Epstein–Barr virus Rta-mediated transactivation of p21 and 14-3-3σ arrests cells at the G1/S transition by reducing cyclin E/CDK2 activity

Abstract

Many herpesviral immediate-early proteins promote their robust lytic phase replications by hijacking the cell cycle machinery. Previously, lytic replication of Epstein-Barr virus (EBV) was found to be concurrent with host cell cycle arrest. In this study, we showed that ectopic expression of EBV immediate-early protein Rta in HEp-2 cells resulted in increased G1/S population, hypophosphorylation of pRb and decreased incorporation of 5-bromo-29-deoxyuridine. In addition, EBV Rta transcriptionally upregulates the expressions of p21 and 14-3-3s in HEp-2 cells, 293 cells and nasopharyngeal carcinoma TW01 cells. Although p21 and 14-3-3s are known targets for p53, Rta-mediated p21 and 14-3-3s transactivation can be detected in the absence of p53. In addition, results from luciferase reporter assays indicated that direct binding of Rta to either promoter sequences is not required for activation. On the other hand, a special class of Sp1-responsive elements was involved in Rta-mediated transcriptional activation on both promoters. Finally, Rta-induced p21 expression diminished the activity of CDK2/cyclin E complex, and, Rta-induced 14-3-3s expression sequestered CDK1 and CDK2 in the cytoplasm. Based on these results, we hypothesize that through the disruption of CDK1 and CDK2 activities, EBV Rta might contribute to cell cycle arrest in EBV-infected epithelial cells during viral reactivation. INTRODUCTIONEpstein-Barr virus (EBV) is classified as a member of the gamma herpesvirinae with a dsDNA genome of 172 kbp. EBV infection is associated with various human malignancies of lymphoid and epithelial cell origin (Crawford et al., 1979;Epstein et al., 1964;Henle & Henle, 1976;Imai et al., 1994;Niedobitek & Young, 1994). Generally, EBV maintains itself as a latent episome in a proliferative cell and converts to a lytic replicating genome when the host exits from the last cell cycle (Feederle et al., 2007;Sun & Thorley-Lawson, 2007;Tao et al., 1995). The reactivation of EBV from latency can be induced by various chemicals (Takada, 1984;zur Hausen et al., 1978) or by ectopic expression of the immediate-early transcription activators Zta (aka BZLF1, ZEBRA and IE1) or Rta (aka BRLF1 and IE2) (Countryman & Miller, 1985;Ragoczy et al., 1998;Zalani et al., 1996). Furthermore, Zta and Rta activate each other and function synergistically to complete a full productive replication cycle (Feederle et al., 2000). Expression of Rta in nasopharyngeal carcinoma (NPC) cells is potentially useful in early diagnosis (Feng et al., 2000).The cell cycle is an ordered, tightly regulated process involving multiple checkpoints that ensure a faithful round of cell duplication. However, these fine-tuned processes are often perturbed by an invading virus once a lytic replication cycle is initiated (Flemington, 2001 limited viral gene products and trigger an S phase cell cycle so that a copious environment for viral replication is guaranteed (Hiscott & Defendi, 1979). On the other hand, large DNA viruses, such as herpesviruses, usually encode their own e...

Kaposi's Sarcoma-Associated Herpesvirus Transactivator Rta Induces Cell Cycle Arrest in G 0 /G 1 Phase by Stabilizing and Promoting Nuclear Localization of p27 kip

“…Our finding that K-Rta arrests cells in G 0 /G 1 phase is in general agreement with recent studies that investigated the role of EBVencoded Rta (E-Rta) in cell cycle regulation (18). The expression of inducible E-Rta in 293 and nasopharyngeal carcinoma (NPC)-derived cells resulted in G 1 arrest (18,19), which was associated with elevated levels of p21 and p27.…”

“…The expression of inducible E-Rta in 293 and nasopharyngeal carcinoma (NPC)-derived cells resulted in G 1 arrest (18,19), which was associated with elevated levels of p21 and p27. However, we did not notice any significant changes in the expression of p21 in K-Rta-expressing cells.…”

“…Previous studies investigating the role of cell cycle in herpesvirus lytic replication suggested that host cell cycle arrest precedes the induction of the lytic cycle and essentially determines whether immediate-early gene expression is initiated or not (16). However, current research increasingly supports the idea that cell cycle arrest follows lytic cycle induction and is a direct consequence of immediate-early gene expression (17)(18)(19). It is hypothesized that arresting cells during early lytic replication is a common evolutionary strategy employed by herpesviruses to avoid competition for resources required for viral DNA replication with the host in the S phase, or it may serve to prevent premature apoptosis during lytic replication (20).…”

Role of ATM in the Formation of the Replication Compartment during Lytic Replication of Epstein-Barr Virus in Nasopharyngeal Epithelial Cells

“…In this study, knockdown of endogenous Sp1 expression suppressed lytic EBV reactivation ( Fig. 4C and D), supporting its role in lytic reactivation of EBV (49,(60)(61)(62)(63). Furthermore, Sp1 localized at replication compartments during lytic reactivation (Fig.…”

Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes

“…In order to validate that EBV Rta does bind to these cellular promoters, chromatin immunoprecipitation (ChIP) assay was performed using specific antibodies against EBV Rta and normal rabbit IgG, which served as a control to estimate nonspecific binding signals. In addition, the promoter region of SFN, which is known to be positively regulated upon Rta binding (12), served as a positive control. The results indicated that EBV Rta bound to the promoter regions of MYC, CCND1, JUN, and SFN at an early time point (12 h) after Dox treatment (Fig.…”