|Epstein-Barr virus transcription factor Zta acts through distal regulatory elements to directly control cellular gene expression. |
Ramasubramanyan, S; Osborn, K; Al-Mohammad, R; Naranjo Perez-Fernandez, IB; Zuo, J; Balan, N; Godfrey, A; Patel, H; Peters, G; Rowe, M; Jenner, RG; Sinclair, AJ
Nucleic acids research
Lytic replication of the human gamma herpes virus Epstein-Barr virus (EBV) is an essential prerequisite for the spread of the virus. Differential regulation of a limited number of cellular genes has been reported in B-cells during the viral lytic replication cycle. We asked whether a viral bZIP transcription factor, Zta (BZLF1, ZEBRA, EB1), drives some of these changes. Using genome-wide chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) we established a map of Zta interactions across the human genome. Using sensitive transcriptome analyses we identified 2263 cellular genes whose expression is significantly changed during the EBV lytic replication cycle. Zta binds 278 of the regulated genes and the distribution of binding sites shows that Zta binds mostly to sites that are distal to transcription start sites. This differs from the prevailing view that Zta activates viral genes by binding exclusively at promoter elements. We show that a synthetic Zta binding element confers Zta regulation at a distance and that distal Zta binding sites from cellular genes can confer Zta-mediated regulation on a heterologous promoter. This leads us to propose that Zta directly reprograms the expression of cellular genes through distal elements.
|Primary B-cell infection with a deltaBALF4 Epstein-Barr virus comes to a halt in the endosomal compartment yet still elicits a potent CD4-positive cytotoxic T-cell response. |
Bernhard Neuhierl,Regina Feederle,Dinesh Adhikary,Birgit Hub,Karsten Geletneky,Josef Mautner,Henri-Jacques Delecluse
Journal of virology
Epstein-Barr virus (EBV) infection is mediated by several viral envelope glycoproteins. We have assessed gp110's functions during the virus life cycle using a mutant that lacks BALF4 (DeltaBALF4). Exposure of various cell lines and primary cell samples of epithelial or lymphoid lineages to the DeltaBALF4 mutant failed to establish stable infections. The DeltaBALF4 virus, however, did not differ from wild-type EBV in its ability to bind and become internalized into primary B cells, in which it elicited a potent T-cell-specific immune reaction against virion constituents. These findings show that DeltaBALF4 viruses can reach the endosome-lysosome compartment and dovetail nicely with the previously identified contribution of gp110 to virus-cell fusion. Other essential steps of the virus life cycle were unaffected in the viral mutant; DNA lytic replication and viral titers were not altered in the absence of gp110, and DeltaBALF4 viruses complemented in trans transformed infected B cells with an efficiency indistinguishable from that observed with wild-type viruses. All of the steps of virus maturation could be observed in lytically induced 293/DeltaBALF4 cells. Induction of lymphoblastoid cells generated with transiently complemented DeltaBALF4 virus led to the production of rare mature virions. We therefore infer that gp110 is not required for virus maturation and egress in 293 cells or in B cells. The DeltaBALF4 virus's phenotypic traits, an inability to infect human cells coupled with potent antigenicity, potentially qualify this mutant as a live vaccine. It will provide a useful tool for the detailed study of EBV-cell interactions in a physiological context.Full Text Article
|Epstein-Barr virus mRNA export factor EB2 is essential for intranuclear capsid assembly and production of gp350. |
Batisse, J; Manet, E; Middeldorp, J; Sergeant, A; Gruffat, H
Journal of virology
Most human herpesviruses, including Epstein-Barr virus (EBV), express a protein which functions primarily as an mRNA export factor. Previously, we deleted the gene for the Epstein-Barr virus mRNA export factor EB2 from the EBV genome and then introduced the mutated genome into 293 cells. Using a transcomplementation assay in which ectopic expression of the transcription factor EB1/ZEBRA was sufficient to induce the EBV productive cycle, we showed that Ori-Lyt-dependent replication of the EBV DNA occurs in the absence of EB2, indicating that EB2 is not essential for the expression and export of early mRNAs. However, in the absence of EB2, no infectious viral particles are produced (H. Gruffat, J. Batisse, D. Pich, B. Neuhierl, E. Manet, W. Hammerschmidt, and A. Sergeant, J. Virol. 76:9635-9644, 2002). In this report, we now show that EB2 is essential for the nuclear export of most, but not all, late mRNAs produced from intronless genes that translate into proteins involved in intranuclear capsid assembly and maturation. As a consequence, we show that EB2 is essential for the proper assembly of intranuclear capsids. Interestingly, the late BLLF1 gene contains an intron, and both unspliced and spliced mRNAs must be exported to the cytoplasm to be translated into gp350 and gp220, respectively (M. Hummel, D. A. Thorley-Lawson, and E. Kieff, J. Virol. 49:413-417, 1984). Our results also demonstrate that although BLLF1 spliced mRNAs are exported to the cytoplasm independently of EB2, EB2 is essential for the nuclear export of unspliced BLLF1 mRNA. In the same assay, herpes simplex virus 1 ICP27 completely inhibited the nuclear export of BLLF1 spliced mRNAs whereas unspliced BLLF1 mRNAs were exported, confirming that in a physiological assay, ICP27 inhibits splicing.Full Text Article
|Glycoprotein gp110 of Epstein-Barr virus determines viral tropism and efficiency of infection. |
Neuhierl, B; Feederle, R; Hammerschmidt, W; Delecluse, HJ
Proceedings of the National Academy of Sciences of the United States of America
The Epstein-Barr virus (EBV) genome has been detected in lymphomas and in tumors of epithelial or mesenchymal origin such as nasopharyngeal carcinoma or leiomyosarcoma. Thus, there is little doubt that EBV can infect cells of numerous lineages in vivo, in contrast to its in vitro infectious spectrum, which appears restricted predominantly to B lymphocytes. We show here that the EBV BALF4 gene product, the glycoprotein gp110, dramatically enhances the ability of EBV to infect human cells. gp110(high) viruses were up to 100 times more efficient than their gp110(low) counterparts in infecting lymphoid or epithelial cells. In addition, gp110(high) viruses infected the carcinoma cell line HeLa and the T cell lymphoma cell line Molt-4, both previously thought to be refractory to EBV infection. Analysis of several virus isolates showed that the amount of BALF4 present within mature virions markedly differed among these strains. In some strains, gp110 was found expressed during lytic replication not only at the nuclear but also at the cellular membrane. Heterologous expression of gp110 during the virus lytic phase neither altered virus concentration nor affected virus binding to cells. It appears that gp110 plays a crucial role after the virus has adhered to its cellular target. gp110 constitutes an important virulence factor that determines infection of non-B cells by EBV. Therefore, the use of gp110(high) viruses will help to determine the range of the target cells of EBV beyond B lymphocytes and provide a useful in vitro model to assess the oncogenic potential of EBV in these cells.