| It has been clear for a number of years that small DNA tumor viruses such as simian virus 40 (SV40) and papilloma virus interact with cell cycle control pathways during lytic replication in a way that promotes entry into the Sphase of the cell cycle. Since these viruses do not code for their own DNA polymerase or other accessory factors that support DNA replication, this strategy is a means of subverting the cell cycle control machinery to support viral DNA replication. In contrast to these viruses, herpesviruses such as EBV contain amuch greater genetic complexity that encodes a viral DNA polymerase, as wellas accessory factors involved in generating nucleotide pools, etc. Indeed, herpesviruses have evolved a distinct viral replication strategy and, unlike SV40 andpapillomavirus, herpesviruses do not require an S-phase environment to support viral replication.The conserved nature of this function across different membersof the herpesvirus family suggests that it is an integral aspect of the herpesvirus replication strategy. Also,regulation of the cell cycle during herpesvirus DNA replication has evolved as a complex series of interactions involving multipleviral factors,further implying an important role for this function in the life cycle of the virus.The EBV is a B lymphotropic gammaherpesvirus,which is one of the most widespread viruses. Studies have shown that EBV infection is found in >90 ~ 95% humans worldwide. A large numbers of clinical studies show that EBV associates with a variety of malignant diseases including Burkitt's lymphoma, nasopharyngeal carcinoma, and gastric cancer. In addition, post-transplant lymphoproliferative disease (PTLD) is also a serious problem due to EBV infecti on. EBV predominantly infects human B cells and epithelial cells. It exists in host cells in two different states, the latent and lytic states.EBV utilizes two separate classes of genes that carry out very distinct functions in its life cycle. The latency-type gene expression patterns are associated with cell proliferation, and many of these genes function, in part, to activate cell cycle pathways leading to cell proliferation. Accordingly, some form of latency gene expression is invariably observed in EBV-associated tumors. Although the association betweenlatency gene expression and cell cycle progression is well established, the relationship between the lytic gene expression program and the cell cycle is less well understood.Nevertheless, previous studies have provided evidence that in contrast to the latency gene expression program, lytic replication process has a greater dependence on EBV-encoded replication proteins. Upon reactivation, the two key EBV immediate-early (IE) lytic genes, BZLF1 and BRLF1, are expressed. These genes encode transactivators that activate viral and certain cellular promoters andlead to an ordered cascade of viral gene expression: activation of early gene expression, followed by the lytic cascade of viral genome replication and late gene expressionthe,lytic replication cycle may be associated with a nonproliferating cellular milieu. In the oral epithelium, lytic replication occurs primarily inthe outer, more differentiated layers. Again, in this system, it was difficult to determine whether the virus responds to growth arrest (and/or differentiation) signals or whether the virus specifically induces growth arrest. Other studies have shown that the immediate-early EBV gene product Zta can induce cell growth arrest, indicating that the EBV lytic program has evolved a mechanism to shutdown cellular DNA synthesis.Cell cycle regulation during activation and progression of the lytic cascade, however, has received only limited attention. This study was designed to study the mechanisms for the EBV reactivation and progression of the cell cycle.In this study, B lymphoma cell line Raji was used and chemical agent, such as TPA/NaB have often been utilized as BZLF1 inducers in latently infecte d cells. It has been demonstrated that the treatment of Epstein–Barr virus (EBV) latently infected Raji cells with TPA/SB caused the cell growth arrest. The Zta positive cells were predominantly enriched in G0/G1 phase of cell cycle. When Zta expression reached a maximal level, a fraction of Zta expressing cell population reentered S phase. However, the approache is somewhat problematic for specifically analyzing the cellular events altered by the EBV lytic program alone, since it is difficult to distinguish between effects of the virus and those that are induced by the treatment itself. For instance, treatment of latently infected cells with most lytic-cycle-inducing agents causes a G0/G1 arrest prior to detectable expression of IE genes. As another and more favorable approach, introduction of the BZLF1 or BRLF1 expression vector, which alone is sufficient to activate the EBV lytic cascade, has been applied.The data is consistent with the finding using transfection system. Analysis of the expression pattern of a key set of cell cycle regulators revealed that the expression of Zta and Rta substantially interfered with the cell cycle regulatory machinery in Raji cells, strongly inhibiting the expression of Rb and p53 and inducing the expression ofE2F1. Down-regulation of Rb was further demonstrated to be mediated byproteasomal degradation, and p53 and p21 affected at transcription level. The data indicate that both Zta and Rta promote entry into S phase of Raji cells. The important roles of Zta and Rta in EBV lytic reactivation were also demonstrated. Our finding suggests that these two transcriptional activators may act synergistically to govern the expression of downstream early and late genes as well as cellular genes and initiation of lytic cycle and manipulation of cell cycle regulatory mechanisms require the joint and interactive contributions of Rta andZta. |
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