRNA were detected by nucleic acid sequence based amplification and found in 86 and 74% of NP brushings, confirming NPC tumour cell presence [23].
• This paper describes the development of two multiplex-nested RT-PCR devised to evaluate latent/immortalizing (EBNA1, EBNA2, LMP1 and LMP2) and lytic [immediate early (Zebra), early, and late (VCA), respectively] Epstein Barr virus (EBV) transcripts [26].
• The expression of EBV nuclear antigen 1 (EBNA1) and LMP1 were determined by immunohistochemistry, and the expression of EBV-encoded RNAs (EBERs) was detected by in situ hybridization [27].
• EBNA-1, LMP-1 and ZEBRA protein expressions were investigated by immunohistochemistry [28].

References