Functional differences between type 1 and type 2 Epstein-Barr virus EBNA-2

Epstein-Barr virus (EBV) establishes a lifelong latent infection in the human host. In vitro EBV immortalises primary B lymphocytes giving rise to latently infected lymphoblastoid cell lines (LCLs). EBV strains are classified as type 1 or type 2 according to the sequence of the EBNA-2 gene, which expresses a transcription factor that induces viral and cell genes important for B cell proliferation. Type 1 EBV transforms primary B lymphocytes into LCLs much more efficiently than type 2 EBV, a difference previously mapped to the EBNA-2 locus. In this study, the greater transforming activity of type 1 EBV was found to correlate 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 EBVBAC recombinant viruses. The enhanced ability of type 1 EBNA-2 to induce LMP-1 and CXCR7 genes was also confirmed in Burkitt’s Lymphoma cell lines. 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 sustain proliferation of EBV-LCLs is mostly determined by the C-terminal region of the protein. Substitution of the C-terminal third of type 1 EBNA-2 into the type 2 protein is sufficient to confer 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-terminal part 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

Helicobacter pylori-derived neutrophil-activating protein increases the lifespan of monocytes and neutrophils

P>An invariable feature of Helicobacter pylori-infected gastric mucosa is the persistent infiltration of inflammatory cells. The neutrophil-activating protein (HP-NAP) has a pivotal role in triggering and orchestrating the phlogistic process associated with H. pylori infection. Aim of this study was to address whether HP-NAP might further contribute to the inflammation by increasing the lifespan of inflammatory cells. We report that HP-NAP is able to prolong the lifespan of monocytes, in parallel with the induction of the anti-apoptotic proteins A1, Mcl-1, Bcl-2 and Bcl-X(L). This effect does not result from a direct action on the apoptotic machinery, but rather it requires the release of endogenous pro-survival factors, such as interleukin-1 beta, which probably acts in synergy with other unidentified mediators. We also report that HP-NAP promotes the survival of Ficoll-purified neutrophils in a monocyte-dependent fashion: indeed, mononuclear cell depletion of Ficoll-purified neutrophils completely abolished the pro-survival effect by HP-NAP. In conclusion, our data reinforce the notion that HP-NAP has a pivotal role in sustaining a prolonged activation of myeloid cells

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

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

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