A single amino acid in EBNA-2 determines superior B lymphoblastoid cell line growth maintenance by Epstein-Barr virus type 1 EBNA-2.

UNLABELLED: Sequence differences in the EBNA-2 protein mediate the superior ability of type 1 Epstein-Barr virus (EBV) to transform human B cells into lymphoblastoid cell lines compared to that of type 2 EBV. Here we show that changing a single amino acid (S442D) from serine in type 2 EBNA-2 to the aspartate found in type 1 EBNA-2 confers a type 1 growth phenotype in a lymphoblastoid cell line growth maintenance assay. This amino acid lies in the transactivation domain of EBNA-2, and the S442D change increases activity in a transactivation domain assay. The superior growth properties of type 1 EBNA-2 correlate with the greater induction of EBV LMP-1 and about 10 cell genes, including CXCR7. In chromatin immunoprecipitation assays, type 1 EBNA-2 is shown to associate more strongly with EBNA-2 binding sites near the LMP-1 and CXCR7 genes. Unbiased motif searching of the EBNA-2 binding regions of the differentially regulated cell genes identified an ETS-interferon regulatory factor composite element motif that closely corresponds to the sequences known to mediate EBNA-2 regulation of the LMP-1 promoter. It appears that the superior induction by type 1 EBNA-2 of the cell genes contributing to cell growth is due to their being regulated in a manner different from that for most EBNA-2-responsive genes and in a way similar to that for the LMP-1 gene. IMPORTANCE: The EBNA-2 transcription factor plays a key role in B cell transformation by EBV and defines the two EBV types. Here we identify a single amino acid (Ser in type 1 EBV, Asp in type 2 EBV) of EBNA-2 that determines the superior ability of type 1 EBNA-2 to induce a key group of cell genes and the EBV LMP-1 gene, which mediate the growth advantage of B cells infected with type 1 EBV. The EBNA-2 binding sites in these cell genes have a sequence motif similar to the sequence known to mediate regulation of the EBV LMP-1 promoter. Further detailed analysis of transactivation and promoter binding provides new insight into the physiological regulation of cell genes by EBNA-2.

C-terminal region of EBNA-2 determines the superior transforming ability of type 1 Epstein-Barr virus by enhanced gene regulation of LMP-1 and CXCR7.

Type 1 Epstein-Barr virus (EBV) strains immortalize B lymphocytes in vitro much more efficiently than type 2 EBV, a difference previously mapped to the EBNA-2 locus. Here we demonstrate that the greater transforming activity of type 1 EBV correlates with a stronger and more rapid induction of the viral oncogene LMP-1 and the cell gene CXCR7 (which are both required for proliferation of EBV-LCLs) during infection of primary B cells with recombinant viruses. Surprisingly, although the major sequence differences between type 1 and type 2 EBNA-2 lie in N-terminal parts of the protein, the superior ability of type 1 EBNA-2 to induce proliferation of EBV-infected lymphoblasts is mostly determined by the C-terminus of EBNA-2. Substitution of the C-terminus of type 1 EBNA-2 into the type 2 protein is sufficient to confer a type 1 growth phenotype and type 1 expression levels of LMP-1 and CXCR7 in an EREB2.5 cell growth assay. Within this region, the RG, CR7 and TAD domains are the minimum type 1 sequences required. Sequencing the C-terminus of EBNA-2 from additional EBV isolates showed high sequence identity within type 1 isolates or within type 2 isolates, indicating that the functional differences mapped are typical of EBV type sequences. The results indicate that the C-terminus of EBNA-2 accounts for the greater ability of type 1 EBV to promote B cell proliferation, through mechanisms that include higher induction of genes (LMP-1 and CXCR7) required for proliferation and survival of EBV-LCLs.

Cellular gene expression that correlates with EBER expression in Epstein-Barr Virus-infected lymphoblastoid cell lines.

Novel Epstein-Barr Virus (EBV) strains with deletion of either EBER1 or EBER2 and corresponding revertant viruses were constructed and used to infect B lymphocytes to make lymphoblastoid cell lines (LCLs). The LCLs were used in microarray expression profiling to identify genes whose expression correlates with the presence of EBER1 or EBER2. Functions of regulated genes identified in the microarray analysis include membrane signaling, regulation of apoptosis, and the interferon/antiviral response. Although most emphasis has previously been given to EBER1 because it is more abundant than EBER2, the differences in cell gene expression were greater with EBER2 deletion. In this system, deletion of EBER1 or EBER2 had little effect on the EBV transformation frequency of primary B cells or the growth of the resulting LCLs. Using the recombinant viruses and novel EBER expression vectors, the nuclear redistribution of rpL22 protein by EBER1 in 293 cells was confirmed, but in LCLs almost all of the cells had a predominantly cytoplasmic expression of this ribosomal protein, which was not detectably changed by EBER1. The changes in LCL gene expression identified here will provide a basis for identifying the mechanisms of action of EBER RNAs.

Regulation of the Epstein-Barr virus Zp promoter in B lymphocytes during reactivation from latency.

Ten novel mutations were introduced into the Zp promoter to test the role of sequences outside the established transcription factor-binding sites in Epstein-Barr virus (EBV) reactivation. Most of these had only small effects, but mutations in the ZID site were shown to reduce Zp activity strongly at early times after induction by anti-immunoglobulin (anti-Ig). The binding of MEF2 transcription factor to ZID was characterized in detail and linked functionally to Zp promoter activity. The presence of XBP-1s, the active form of XBP-1, after administration of anti-Ig to Akata Burkitt's lymphoma cells is consistent with a role for this factor in reactivation of the EBV lytic cycle, although signalling through MEF2D was quantitatively much more significant in activation of Zp. Silencing of Zp during latency is thought to be primarily a consequence of a repressive chromatin structure on Zp, and this aspect of Zp regulation can be observed in the Akata genome through protection of Zp from activation by BZLF1 in the absence of signalling from the B-cell receptor.

Epstein-Barr virus BART gene expression.

Introns from the Epstein-Barr virus (EBV) BART RNAs produce up to 20 micro RNAs (miRNAs) but the spliced exons of the BART RNAs have also been investigated as possible mRNAs, with the potential to express the RPMS1 and A73 proteins. Recombinant RPMS1 and A73 proteins were expressed in Escherichia coli and used to make new monoclonal antibodies that reacted specifically with artificially expressed RPMS1 and A73. These antibodies did not detect endogenous expression of A73 and RPMS1 proteins in a panel of EBV-infected cell lines representing the different known types of EBV infection. BART RNA could not be detected on Northern blots of cytoplasmic poly(A)(+) RNA from the C666.1 NPC cell line and BART RNA was found to be mainly in the nucleus of C666.1 cells, arguing against an mRNA role for BART RNAs. In contrast, some early lytic cycle EBV mRNAs were found to be expressed in C666.1 cells. Artificially expressed A73 protein was known to be able to bind to the cellular RACK1 protein and has now also been shown to be able to regulate calcium flux, presumably via RACK1. Overall, the results support the conclusion that the miRNAs are functionally important products of BART transcription in the cell lines studied because the A73 and RPMS1 proteins could not be detected in natural EBV infections. However, the possibility remains that A73 and RPMS1 might be expressed in some situations because of the clear potential relevance of their biochemical functions.

Cell target genes of Epstein-Barr virus transcription factor EBNA-2: induction of the p55alpha regulatory subunit of PI3-kinase and its role in survival of EREB2.5 cells.

Microarray analysis covering most of the annotated RNAs in the human genome identified a panel of genes induced by the Epstein-Barr virus (EBV) EBNA-2 transcription factor in the EREB2.5 human B-lymphoblastoid cell line without the need for any intermediate protein synthesis. Previous data indicating that PIK3R1 RNA (the alpha regulatory subunit of PI3-kinase) was induced were confirmed, but it is now shown that it is the p55alpha regulatory subunit that is induced. Several EBV-immortalized lymphoblastoid cell lines were shown to express p55alpha. Expression of PI3-kinase p85 regulatory and p110 catalytic subunits was not regulated by EBNA-2. Proliferation of EREB2.5 lymphoblastoid cells was inhibited by RNAi knock-down of p55alpha protein expression, loss of p55alpha being accompanied by an increase in apoptosis. p55alpha is thus a functional target of EBNA2 in EREB2.5 cells and the specific regulation of p55alpha by EBV will provide an opportunity to investigate the physiological function of p55alpha in this human cell line.

Transcriptional cross-regulation of RUNX1 by RUNX3 in human B cells.

RUNX transcription factors are important in development and in numerous types of human cancer. They act as either transcriptional activators or repressors and can be proto-oncogenes or tumour suppressors. Understanding their regulation and interaction may explain how RUNX factors contribute to such different and often opposing biological processes. We show that RUNX3 regulates RUNX1 expression, contributing to the mutually exclusive expression of RUNX3 and RUNX1 in human B lymphoid cell lines. RUNX3 repressed the RUNX1 P1 promoter by binding specifically to conserved RUNX sites near the transcription start of the promoter. siRNA inhibition of RUNX3 in lymphoblastoid cells resulted in increased RUNX1 expression, indicating that continuous expression of physiological levels of RUNX3 is required to maintain repression. Furthermore, expression of RUNX3 was required for efficient proliferation of B cells immortalized by Epstein-Barr virus. Cross-regulation between different RUNX family members is therefore a means of controlling RUNX protein expression and must now be considered in the interpretation of pathological changes due to loss of RUNX3 tumour suppressor function or following gene duplication or translocation events.

Lytic cycle gene regulation of Epstein-Barr virus.

Episomal reporter plasmids containing the Epstein-Barr virus (EBV) oriP sequence stably transfected into Akata Burkitt's lymphoma cells were used to analyze EBV lytic cycle gene regulation. First, we found that the Zp promoter of EBV, but not the Rp promoter, can be activated in the absence of protein synthesis in these oriP plasmids, casting doubt on the immediate early status of Rp. An additional level of regulation of Zp was implied by analysis of a mutation of the ZV element. Second, our analysis of late lytic cycle promoters revealed that the correct relative timing, dependence on ori lyt in cis, and sensitivity to inhibitors of DNA replication were reconstituted on the oriP plasmids. Late promoter luciferase activity from oriP plasmids also incorporating replication-competent ori lyt was phosphonoacetic acid sensitive, a hallmark of EBV late genes. A minimal ori lyt, which only replicates weakly, was sufficient to confer late timing of expression specifically on late promoters. Finally, deletion analysis of EBV late promoter sequences upstream of the transcription start site confirmed that sequences between -49 and +30 are sufficient for late gene expression, which is dependent on ori lyt in cis. However, the TATT version of the TATA box found in many late genes was not essential for late expression.