Inhibition of the synthesis of proteins needed for Epstein-Barr virus replication by antisense RNA against the zta gene

Antisense RNA complementary to the Epstein-Barr virus (EBV) Zta gene, an immediate-early gene encoding a transactivator, was applied to inhibit EBV protein synthesis during its lytic cycle. A DNA fragment containing the Zta gene sequence was inserted into an expression vector, pMAMneo, in a sense and antisense direction under a dexamethasone-inducible murine mammary tumor virus LTR promoter, resulting in the construction of plasmids pZ(+) and pZ(–), respectively. Synthesis of Zta protein was reduced in pZ(–)-transfected cells upon dexamethasone induction. Because D-form early antigen and DNA polymerase are essential for viral DNA replication, the contents of these two viral proteins were examined. Amounts of the two lytic proteins were observed to be significantly repressed in pZ(–)-transfected cells. In contrast, both proteins were normally expressed in the sense plasmid pZ(+) or cells transfected with vector alone. Above results demonstrate that Zta antisense RNA can reduce the production of Zta protein and the other lytic proteins, possibly resulting in the inhibition of EBV replication.     

Cell cycle analysis of Epstein-Barr virus-infected cells following treatment with lytic cycle-inducing agents

While Epstein-Barr virus (EBV) latency-associated gene expression is associated with cell cycle progression, the relationship between the EBV lytic program and the cell cycle is less clear. Using four different EBV lytic induction systems, we address the relationship between lytic cycle activation and the cell cycle. In three of these systems, G0 or G1 cell growth arrest signaling is observed prior to detection of the EBV immediate-early gene product Zta. In tetradecanoyl phorbol acetate-treated P3HR1 cultures and in 5-iodo-2'-deoxyuridine-treated NPC-KT cultures, cell cycle analysis of Zta-expressing cell populations showed a significant G1 bias during the early stages of lytic cycle progression. In contrast, treatment of the cell line Akata with anti-immunoglobulin (Ig) results in rapid induction of immediate-early gene expression, and accordingly, activation of the immediate-early gene product Zta precedes significant anti-Ig-induced cell cycle effects. Nevertheless, cell cycle analysis of the Zta-expressing population following anti-Ig treatment shows a bias for cells in G1, indicating that anti-Ig-mediated induction of Zta occurs more efficiently in cells traversing G1. Last, although 5-azacytidine treatment of Rael cells results in a G1 arrest in the total cell population which precedes the induction of Zta, cell cycle analysis of the Zta-expressing population shows a significant bias for cells with an apparent G2/M DNA content. This bias may result, in part, from activation of Zta expression following demethylation of the Zta promoter during S-phase. Together, these studies indicate that induction of Zta occurs through several distinct mechanisms, some of which may involve checkpoint signaling.     

Analysis of global methylation using a Zta-expressing nasopharyngeal carcinoma cell line

EBV infects more than 90% of the human population and persists in most individuals as a latent infection where the viral genome is silenced by host-driven methylation. The lytic cycle is initiated when the viral protein Zta binds to methylated BRLF1 and BRRF1 promoters. Although studies reveal the role of Zta and methylation changes in the viral genome upon EBV infection to reactivation, whether Zta plays any role in alteration of methylation in the host genome remains unknown. Using an inducible model, we demonstrate that global DNA methylation, based on whole-genome 5-methylcytosine content, and regional DNA methylation in repetitive elements, imprinting genes and the X chromosome, remains unchanged in response to Zta expression. Expression of DNA methyltransferases was also unaffected by ectopically expressed Zta. Our data imply that alteration of host gene expression following EBV reactivation may reflect methylation-independent Zta-mediated gene activation and not epigenetic modification of the host genome.     

Interaction with the Epstein-Barr virus helicase targets Zta to DNA replication compartments

Zta has a dual role in the Epstein-Barr virus (EBV) lytic cycle, acting as a key regulator of EBV lytic gene expression and also being essential for lytic viral DNA replication. Zta's replication function is mediated in part through interactions with the core viral replication proteins. We now show interaction between Zta and the helicase (BBLF4) and map the binding region to within amino acids (aa) 22 to 86 of the Zta activation domain. In immunofluorescence assays, green fluorescent protein (GFP)-tagged BBLF4 localized to the cytoplasm of transfected cells. Cotransfection of Zta resulted in translocation of BBLF4-GFP into the nucleus indicating interaction between these two proteins. However, Zta with a deletion of aa 24 to 86 was unable to mediate nuclear translocation of BBLF4-GFP. Results obtained with Zta variants carrying deletions across the aa 24 to 86 region indicated more than one contact site for BBLF4 within this domain, and this was reinforced by the behavior of the four-point mutant Zta (m22/26,74/75), which was severely impaired for BBLF4 interaction. Binding of BBLF4 to Zta was confirmed using GST affinity assays. In both cotransfection-replication assays and replication assays performed in EBV-positive P3HR1 cells, the Zta (m22/26,74/75) mutant was replication defective. In Zta-transfected D98-HR1 cells, replication compartments could be detected by immunofluorescence staining using anti-BMRF1 monoclonal antibody. Cells transfected with Zta variants that were defective for helicase binding still formed replication compartments, but Zta was excluded from these compartments. These experiments reveal a role for the Zta-helicase interaction in targeting Zta to sites of viral DNA replication.     

trans-acting requirements for replication of Epstein-Barr virus ori-Lyt.

Epstein-Barr virus (EBV) utilizes a completely different mode of DNA replication during the lytic cycle than that employed during latency. The latency origin of replication, ori-P, which functions in the replication of the latent episomal form of the EBV genome, requires only a single virally encoded protein, EBNA-1, for its activity. During the lytic cycle, a separate origin, ori-Lyt, is utilized. Relatively little is known about the trans-acting proteins involved in ori-Lyt replication. We established a cotransfection-replication assay to identify EBV genes whose products are required for replication of ori-Lyt. In this assay, a BamHI-H plasmid containing ori-Lyt was replicated in Vero cells cotransfected with the BamHI-H target, the three EBV lytic-cycle transactivators Zta, Rta, and Mta, and the EBV genome provided in the form of a set of six overlapping cosmid clones. By removing individual cosmids from the cotransfection mixture, we found that only three of the six cosmids were necessary for ori-Lyt replication. Subcloning of the essential cosmids led to the identification of six EBV genes that encode replication proteins. These genes and their functions (either known or predicted on the basis of sequence comparison with herpes simplex virus) are BALF5, the DNA polymerase; BALF2, the single-stranded DNA-binding protein homolog; BMRF1, the DNA polymerase processivity factor; BSLF1 and BBLF4, the primase and helicase homologs; and BBLF2/3, a potential homolog of the third component of the helicase-primase complex. In addition, ori-Lyt replication in this cotransfection assay was also dependent on one or more genes provided by the EBV SalI-F fragment and on the three lytic-cycle transactivators Zta, Rta, and Mta.     

Different activation of Epstein-Barr virus immediate-early and early genes in Burkitt lymphoma cells and lymphoblastoid cell lines.

Specific expression of the Epstein-Barr virus (EBV) immediate-early and early gene products Zta, Rta, I'ta, and MSta by a recombinant vaccinia virus system allowed us to analyze the first steps in the induction of the lytic cycle in EBV-infected Burkitt lymphoma (BL) cells and lymphoblastoid cell lines (LCLs). Significant differences in the induction of early genes were found between these cell types: whereas in BL cells the trans activator Zta was found to induce key steps of the early lytic cycle, only minor activities of Zta were noted in LCLs. Contrary to Zta, the trans activator Rta was found to be highly effective in LCLs. These observations suggest that Rta may play an important role in the activation of the early lytic cycle in LCLs, although it cannot be activated by Zta. The latter may be a reason for the lower tendency of LCLs to switch into the lytic cycle compared with BL cells or differentiated epithelial cells.