Reactivation of lytic replication from B cells latently infected with Epstein-Barr virus occurs with high S-phase cyclin-dependent kinase activity while inhibiting cellular DNA replication

Productive infection and replication of herpesviruses usually occurs in growth-arrested cells, but there has been no direct evidence in the case of Epstein-Barr virus (EBV), since an efficient lytic replication system without external stimuli does not exist for the virus. Expression of the EBV lytic-switch transactivator BZLF1 protein in EBV-negative epithelial tumor cell lines, however, is known to arrest the cell cycle in G(0)/G(1) by induction of the tumor suppressor protein p53 and the cyclin-dependent kinase (CDK) inhibitors p21(WAF-1/CIP-1) and p27(KIP-1), followed by the accumulation of a hypophosphorylated form of the Rb protein. In order to determine the effect of the onset of lytic viral replication on cellular events in latently EBV-infected B LCLs, a tightly controlled induction system of the EBV lytic-replication program by inducible BZLF1 protein expression was established in B95-8 cells. The induction of lytic replication completely arrested cell cycle progression and cellular DNA replication. Surprisingly, the levels of p53, p21(WAF-1/CIP-1), and p27(KIP-1) were constant before and after induction of the lytic program, indicating that the cell cycle arrest induced by the lytic program is not mediated through p53 and the CDK inhibitors. Furthermore, although cellular DNA replication was blocked, elevation of cyclin E/A expression and accumulation of hyperphosphorylated forms of Rb protein were observed, a post-G(1)/S phase characteristic of cells. Thus, while the EBV lytic program promoted specific cell cycle-associated activities involved in the progression from G(1) to S phase, it inhibited cellular DNA synthesis. Such cellular conditions appear to especially favor viral lytic replication.     

The Epstein-Barr Virus Protein BRLF1 Activates S Phase Entry through E2F1 Induction

The Epstein-Barr Virus (EBV) immediate-early protein BRLF1 is one of two transactivators which mediate the switch from latent to lytic replication in EBV-infected cells. DNA viruses often modulate the function of critical cell cycle proteins to maximize the efficiency of virus replication. Here we have examined the effect of BRLF1 on cell cycle progression. A replication-deficient adenovirus expressing BRLF1 (AdBRLF1) was used to infect normal human fibroblasts and various epithelial cell lines. BRLF1 expression induced S phase entry in contact-inhibited fibroblasts and in the human osteosarcoma cell line U-2 OS. AdBRLF1 infection produced a dramatic increase in the level of E2F1 but not E2F4. In contrast, the levels of Rb, p107, and p130 were decreased in AdBRLF1-infected cells. Electrophoretic mobility shift assays confirmed an increased level of free E2F1 in the AdBRLF1-infected human fibroblasts. Consistent with the previously described effect of E2F1, AdBRLF1-infected fibroblasts had increased levels of p53 and p21 and died by apoptosis. BRLF1-induced activation of E2F1 may be required for efficient EBV lytic replication, since at least one critical viral replication gene (the viral DNA polymerase) is activated by E2F (C. Liu, N. D. Sista, and J. S. Pagano, J. Virol. 70:2545-2555, 1996).     

Inhibition of benzo(a)pyrene-induced cell cycle progression by all-trans retinoic acid partly through cyclin D1/E2F-1 pathway in human embryo lung fibroblasts

Benzo(a)pyrene [B(a)P] is a potent environmental carcinogen, which induces cell cycle changes. All-trans retinoic acid (ATRA) is a promising agent in prevention and treatment of human cancers. In the present study, we investigated the inhibition of B(a)P-induced cell cycle progression by ATRA in human embryo lung fibroblast (HELF). Our results showed that after treatment with B(a)P, the expression of cyclin D1 and E2F-1 were both increased significantly in HELF. There were almost no changes of CDK4 and E2F-4 expression by treatment with B(a)P. As expected, pretreatment with ATRA could efficiently decrease B(a)P-induced overexpression of cyclin D1 and E2F-1. In a further study, we stably transfected antisense cyclin D1 and antisense CDK4 plasmid into HELF. The inhibition of cyclin D1 expression and the inhibition of CDK4 expression significantly impaired the B(a)P-induced overexpression of E2F-1 respectively. Pretreatment with ATRA, cells expressing antisense cyclinD1 or antisense CDK4 showed a lesser decrease of B(a)P-induced overexpression of E2F-1 compared with similarly treated HELF. Furthermore, flow cytometry analysis showed that B(a)P promoted cell cycle progression from G(1) phase to S phase, while pretreatment with ATRA could inhibit B(a)P-induced cell cycle progression by an accumulation of cells in the G(1) phase. It was suggested that ATRA could block B(a)P-induced cell cycle promotion partly through the cyclin D1/E2F-1 pathway in HELF.     

The lytic cycle of Epstein-Barr virus is associated with decreased expression of cell surface major histocompatibility complex class I and class II molecules

Human herpesviruses utilize an impressive range of strategies to evade the immune system during their lytic replicative cycle, including reducing the expression of cell surface major histocompatibility complex (MHC) and immunostimulatory molecules required for recognition and lysis by virus-specific cytotoxic T cells. Study of possible immune evasion strategies by Epstein-Barr virus (EBV) in lytically infected cells has been hampered by the lack of an appropriate permissive culture model. Using two-color immunofluorescence staining of cell surface antigens and EBV-encoded lytic cycle antigens, we examined EBV-transformed B-cell lines in which a small subpopulation of cells had spontaneously entered the lytic cycle. Cells in the lytic cycle showed a four- to fivefold decrease in cell surface expression of MHC class I molecules relative to that in latently infected cells. Expression of MHC class II molecules, CD40, and CD54 was reduced by 40 to 50% on cells in the lytic cycle, while no decrease was observed in cell surface expression of CD19, CD80, and CD86. Downregulation of MHC class I expression was found to be an early-lytic-cycle event, since it was observed when progress through late lytic cycle was blocked by treatment with acyclovir. The immediate-early transactivator of the EBV lytic cycle, BZLF1, did not directly affect expression of MHC class I molecules. However, BZLF1 completely inhibited the upregulation of MHC class I expression mediated by the EBV cell-transforming protein, LMP1. This novel function of BZLF1 elucidates the paradox of how MHC class I expression can be downregulated when LMP1, which upregulates MHC class I expression in latent infection, remains expressed in the lytic cycle.     

Ultraviolet light induces the sustained unscheduled expression of cyclin E in the absence of functional p53

Cell cycle progression is regulated through changes in the activity of cyclin-dependent kinases that are, in turn, regulated by the expression of their respective cyclin partners. In primary cells, cyclin E expression increases through the G1 phase of the cell cycle and peaks near the G1/S boundary. The unscheduled expression of cyclin E in primary human fibroblasts leads to chromosomal instability that is greatly increased by loss of the p53 tumour suppressor. Intriguingly, ultraviolet light (UV), the most prevalent environmental carcinogen, is similarly known to induce chromosomal instability more dramatically in the absence of p53. Here we report that UV light transiently increased the expression of cyclin E in normal human fibroblasts. Strikingly, cyclin E levels remained elevated for an extended period of time in the absence of functional p53. UV-induced cyclin E expression was not restricted to the G1/S boundary but remained elevated throughout S phase and this correlated with a massive accumulation of p53-deficient fibroblasts in this phase of the cell cycle. Forced expression of cyclin E alone was insufficient to cause a similar S phase arrest but forced expression of cyclin E led to an increase in the proportion of UV-irradiated cells in S phase. The present work suggests that p53 affects S phase progression following UV exposure by preventing the sustained unscheduled expression of cyclin E and that this may limit the clastogenic and carcinogenic effects of UV light.     

The loss of PIN1 deregulates cyclin E and sensitizes mouse embryo fibroblasts to genomic instability

During the G0/G1-S phase transition, the timely synthesis and degradation of key regulatory proteins is required for normal cell cycle progression. Two of these proteins, c-Myc and cyclin E, are recognized by the Cdc4 E3 ligase of the Skp1/Cul1/Rbx1 (SCF) complex. SCF(Cdc4) binds to a similar phosphodegron sequence in c-Myc and cyclin E proteins resulting in ubiquitylation and degradation of both proteins via the 26 S proteosome. Since the prolyl isomerase Pin1 binds the c-Myc phosphodegron and participates in regulation of c-Myc turnover, we hypothesized that Pin1 would bind to and regulate cyclin E turnover in a similar manner. Here we show that Pin1 regulates the turnover of cyclin E in mouse embryo fibroblasts. Pin1 binds to the cyclin E-Cdk2 complex in a manner that depends on Ser384 of cyclin E, which is phosphorylated by Cdk2. The absence of Pin1 results in an increased steady-state level of cyclin E and stalling of the cells in the G1/S phase of the cell cycle. The cellular changes that result from the loss of Pin1 predispose Pin1 null mouse embryo fibroblasts to undergo more rapid genomic instability when immortalized by conditional inactivation of p53 and sensitizes these cells to more aggressive Ras-dependent transformation and tumorigenesis.     

Identification of an arginine-rich motif in human papillomavirus type 1 E1;E4 protein necessary for E4-mediated inhibition of cellular DNA synthesis in vitro and in cells

Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G(2)-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1;E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.     

Epstein-Barr virus immediate-early protein BZLF1 inhibits tumor necrosis factor alpha-induced signaling and apoptosis by downregulating tumor necrosis factor receptor 1

Tumor necrosis factor alpha (TNF-alpha) is a key mediator of host immune and inflammatory responses and inhibits herpesvirus replication by cytolytic and noncytolytic mechanisms. TNF-alpha effects are primarily mediated through the major TNF-alpha receptor, TNF-R1, which is constitutively expressed in most cell types. Here we show that the Epstein-Barr virus (EBV) immediate-early protein BZLF1 prevents TNF-alpha activation of target genes and TNF-alpha-induced cell death. These effects are mediated by down-regulation of the promoter for TNF-R1. Additionally, we demonstrate that expression of TNF-R1 is downregulated during the EBV lytic replication cycle. Thus, EBV has developed a novel mechanism for evading TNF-alpha antiviral effects during lytic reactivation or primary infection.     

Use of adenovirus vectors expressing Epstein-Barr virus (EBV) immediate-early protein BZLF1 or BRLF1 to treat EBV-positive tumors

The Epstein-Barr virus (EBV) genome is present in a variety of tumor types, including virtually all undifferentiated nasopharyngeal carcinomas (NPC) and a portion of gastric carcinomas. The uniform presence of the EBV genome in certain tumors (versus only a very small number of normal B cells) suggests that novel therapies which specifically target EBV-positive cells for destruction might be effective for treating such tumors. Although the great majority of EBV-positive tumor cells are infected with one of the latent forms of EBV infection, expression of either viral immediate-early protein (BZLF1 or BRLF1) is sufficient to convert the virus to the lytic form of infection. Induction of the lytic form of EBV infection could potentially result in death of the tumor cell. Here we have examined the efficacy of adenovirus vectors expressing the BZLF1 or BRLF1 proteins for treatment of EBV-positive epithelial tumors. The BZLF1 and BRLF1 vectors induced preferential killing of EBV-positive, versus EBV-negative, gastric carcinoma cells in vitro. Infection of C18 NPC tumors (grown in nude mice) with either the BZLF1 or BRLF1 vector, but not a control adenovirus vector, induced expression of early lytic EBV genes in tumor cells. Injection of C18 tumors with the BZLF1 or BRLF1 adenovirus vector, but not the control vector, also significantly inhibited growth of the tumors in nude mice. The addition of ganciclovir did not significantly affect the antitumor effect of the BZLF1 and BRLF1 adenovirus vectors. These results suggest a potential cancer therapy against EBV-related tumors.     

Cdc42 promotes G1 progression through p70 S6 kinase-mediated induction of cyclin E expression

The Rho family GTPase Cdc42 is recognized for its role in cellular proliferation and transformation. However, the mechanism by which it promotes cell cycle progression has remained undefined. Using an inducible expression system, we show that constitutively active Cdc42 (Cdc42V12) is sufficient by itself to induce anchorage-independent but not mitogen-independent growth in NIH3T3 cells. However, Cdc42V12 markedly accelerates activation of cyclin E-Cdk2 in response to mitogen. These effects were highly specific, as the kinetics of cyclin D-Cdk4 activation was unaltered. Cdc42V12 promotes Cdk2 activation by selectively inducing cyclin E expression without affecting other regulatory proteins such as the p27 Cdk inhibitor or Cdc25A. Furthermore, Cdc42V12 was able to activate a reporter gene driven by the cyclin E promoter in the absence of exogenous mitogen or adhesion. Cyclin E induction was sensitive to rapamycin but not inhibitors of mitogen-activated protein kinases, implicating p70 S6 kinase (p70S6k) as the relevant mediator. Consistent with this notion, wild type and constitutively active alleles of p70S6k were sufficient to activate the cyclin E promoter. In sum, these studies provide novel insights into the mechanism by which Cdc42 promotes G1 progression.     

Deregulated expression of cloned transcription factor E2F-1 in Chinese hamster ovary cells shifts protein patterns and activates growth in protein-free medium

Engineering of the cell cycle can be an effective means for bypassing growth factor requirements of animal cells. Cloned human E2F-1 from Nalm 6 cells was subcloned into pRc/CMV and transfected into Chinese hamster ovary (CHO) cells. Ten stable transfectant clones isolated from cells cultured under neomycin-resistance selection pressure all expressed significantly higher amounts of E2F-1 than control cells as determined by Western analysis. Confocal immunofluorescent microscopy and Southern analysis of several clones also provided evidence for the expression of cloned E2F-1 in these cells. CHO K1:E2F-1 cells are able to proliferate on well-defined serum- and protein-free basal medium and exhibit an S-phase extended by 65% compared to CHO K1 cells mitogenically stimulated by basic fibroblast growth factor (bFGF). Two-dimensional electrophoresis of the intracellular proteins of E2F-1 clones shows an increase in 236 gene products compared to CHO K1 control cells, further verifying a functional regulatory role of cloned E2F-1 in CHO cells. Among these upregulated species is the cell cycle regulatory protein, cyclin A, which has already been shown to be regulated by E2F-1 in human fibroblasts. Overexpression of cloned E2F-1 in CHO cells is a potentially useful new strategy for bypassing serum requirements in mammalian cell culture. Furthermore, such cell cycle control stimulus-protein pattern response data can contribute to a clearer understanding of complex multigene networks involved in mammalian cell cycle regulation. (c) 1996 John Wiley & Sons, Inc.