Disease relevance of EBNA-2

• Two Epstein-Barr virus (EBV) types are known, EBV1 and EBV2, which possess substantially diverged alleles for latency genes EBNA-2, EBNA-3A, EBNA-3B and EBNA-3C but are thought to be otherwise similar [1].
• MAIN OUTCOME MEASURES: Antibodies including IgA against EBV viral capsid antigen (VCA) and IgG against VCA, nuclear antigens (EBNA complex, EBNA-1, and EBNA-2), diffuse and restricted early antigens, and cytomegalovirus [2].
• In the present investigation we show that the resistance to the anti-proliferative effect of IFN class I on certain EBV-carrying Burkitt lymphoma cell lines is connected to the presence of the EBNA-2 gene and parts of the EBNA-5 gene of the EBV genome [3].
• The demonstration of multiple variably sized BamHI H fragments on Southern blot analysis and cloning of the EBNA-2 gene coding region also suggested the presence of multiple viral strains or variants coinfecting hairy leukoplakia [4].
• To determine whether another transcriptional activator can substitute for this function, a chimeric virus was constructed that contained a portion of the transcriptional activation domain from the herpes simplex virus VP16 protein inserted in place of the 14-amino acid domain of EBNA-2 [5].

High impact information on EBNA-2

• Epstein-Barr virus-associated Burkitt lymphomagenesis selects for downregulation of the nuclear antigen EBNA2 [6].
• Here we identify a subset of BL tumors in which the Latency III-associated EBNA promoter Wp is active and most EBNAs are expressed, but where a gene deletion has specifically abrogated the expression of EBNA2 [6].
• The malignant cells latently infected with EBV typically express the transcription factor EBNA2 as one of nine latent viral genes [7].
• We tested whether an EBNA2-responsive EBV promoter may selectively target EBV-related lymphoma cells by virus-regulated expression of a suicide gene [7].
• Using the BamC promoter driving a hygromycin-thymidine kinase fusion gene or controls, we demonstrated that sensitivity to ganciclovir was selectively enhanced in cells expressing EBNA2 [7].

Chemical compound and disease context of EBNA-2

• Furthermore, 5-Azacytidine induced upregulation of the latent EBV genes LMP2A, LMP2B, and EBNA2 in a similar fashion as observed following switching of latent to lytic EBV upon cross-linking of the B-cell receptor [8].
• Only the wild-type EBNA-2 HYG gene EBV recombinant transformed primary B lymphocytes [9].
• One of the novel genes, C5, is a positional homologue for the transformation-essential EBV gene EBNA-2 [10].
• Epstein-Barr virus nuclear antigen 3C putative repression domain mediates coactivation of the LMP1 promoter with EBNA-2 [11].
• A conditional mutant of Epstein-Barr virus nuclear antigen 2 (EBNA2) regulated by estrogen was employed to study the effect of EBNA2 on the cellular phenotype [12].

Biological context of EBNA-2

• Here, we report that CTCF, a chromatin boundary factor with well-established enhancer-blocking activity, binds to EBV sequences between the OriP and the RBP-Jkappa response elements of the C promoter (Cp) and regulates transcription levels of EBNA2 mRNA [13].
• 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].
• Deletion of this CTCF binding site in a recombinant EBV bacterial artificial chromosome (BAC) increased EBNA2 transcription by 3.5-fold compared to a wild-type EBV BAC [13].
• EBV signaling (through EBNA2, LMP1 and LMP2A) and NF-kB activation correlated with upregulation of target proteins: cMYC, JunB, CCL22, TRAF1 and IRF4 [14].
• Epstein-Barr virus nuclear antigen 2 (EBNA-2) plays a crucial role in B cell immortalization by Epstein-Barr virus (EBV), most probably by its ability to transactivate several cellular and viral genes [15].

Anatomical context of EBNA-2

• 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].
• We also show that c-MYC, as well as EBNA2, can stimulate CTCF mRNA levels, suggesting that CTCF levels may contribute to B-cell differentiation as well as EBV latency type determination [13].
• Thus, EBNA-2 is an essential molecule in lymphocyte growth transformation by EBV and a major determinant of the differences between EBV-1 and EBV-2 in lymphocyte growth transformation [17].
• Lack of the expression of EBNA-2 and LMP-1 in T-cell neoplasms possessing Epstein-Barr virus [18].
• Group 2 isolates (HD, n = 1; AILD, n = B-NHL, n = 1; tonsils of EBV-seropositive children, n = 9; IM, n = 20 showed a nucleotide change at position 49095 in the EBNA-2 gene, leading to an amino acid substitution (Pro-->Ser), and EBV type-2 sequences in the EBER region [19].

Associations of EBNA-2 with chemical compounds

• A glutathione S-transferase-EBNA-2 fusion protein containing aa 252-425 of EBNA-2 interacted with CBF1 to yield a slowly migrating complex in an electrophoretic mobility shift assay [20].
• Mutation of a tryptophan within the 14-amino acid domain of EBNA-2 completely abolished transcriptional activation and B-cell transformation [5].
• EBNA-2 is an acidic transcriptional transactivator that is brought to virus and cell EBNA-2 response elements by interaction with a factor that recognizes the double-stranded sequence MNYYGTGGGAA, where M is A or C, N is any nucleotide, and Y is a pyrimidine [21].
• Intrastrain recombination within the HLP lesion also generated variation within the EBNA-2 polyproline region [22].
• (ii) Substitutions of this site in each W2 repeat domain with alanine markedly reduced the ability of the protein to induce LMP-1 expression in combination with EBNA-2 in Akata cells [23].

Enzymatic interactions of EBNA-2

• The mutant EBNA-2 genes were assayed for lymphocyte transformation after recombination with the EBNA-2-deleted P3HR-1 EBV genome and for LMP-1 transactivation following transfection into P3HR-1-infected B-lymphoma cells [24].

Regulatory relationships of EBNA-2

• Immunoblotting of LGL protein extracts established that, of the EBV gene products, EBV nuclear antigen-1 (EBNA-1) was expressed but EBNA-2 and the latent membrane protein (LMP-1) were not detectable in the leukemic cells [25].
• Expression of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) oncogene is regulated by the EBV nuclear protein 2 (EBNA-2) transactivator [26].
• EBNA-LP also stimulates EBNA-2 activation of a multimerized regulatory element from the BamC EBNA promoter [27].
• A few EBV-infected B cells expressed BZLF1 (an EBV immediate-early gene product) while none expressed EBNA-2 or latent membrane protein 1 [28].
• In a transient-expression assay, the EBNA2-activated transcription was found to be downregulated by EBNA3A, EBNA3B, and EBNA3C [29].
• Since BCL6 is indispensable for GC formation and somatic hypermutations (SHM), we suggest that the previously reported lack of SHM seen in EBNA2-expressing GC cells from infectious mononucleosis tonsils could be due to negative regulation of BCL6 by EBNA2 [30].

Other interactions of EBNA-2

• 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 [31].
• RPMS1 can inhibit the transcription activation induced through CBF1 by NotchIC or EBNA-2 [32].
• Vectors encoding two viral genes, EBNA-2 and EBNA-LP, are sufficient to activate the expression of cyclin D2 in this system [33].
• Expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) and the EBNA-3 genes is variable relative to that of EBNA-1, as is characteristic of some naturally infected Burkitt tumor cells [34].
• These cases also expressed LMP-1, EBNA-2 and BZLF-1, but at differing levels [35].

Analytical, diagnostic and therapeutic context of EBNA-2

• The primers designed to amplify these three gene loci encompass either type-specific deletion sequences (EBNA-2 and EBNA-3C) or type-specific point mutations (EBER) that identify the virus strain based on the sizes of the polymerase chain reaction (PCR)-amplified products or the mobility shifts in single-strand conformation polymorphism analysis [36].
• Western blot analysis for existence of size polymorphisms in three classes of Epstein-Barr nuclear antigens (EBNA-1, EBNA-2, and EBNA-3) confirmed the DNA results [37].
• EBNA-2-positive cells were detected by anti-complement immunofluorescence staining just 12 h after addition of 4 microM 5-AzaC and reached a maximum number at 72 h, when up to 75% of the population was positive [38].
• We have shown by both gel filtration and sucrose gradient analysis that the purified material corresponds to a multimer containing eight EBNA-2 subunits [39].
• Although EBNA-2 and EBNA-3C are extensively associated with RBPJkappa, a fraction of RBPJkappa appears to be free of EBNAs after repeated immunoprecipitations with anti-EBNA, Epstein-Barr virus-immune, human antibody [40].

References