Epstein-Barr virus nuclear antigen 3C regulates cyclin A/p27 complexes and enhances cyclin A-dependent kinase activity

Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is essential for primary B-cell transformation. In this report we show that cyclin A, an activator of S phase progression, bound tightly to EBNA3C. EBNA3C interacted with cyclin A in vitro and associated with cyclin A complexes in EBV-transformed lymphoblastoid cell lines. Importantly, EBNA3C stimulated cyclin A-dependent kinase activity and rescued p27-mediated inhibition of cyclin A/Cdk2 kinase activity by decreasing the molecular association between cyclin A and p27 in cells. Additionally, phosphorylation of the retinoblastoma protein, a major regulator of cell cycle progression, was enhanced both in vitro and in vivo in the presence of EBNA3C. Cyclin A interacted with a region of the carboxy terminus of EBNA3C, shown to be important both for stimulation of cyclin A-dependent kinase activity and for cell cycle progression. This provides the first evidence of an essential EBV latent antigen's directly targeting a cell cycle regulatory protein and suggests a novel mechanism by which EBV deregulates the mammalian cell cycle, which is of critical importance in B-cell transformation.     

Enhancement of hepatitis C virus replication by Epstein-Barr virus-encoded nuclear antigen 1.

Based on our recent observation that Epstein-Barr virus (EBV) is detected in 37% of the tissues of hepatocellular carcinoma, and especially frequently in cases with hepatitis C virus (HCV), the effect of EBV infection on the replication of HCV was investigated. EBV-infected cell clones and their EBV-uninfected counterparts in cell lines MT-2 (a human T-lymphotropic virus type I-infected T-cell line), HepG2 (a hepatoblastoma cell line) and Akata (a Burkitt's lymphoma cell line) were compared in terms of their permissiveness for HCV replication following inoculation of HCV derived from patients who were HCV carriers. The results indicated that EBV-infected cell clones, but not their EBV-uninfected counterparts, promoted HCV replication. EBV-encoded nuclear antigen 1 (EBNA1), which is invariably expressed in EBV-infected cells, supported HCV replication. Deletion analysis of the EBNA1 gene showed good correlation between transactivation activity and the activity supporting HCV replication. The present findings suggest that EBV acts as a helper virus for HCV replication.     

Herpes simplex type 1 activation by Epstein-Barr virus nuclear antigen 1

We have investigated the effect of Epstein-Barr virus nuclear antigen 1 (EBNA-1), a nuclear protein encoded by EBV, on herpes simplex virus type 1 (HSV-1) infection either in cells constitutively expressing EBNA-1 or in transient expression assays. Rat-1 cells and rat embryo fibroblasts (REF) immortalized by c-myc or E1A were transfected with a specific EBV DNA fragment coding for EBNA-1. Cloned cell lines which constitutively expressed this antigen were infected with HSV-1. Our results indicate that in EBNA-1-expressing cells, virus growth was higher than in control cells for different virus strains or rodent cell lines. This increase was maximal when cells were infected at low multiplicity, as determined by virus growth, and correlated with the stimulation of viral DNA synthesis. REF + c-myc and Vero cells were contransfected by an EBNA-1 expression vector driven by Moloney murine leukaemia virus LTR and HSV-1 immediate-early (α0) or early thymidine kinase upstream promoter regulatory regions linked to chloramphenicol acetyltransferase (CAT) coding sequences as effectors. In both cell lines, stimulation of CAT expression by EBNA-1 was observed only with the immediate-early promoter. These results suggest that EBNA-1 can transactivate immediate-early HSV-1 expression.     

Epstein-Barr virus nuclear antigen EBNA3C/6 expression maintains the level of latent membrane protein 1 in G1-arrested cells.

The Epstein-Barr virus in the Burkitt lymphoma-derived cell line Raji has a deletion in the EBNA3C gene. When Raji cells are allowed to grow to high density and most of the cells become growth arrested in the G1 phase of the cell cycle, the level of detectable latent membrane protein 1 (LMP1) is substantially reduced. After dilution of the cells with fresh growth medium, within 8 h, there is a large increase in LMP1 mRNA, and by 12 h, LMP1 is expressed to a high level (H. Boos, M. Stoehr, M. Sauter, and N. Mueller-Lantzch, J. Gen. Virol. 71:1811-1815, 1990). Here we show that in Raji cells which constitutively express a transfected EBNA3C gene, the down-regulation of LMP1 in growth-arrested cells does not take place. Furthermore, we show that in wild-type Raji cells, low-level LMP1 expression occurs when most of the cells are arrested at a point(s) early in G1 (or G0) when the product of the retinoblastoma gene, pRb, is hypophosphorylated. The dramatic synthesis of LMP1 coincides with the progression of these cells to late G1 when pRb becomes hyperphosphorylated. Thus, in Raji cells, the LMP1 gene is apparently regulated in a cell cycle- or proliferation-dependent manner, but when EBNA3C is present, sustained LMP1 expression occurs as it does in a lymphoblastoid cell line. EBNA3C appears to either relieve the apparent repression of LMP1 in cells progressing through early G1 or possibly alter the stage at which the cells growth arrest to one where they are permissive for LMP1 expression.     

Migratory localization of cyclin D2-Cdk4 complex suggests a spatial regulation of the G1-S transition

The association of the cyclin D-Cdk (DC) complex with retinoblastoma protein (pRb) is required for the G1-S transition of the cell cycle. Cyclin synthesis, nuclear localization and degradation are control mechanisms for the transition, but regulation of the DC complex nuclear import also contributes to the transition. Analysis of the timing of the G1-S transition in mammalian cell lines revealed acceleration with overexpression of cyclin D2 and Cdk4. Immunolocalization assays revealed that cyclin D2 and Cdk4 formed a complex in the cytoplasm and approached the nucleus. They accumulated on the cytosolic surfaces of the nuclear pores and then were arrested at the nuclear membrane before the nucleus reached a critical size. Finally, the complex was released into the nucleus and colocalized with pRb there, which led to pRb phosphorylation and DNA synthesis. The translocalization depended on the G1-S transition. In contrast, a truncated cyclin D2 that was not able to fully associate with Cdk4 lost the ability for release into the nucleus. This pattern of translocalization suggests a spatial separation of the cyclin D-Cdk complex from pRb and DNA in the nucleus to regulate the G1-S transition.     

Regulation of the cell cycle at the G1-S transition by proteolysis of cyclin E and p27Kip1

The transition from G1 phase to S phase of the mammalian cell cycle is controlled by many positive and negative regulators, among which cyclin E and p27Kip1, respectively, undergo the most marked changes in concentration at this transition. The abundance of both cyclin E and p27Kip1 is regulated predominantly by posttranslational mechanisms, in particular by proteolysis mediated by the ubiquitin-proteasome pathway. Cyclin E and p27Kip1 each bind to and undergo polyubiquitination by the same ubiquitin ligase, known as SCF(Skp2). The degradation of cyclin E and p27Kip1 is greatly impaired in Skp2-deficient mice, resulting in intracellular accumulation of these proteins. In this article, recent progress in characterization of the molecular mechanisms that control the proteolysis of cyclin E and p27Kip1 is reviewed.     

Mitosis-specific hyperphosphorylation of Epstein-Barr virus nuclear antigen 2 suppresses its function

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is a key gene expressed in EBV type III latent infection that can transactivate numerous promoters, including those for all the other type III viral latency genes as well as cellular genes responsible for cell proliferation. EBNA-2 is essential for EBV-mediated immortalization of primary B lymphocytes. We now report that EBNA-2, a phosphoprotein, is hyperphosphorylated specifically in mitosis. Evidence that the cyclin-dependent kinase p34(cdc2) may be involved in this hyperphosphorylation includes (i) coimmunoprecipitation of EBNA-2 and p34(cdc2), suggesting physical association; (ii) temporal correlation between hyperphosphorylation of EBNA-2 and an increase in p34(cdc2) kinase activity; and (iii) ability of purified p34(cdc2)/cyclin B1 kinase to phosphorylate EBNA-2 in vitro. Hyperphosphorylation of EBNA-2 appears to suppress its ability to transactivate the latent membrane protein 1 (LMP-1) promoter by about 50%. The association between EBNA-2 and PU.1 is also decreased by about 50% in M-phase-arrested cells, as shown by coimmunoprecipitation from cell lysates, suggesting that hyperphosphorylation of EBNA-2 impairs its affinity for PU.1. Finally, endogenous LMP-1 mRNA levels in M phase are around 55% of those in asynchronously growing cells. These results suggest that regulation of gene expression during type III latency may be regulated in a cell-cycle-related manner.     

The DNA segregation mechanism of Epstein-Barr virus nuclear antigen 1

Latent Epstein–Barr virus (EBV) genomes are maintained in human cells as low copy number episomes that are thought to be partitioned by attachment to the cellular mitotic chromosomes through the viral EBNA1 protein. We have identified a human protein, EBP2, which interacts with the EBNA1 sequences that govern EBV partitioning. Here we show that, in mitosis, EBP2 localizes to the condensed cellular chromosomes producing a staining pattern that is indistinguishable from that of EBNA1. The localization of EBNA1 proteins with mutations in the EBP2 binding region was also examined. An EBNA1 mutant (Δ325–376) disrupted for EBP2 binding and segregation function was nuclear but failed to attach to the cellular chromosomes in mitosis. Our results indicate that amino acids 325–376 mediate the binding of EBNA1 to mitotic chromosomes and strongly suggest that EBNA1 mediates EBV segregation by attaching to EBP2 on the cellular mitotic chromosomes.     

Mutational analysis of Epstein-Barr virus nuclear antigen 1 (EBNA 1).

We have constructed a set of nonsense mutants in the EBNA 1 gene of Epstein-Barr virus by inserting a synthetic oligonucleotide, which has translational termination codons in all three reading frames, at various positions in a cloned copy of the EBNA 1 gene. The EBNA 1 proteins encoded by these mutants and three deletion mutants were analyzed using several functional assays. It was determined that there are two separable phosphorylation domains in the carboxy half of the molecule. The carboxy half of the molecule was also found to contain a region between the unique Sac I and Sac II sites that is required for transactivation of the EBNA 1-specific enhancer element found within ori P. The mutants also served to identify a 248 bp region that affects the pattern of intranuclear localization of the protein. Correlations between the functional domains established by these studies and other properties of EBNA 1 are discussed.     

Phosphorylation of the Epstein-Barr virus nuclear antigen 2.

A major in vivo phosphorylation site of the Epstein-Barr virus nuclear antigen 2 (EBNA-2) was found to be localized at the C-terminus of the protein. In vitro phosphorylation studies using casein kinase 1 (CK-1) and casein kinase 2 (CK-2) revealed that EBNA-2 is a substrate for CK-2, but not for CK-1. The CK-2 specific phosphorylation site was localized in the 140 C-terminal amino acids using a recombinant trpE-C-terminal fusion protein. In a similar experiment, the 58 N-terminal amino acids expressed as a recombinant trpE-fusion protein were not phosphorylated. Phosphorylation of a synthetic peptide corresponding to amino acids 464-476 of EBNA-2 as a substrate led to the incorporation of 0.69 mol phosphate/mol peptide indicating that only one of three potential phosphorylation sites within the peptide was modified. The most likely amino acid residues for phosphorylation by CK-2 are Ser469 and Ser470.