Differential methylation of Epstein-Barr virus latency promoters facilitates viral persistence in healthy seropositive individuals

Epstein-Barr virus (EBV) establishes a life-long infection in humans, with distinct viral latency programs predominating during acute and chronic phases of infection. Only a subset of the EBV latency-associated antigens present during the acute phase of EBV infection are expressed in the latently infected memory B cells that serve as the long-term EBV reservoir. Since the EBV immortalization program elicits a potent cellular immune response, downregulation of viral gene expression in the long-term latency reservoir is likely to facilitate evasion of the immune response and persistence of EBV in the immunocompetent host. Tissue culture and tumor models of restricted EBV latency have consistently demonstrated a critical role for methylation of the viral genome in maintaining the restricted pattern of latency-associated gene expression. Here we extend these observations to demonstrate that the EBV genomes in the memory B-cell reservoir are also heavily and discretely methylated. This analysis reveals that methylation of the viral genome is a normal aspect of EBV infection in healthy immunocompetent individuals and is not restricted to the development of EBV-associated tumors. In addition, the pattern of methylation very likely accounts for the observed inhibition of the EBV immortalization program and the establishment and maintenance of a restricted latency program. Thus, EBV appears to be the first example of a parasite that usurps the host cell-directed methylation system to regulate pathogen gene expression and thereby establish a chronic infection.     

Epstein-Barr virus with heterogeneous DNA disrupts latency.

By cloning the HR-1 Burkitt lymphoma line, we previously uncovered two distinct biological variants of nontransforming Epstein-Barr virus (EBV). The most commonly cloned variant has a low rate of spontaneous viral synthesis and is unable to induce early antigen in Raji cells (EAI-). A rare variant spontaneously releases virus which is capable of inducing early antigen in Raji cells (EAI+). Since EAI- virus lacks heterogeneous DNA (het-) and EAI+ virus contains heterogeneous DNA (het+), we suggested that spontaneous viral synthesis and induction of early antigen are biological properties which correlate with the presence of het sequences. The present experiments provide three new lines of experimental evidence in favor of this hypothesis. (i) Revertant subclones of the EAI+ het+ variant which have lost the het DNA concomitantly lost EAI ability. Thus, het DNA is not stably associated with the cells as are the episomes. (ii) het DNA was acquired by two het- subclones of the HR-1 line after superinfection with EAI+ virus. After superinfection, these clones synthesized EAI+ het+ virus. Thus, het DNA may be maintained in the HR-1 line by cell-to-cell spread. (iii) Virus with het DNA activated full expression of endogenous latent EBV of the transforming phenotype in a line of immortalized neonatal lymphocytes designated X50-7. By use of restriction endonuclease polymorphisms unique to both the superinfecting and endogenous genomes, we show that the genome of the activated virus resembles that of the virus which was endogenous to X50-7 cells. This result suggests that het sequences result in transactivation of the latent EBV. het DNA had homology with EBV sequences which are not normally contiguous on the physical map of the genome. het DNA was always accompanied by the presence of DNA of nonheterogenous HR-1. Thus, het DNA is a form of "defective" EBV DNA. However, the biological effect of this defective DNA is to enhance rather than to interfere with EBV replication. This is a novel property of defective virus.     

Cocaine potentiates the switch between latency and replication of Epstein-Barr virus in Raji cells.

This paper shows that cocaine amplifies Epstein-Barr virus (EBV) reactivation in Raji cells. Its effect on early viral protein synthesis was maximal when it was added with 12-O-tetradecanoyl phorbol-13-acetate (TPA) plus n-butyrate, but nil when added alone. The enhancing effect of cocaine on early replicative stages of latent EBV was associated with an increase of Ca(2+) mobilization induced by the drug and with an induction of cellular protein phosphorylation in chemicals and cocaine-treated Raji cells. Cocaine also acted synergistically with TPA and n-butyrate to induce Z Epstein-Barr replication activator (ZEBRA), a nuclear phosphoprotein responsible for the activation of early viral gene expression. These findings provide the first evidence that cocaine may represent an important co-factor in the reactivation of early stages of latent EBV infection.     

Activated mouse Notch1 transactivates Epstein-Barr virus nuclear antigen 2-regulated viral promoters

Epstein-Barr virus nuclear antigen 2 (EBNA2) is essential for B-cell immortalization by EBV, most probably by its ability to transactivate a number of cellular and viral genes. EBNA2-responsive elements (EBNA2REs) have been identified in several EBNA2-regulated viral promoters, each of them carrying at least one RBP-Jkappa recognition site. RBP-Jkappa recruits EBNA2 to the EBNA2RE and, once complexed to EBNA2, is converted from a repressor into an activator. An activated form of the cellular receptor Notch also interacts with RBP-Jkappa, providing a link between EBNA2 and Notch signalling. To determine whether activated Notch is able to transactivate EBNA2-responsive viral promoters, we performed cotransfection experiments with activated mouse Notch1 (mNotch1-IC) and luciferase constructs of the BamHI C, LMP1, and LMP2A promoters. We present here evidence that mNotch1-IC transactivates viral promoters known to be regulated by EBNA2. As shown for EBNA2, mutations or deletions of the RBP-Jkappa sites diminish or eliminate mNotch1-IC-mediated transactivation of the promoters, pointing to an essential role for Notch-RBP-Jkappa interaction. In addition to RBP-Jkappa, other cellular factors may bind within the EBNA2REs of viral promoters. While some factors appear to play an important role in both EBNA2- and mNotch1-IC-mediated transactivation, others are only important for the activity of either EBNA2 or mNotch1-IC. We could observe specific mNotch1-IC-responsive regions, thereby throwing more light upon which cofactors interact with EBNA2 and mNotch1-IC, thus enabling them to become functionally transactivators in vivo.     

Latent and lytic cycle promoters of Epstein-Barr virus

Four RNA polymerase II promoters have been mapped in the DNA sequence of the EcoRI-H and -Dhet fragments of B95-8 Epstein-Barr virus. RNAs transcribed from three of these promoters are dramatically induced by treatment of B95-8 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA). The other promoter is active with or without TPA treatment of the cells and is thus active in the latent virus cycle. Deletion mapping suggests that DNA sequence homologies between some of the promoters lie in the same region as essential upstream promoter elements.     

Induction of Epstein-Barr virus lytic replication by recombinant adenoviruses expressing the zebra gene with EBV specific promoters

The latent Epstein-Barr virus (EBV) is found in the cells of many tumors. For example, EBV is detectable in almost all cases, and in almost all tumor cells, of non-keratinizing nasopharyngeal carcinoma.Activating the latent virus, which will result in its lytic replication and the death of tumor cells, is a potential approach for the treatment of EBV-associated cancers. In this study, three recombinant adenoviruses were constructed to express the Zebra gene, an EBV gene responsible for switching from the latent state to lytic replication. EBV-specific promoters were used in order to limit Zebra expression in EBV-positive cells, and reduce the potential side effects. The EBV promoters used were Cp, Zp and a dual promoter combining both promoters, CpZp. The Zebra protein was detected in HEK293 cells as well as the EBV-positive D98-HR1 cells infected with recombinant viruses. An EBV lytic replication early antigen, EA-D, was also detected in infected D98-HR1, implying the initiation of lytic replication. In the cell viability assay, Zebra-expressing adenoviruses had little effect on EBV-negative HeLa cells, while significantly reducing the cell viability and proliferation of D98-HR1 cells. The results indicate that EBV virus promoters can be used in adenovirus vectors to express the Zebra gene and induce EBV lytic replication in D98-HR1 cells.     

Viral latency and transformation: the strategy of Epstein-Barr virus

Evidence that GAP is an effector of ras action can be summarized as follows: GAP interacts at a site on p21 defined genetically as the effector binding site. Regions of p21 that are nonessential for biological activity are nonessential for GAP interaction. GAP interacts with all known types of p21. (Upstream factors are expected to be specific for individual types). GAP interacts with p21 proteins (normal and mutant) in a GTP-dependent fashion. None of these constitute proof. It remains possible that GAP simply regulates p21-GTP levels, and binds to the same site as the true effector without transmitting a downstream signal. If indeed GAP mediates ras action, the question immediately arises as to the biochemical function of GAP itself. The requirement of ras proteins for membrane localization to exert their effects may be a valuable clue in the search for this function. Perhaps GAP is an enzyme (or is bound to an enzyme) that acts on membrane components in a p21-GTP-dependent manner and in doing so transmits signals to other downstream effectors. The ability of GAP to interact with many members of the ras family would allow many upstream signals to feed into this downstream pathway. Clearly, proof (or disproof) that GAP is downstream of ras is the next step toward clarification of this aspect of ras action; identification of biochemical activities associated with GAP (or the true ras effector) will, we hope, follow soon.     

Histone acetylation and reactivation of Epstein-Barr virus from latency

Induction of the viral BZLF1 gene has previously been shown to be one of the first steps in the reactivation of Epstein-Barr virus (EBV). Using an EBV oriP episomal vector system, we have reconstituted the regulation of the promoter for BZLF1 on stably transfected episomes, mapped promoter elements required for that regulation, and investigated mechanisms that may control the switch between latency and the lytic cycle. Changes in histone acetylation at the promoter for the BZLF1 gene appear to be a key part of the reactivation mechanism of this herpesvirus.     

Targeted methylation of CMV and E1A viral promoters

Recombinant thermosomes from the Acidianus tengchongensis strain S5^T were purified to homogeneity and assembled in vitro into homo-oligomers (rATcpnα or rATcpnβ) and hetero-oligomers (rATcpnαβ). The symmetries of these complexes were determined by electron microscopy and image analysis. The rATcpnα homo-oligomer was shown to possess 8-fold symmetry while both rATcpnβ and rATcpnαβ oligomers adopted 9-fold symmetry. rATcpnαβ oligomers were shown to contain the α and β subunits in a 1:2 ratio. All of the complexes prevented the irreversible inactivation of yeast alcohol dehydrogenase at 55 °C and completely prevented the formation of aggregates during thermal inactivation of citrate synthase at 45 °C. All rATcpn complexes showed trace ATP hydrolysis activity. Furthermore, rATcpnβ sequestered fully chemically denatured substrates (GFP and thermophilic malic dehydrogenase) in vitro without refolding them in an ATP-dependent manner. This property is similar to previously reported properties of chaperonins from Sulfolobus tokodaii and Sulfolobus acidocaldarius. These features are consistent with the slow growth rates of these species of archaea in their native environment.     

Conservation and progressive methylation of Epstein-Barr viral DNA sequences in transformed cells.

The structure of intracellular viral DNA from a number of cell lines arising by clonal transformation of human lymphocytes in vitro with Epstein-Barr virus was analyzed. Intracellular viral DNAs were partially purified and digested with several restriction endonucleases, and the products of digestion were separated by electrophoresis in agarose gels. The viral fragments were detected by transferring the DNA from the gel to nitrocellulose sheets, hybridizing radiolabeled recombinant vectors carrying fragments of viral DNA to those transfers, and visualizing the hybrids by autoradiography. These analyses indicated that: (i) regions of repetitious viral DNA do undergo expansion and contraction although one size predominates; (ii) novel sequence arrangements appear in the intracellular viral DNA of different clones but are not found in clones analyzed serially and propagated extensively; (iii) the viral DNA is increasingly methylated upon cell propagation. We have not identified a transformed cell phenotype or a viral phenotype that segregates with the observed progressive methylation. We have not detected in Epstein-Barr viral plasmids analogs of the gross rearrangements of viral DNAs observed after lytic infections with high multiplicities of papova-, adeno-, or herpes simplex viruses.     

Functional and physical interaction between p53 and BZLF1: implications for Epstein-Barr virus latency.

The p53 tumor suppressor protein, which is commonly mutated in human cancers, has been shown to interact directly with virally encoded from papillomavirus, adenovirus, and simian virus 40. The disruption of p53 function may be required for efficient replication of certain viruses and may also play a role in the development of virally induced malignancies. Infection with Epstein-Barr virus (EBV) has been associated with the development of B-cell lymphomas and nasopharyngeal carcinoma. Here we show that the EBV immediate-early protein, BZLF1 (Z), which is responsible for initiating the switch from latent to lytic infection, can interact directly in vitro and in vivo with the tumor suppressor protein, p53. This interaction requires the coiled-coil dimerization domain of the Z protein and the carboxy-terminal portion of p53. Overexpression of wild-type p53 inhibits the ability of Z to disrupt viral latency. Likewise, Z inhibits p53-dependent transactivation in lymphoid cells. The direct interaction between Z and p53 may play a role in regulating the switch from latent to lytic viral infection.