Epstein-Barr virus (EBV) nuclear antigen 1 colocalizes with cellular replication foci in the absence of EBV plasmids

Epstein-Barr virus (EBV) EBNA-1 is the only EBV-encoded protein that is essential for the once-per-cell-cycle replication and maintenance of EBV plasmids in latently infected cells. EBNA-1 binds to the oriP region of latent EBV plasmids and cellular metaphase chromosomes. In the absence of oriP-containing plasmids, EBNA-1 was highly colocalized with cellular DNA replication foci that were identified by immunostaining S-phase cells for proliferating cell nuclear antigen and replication protein A (RP-A) in combination with DNA short pulse-labeling. For the association of EBNA-1 with the cellular replication focus areas, the EBNA-1 regions of amino acids (aa) 8 to 94 and/or aa 315 to 410, but not the RP-A-interacting carboxy-terminal region, were necessary. These results suggest a new aspect of latent virus-cell interactions.     

Up regulation of the Epstein-Barr virus (EBV)-encoded membrane protein LMP in the Burkitt's lymphoma line Daudi after exposure to n-butyrate and after EBV superinfection.

The Burkitt's lymphoma line Daudi carries a nontransforming Epstein-Barr virus (EBV) strain that has a deletion in the BamHI WYH region of the genome coding for the EBV nuclear antigen 2 (EBNA-2). Daudi cells fail to express the EBV-encoded latent membrane protein (LMP) (D. Ghosh and E. Kieff, J. Virol. 64:1855-1858, 1990). We show that LMP expression can be up regulated by exposure to n-butyrate and by superinfection with the B95-8 (B virus)- and P3HR1 (P virus)-derived EBV strains. Two LMP polypeptides of 60 and 48 kilodaltons (kDa) were detected in immunoblots of Daudi cells that had been exposed to 3 mM n-butyrate for 24 h. The intensity of the 48-kDa LMP increased during 72 h, in parallel with the appearance of early antigen-positive cells. The 60-kDa LMP was expressed at a low level and remained constant. Superinfection of Daudi cells with B and P virus induced the 60-kDa LMP within 3 h. In addition, P virus induced the 48-kDa LMP at a low level. The B virus-encoded EBNA-2 and EBNA-5 were detected 12 h after superinfection. The B virus-encoded 63-kDa LMP was coexpressed with the endogenous LMP after 48 h. Inactivation of the virus by UV illumination abolished the expression of the B virus-encoded antigens but did not affect the induction of the endogenous LMP. The B-cell activation marker CD23 was up regulated by B virus superinfection but not by n-butyrate exposure. CD23 was also expressed at a higher level in a stable B virus-converted subline, E95A-Daudi, that was EBNA-2 positive and coexpressed the Daudi virus- and B virus-encoded LMP. The results suggest that LMP expression is regulated by the interaction of cellular and viral factors. Binding of the virus to its membrane receptor might be involved in the triggering of cellular control mechanisms. Viral gene products are not directly involved in this function but may contribute to create a permissive cellular environment for LMP expression.     

Identification of cellular factors that bind specifically to the Epstein-Barr virus origin of DNA replication.

The specific binding of HeLa cell factors to DNA sequences at the Epstein-Barr virus (EBV) latent origin of DNA replication was detected by gel shift experiments and DNase I footprinting analysis. These cellular proteins protected at least five discrete regions of the DNA replication origin. The viral protein required for EBV plasmid replication, EBV nuclear antigen 1 (EBNA-1), binds to specific sequences within the origin region. The HeLa cell proteins competed with EBNA-1 for binding to EBV origin DNA in vitro, leading to the possibility that these cellular proteins regulate EBV DNA replication by displacing EBNA-1 at the origin sites.     

EBV interferes with the sensitivity of Burkitt lymphoma Akata cells to etoposide

Burkitt lymphoma (BL) commonly exhibits Epstein-Barr virus (EBV) positivity associated with latent chronic infection. Models of acute EBV infection have been associated with cellular resistance to apoptosis. However, the effect of latent long-term EBV infection on apoptosis induced by drugs is not well defined. To determine this, we have studied the response of the Akata EBV+ cell line (type I latency) to etoposide, before and after downregulating EBV gene expression. We observed that downregulating EBV nuclear antigen-1 (EBNA-1) expression with siRNAs reverted cellular sensitivity to etoposide. In accordance with this finding, Akata EBV+ cells showed increased sensitivity to etoposide, when compared to the Akata EBV- cells. We also observed that Akata EBV+ cells presented increased apoptosis levels and decreased Bcl-xL mRNA and protein levels, when compared to the Akata EBV- cells. In addition, Akata EBV+ cells contained less endoplasmic reticulum (ER) than EBV- cells. Finally, downregulation of EBV with EBNA-1 siRNAs caused an increase in the expression of Bcl-xL indicating that EBV is responsible for the differences found between the Akata EBV+ and EBV- cell lines.     

Long-term stability of large insert genomic DNA episomal shuttle vectors in human cells

We have constructed an episomal shuttle vector which can transfer large (>100 kb) human genomic DNA inserts back and forth between bacteria and human cells and which can be tracked in rapidly dividing human cells using a live cell assay. The vector (p5170) is based on the F factor-derived bacterial artificial chromosome cloning vector used in Escherichia coli, with the addition of the family of repeats element from the Epstein-Barr virus (EBV) latent origin of replication. This element provides nuclear retention in cells expressing the EBV protein EBNA-1. We have subcloned a series of genomic DNA inserts into p5170 and transfected the constructs into an EBNA-1(+) human cell line. Episomal mitotic stability was quantitatively analysed using flow cytometry. The episomes were also tracked by time course photography of expanding colonies. A 117 kb episome was retained at approximately 2 copies/cell and could be shuttled unrearranged from the human cells into bacterial cells after 15 months of continuous cell growth. Furthermore, the episome could still be rescued from human cells cultured in the absence of selection for 198 days. Such a trackable E.coli /human cell line shuttle vector system capable of carrying >100 kb of genomic DNA in human cells could prove a valuable tool in gene expression studies.     

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.     

Efficient expression of an Epstein-Barr nuclear antigen in Drosophila cells transfected with Epstein-Barr virus DNA.

In a search for exogenous promoters which function in cultured Drosophila cells, we have co-transfected a D. melanogaster cell line with an Epstein-Barr virus (EBV) cosmid clone which encodes the Epstein-Barr nuclear antigen (EBNA-1). Here we report that Drosophila cells containing stably integrated copies of EBNA-1 encoding DNA synthesise a polypeptide of mol. wt. identical to that of authentic EBNA-1, which is detectable with EBNA-positive but not EBNA-negative human serum. As in EBV-transformed lymphoblastoid cells, this neo-antigen is associated with the nucleus of transfected cells suggesting that cellular localisation signals which operate in mammalian cells are also recognised in insect cells.     

Two families of sequences in the small RNA-encoding region of Epstein-Barr virus (EBV) correlate with EBV types A and B

DNA sequence analysis was carried out on the 1-kilobase SacI-EcoRI region of the EcoRI J fragment of four strains of Epstein-Barr virus (EBV) (MABA, P3HR-1, FF41, and NPC-5), and the sequences were compared with the prototype sequence from strain B95-8. Ten single-base changes which grouped the strains into two families (1 and 2) were found. Restriction endonuclease polymorphisms predicted from the sequences were used to classify the EBV DNA from a further 26 EBV-positive cell lines into these two families. The EBNA-2 types (A or B) of the strains were found to correlate with the J region type; EBNA-2 type A DNA regularly contained J region sequence type 1, while EBNA-2 type B DNA generally carried J region sequence type 2. These data are consistent with the notion of there being two distinct families of EBV with discrete, conserved differences in DNA sequence.     

Epstein-Barr virus nuclear antigen 2 transactivates latent membrane protein LMP1.

Several lines of evidence are compatible with the hypothesis that Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) or leader protein (EBNA-LP) affects expression of the EBV latent infection membrane protein LMP1. We now demonstrate the following. (i) Acute transfection and expression of EBNA-2 under control of simian virus 40 or Moloney murine leukemia virus promoters resulted in increased LMP1 expression in P3HR-1-infected Burkitt's lymphoma cells and the P3HR-1 or Daudi cell line. (ii) Transfection and expression of EBNA-LP alone had no effect on LMP1 expression and did not act synergistically with EBNA-2 to affect LMP1 expression. (iii) LMP1 expression in Daudi and P3HR-1-infected cells was controlled at the mRNA level, and EBNA-2 expression in Daudi cells increased LMP1 mRNA. (iv) No other EBV genes were required for EBNA-2 transactivation of LMP1 since cotransfection of recombinant EBNA-2 expression vectors and genomic LMP1 DNA fragments enhanced LMP1 expression in the EBV-negative B-lymphoma cell lines BJAB, Louckes, and BL30. (v) An EBNA-2-responsive element was found within the -512 to +40 LMP1 DNA since this DNA linked to a chloramphenicol acetyltransferase reporter gene was transactivated by cotransfection with an EBNA-2 expression vector. (vi) The EBV type 2 EBNA-2 transactivated LMP1 as well as the EBV type 1 EBNA-2. (vii) Two deletions within the EBNA-2 gene which rendered EBV transformation incompetent did not transactivate LMP1, whereas a transformation-competent EBNA-2 deletion mutant did transactivate LMP1. LMP1 is a potent effector of B-lymphocyte activation and can act synergistically with EBNA-2 to induce cellular CD23 gene expression. Thus, EBNA-2 transactivation of LMP1 amplifies the biological impact of EBNA-2 and underscores its central role in EBV-induced growth transformation.     

Prediction and demonstration of a novel Epstein-Barr virus nuclear antigen.

The protein sequence predicted by the Epstein Barr virus (EBV) BERF4 open reading frame includes a tetrapeptide, Lys-Arg-Pro-Arg (KRPR), shown for other proteins to be a component of a signal for rapid nuclear localization. A subgenomic fragment of EBV DNA containing BERF4 has been incorporated into an expression vector, transfected onto primate cells and the nuclear distribution of the resulting protein established by immunofluorescence using EBV positive human sera. These sera contained high titres of antibodies to a fusion protein, produced in E. coli, consisting of beta-galactosidase and the C-terminal 167 amino acids of BERF4. Immunoaffinity purified antibodies reactive with the EBV component of the fusion show the molecular weight of this antigen in EBV immortalized B-cell lines to be about 160 kD. The demonstration that BERF4 contains an exon encoding a nuclear protein identifies a new EBNA gene (EBNA-6) and suggests that KRPR is a signal sequence common to a number of viral and cellular nuclear polypeptides which bind to nucleic acids and may therefore be of predictive value in identifying karyophilic proteins.     

The average number of molecules of Epstein-Barr nuclear antigen 1 per cell does not correlate with the average number of Epstein-Barr virus (EBV) DNA molecules per cell among different clones of EBV-immortalized cells.

Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein required to support latent replication of Epstein-Barr virus (EBV). To assess the likelihood that EBNA-1 regulates the amount of EBV DNA in a cell, we measured the average numbers of EBNA-1 molecules and EBV DNA molecules per cell in different clones of cells. The amount of EBNA-1 protein present in recently established lymphoblastoid cell lines was measured with affinity-purified anti-EBNA-1 antibodies, and viral DNA was measured by nucleic acid hybridization. The average levels of EBNA-1 protein varied little between these cell lines, whereas the average amount of viral DNA present varied substantially; consequently, these numbers were not correlated. There is no apparent relationship between amounts of EBNA-1 and viral DNA.     

The plasmid replicon of EBV consists of multiple cis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element.

Plasmids containing oriP, the plasmid origin of Epstein-Barr virus (EBV), are replicated stably in human cells that express a single viral trans-acting factor, EBNA-1. Unlike plasmids of other viruses, but akin to human chromosomes, oriP plasmids are synthesized once per cell cycle, and are partitioned faithfully to daughter cells during mitosis. Although EBNA-1 binds multiple sites within oriP, its role in DNA synthesis and partitioning has been obscure. EBNA-1 lacks enzymatic activities that are present in the origin-binding proteins of other mammalian viruses, and does not interact with human cellular proteins that provide equivalent enzymatic functions. We demonstrate that plasmids with oriP or its constituent elements are synthesized efficiently in human cells in the absence of EBNA-1. Further, we show that human cells rapidly eliminate or destroy newly synthesized plasmids, and that both EBNA-1 and the family of repeats of oriP are required for oriP plasmids to escape this catastrophic loss. These findings indicate that EBV's plasmid replicon consists of genetic elements with distinct functions, multiple cis-acting elements that facilitate DNA synthesis and viral cis/trans elements that permit retention of replicated DNA in daughter cells. They also explain historical failures to identify mammalian origins of DNA synthesis as autonomously replicating sequences.     

Mammalian assay for site-specific DNA damage processing using the human H-ras proto-oncogene.

The human genomic H-ras proto-oncogene was inserted into an Epstein-Barr virus (EBV) vector (p220.2) that replicates synchronously with the cell cycle. Unique restriction enzyme sites, 30 bp apart, were created on either side of codon 12 to enable the construction of gapped heteroduplex (GHD) DNA. Depending upon experimental protocol, the gap could be located either on the coding (non-transcribed) strand or the non-coding (transcribed) strand. GHD DNA was created using a 1.8 kb segment of H-ras DNA containing exon 1, into which a synthetic 30 nucleotide oligomer containing a strand- and site-specific mismatched nucleotide was annealed. The 1.8 kb segment of H-ras DNA containing a codon 12; middle G:T, A:C or T:C mismatch has been religated with high efficiency into the EBV vector and transfected into NIH 3T3 cells using a mild liposome-mediated protocol. Subsequent hygromycin resistant NIH 3T3 colonies have been PCR amplified and sequenced. In this study, codon 12; middle nucleotide mismatch correction rates to wild-type G:C during replication in NIH 3T3 cells were 96.4% of G:T mismatches, 87.5% of A:C mismatches and 67% of T:C mismatches.     

Monoclonal and polyclonal antibodies against Epstein-Barr virus nuclear antigen 5 (EBNA-5) detect multiple protein species in Burkitt''s lymphoma and lymphoblastoid cell lines.

The Epstein-Barr virus nuclear antigen 5 (EBNA-5) is encoded by highly spliced mRNA from the major IR1 (BamHI-W) repeat region of the virus genome. A mouse monoclonal antibody, JF186, has been raised against a synthetic 18-amino-acid peptide deduced from the EBNA-5 message of B95-8 and Raji cells. The antibody showed characteristic coarse nuclear granules by indirect immunofluorescence and revealed multiple EBNA-5 species by immunoblotting and immunoprecipitation. The B95-8 line itself and all B95-8 virus-carrying cells, whether lymphoblastoid cell lines or in vitro-converted sublines of Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL) lines, were EBNA-5 positive. Among 36 cell lines carrying different EBV strains, only 10 expressed the B95-8-Raji-prototype EBNA-5 recognized by JF186; this was probably due to genetic variation in the epitope recognized by JF186, as shown for P3HR-1. Human antibodies, affinity purified against EBNA-5-JF186 immunoprecipitates, detected EBNA-5 in the majority of EBV-positive BL lines and in all lymphoblastoid cell lines containing the BL-derived viruses. Thus, EBNA-5 can be expressed by all virus isolates examined, but is down-regulated, together with other latent gene products, in a minority of BL lines which have a particular cellular phenotype. EBNA-5 was detected as a ladder of protein species of 20 to 130 kilodaltons (kDa), with a regular spacing of 6 to 8 kDa, consistent with the coding capacity of the combined BamHI-W 66- and 132-base-pair exons, together with shifts of 2 to 4 kDa, consistent with the size of the separate 66- and 132-base-pair exons. Multiple EBNA-5 proteins can be expressed by the single cell as shown by cloning of newly infected cells.     

Variable expression of latent membrane protein in nasopharyngeal carcinoma can be related to methylation status of the Epstein-Barr virus BNLF-1 5''-flanking region.

Seven virus-coded proteins, the nuclear proteins EBNA-1 to EBNA-6 and the latent membrane protein (LMP), are regularly expressed in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines. In nasopharyngeal carcinoma (NPC), only EBNA-1 is regularly expressed; LMP is detected in about 65% of the tumors. In Burkitt's lymphoma tumors only EBNA-1 is expressed. We have recently shown that the methylation patterns of the EBV genome varied between these cell types. In virally transformed lymphoblastoid cell lines of normal origin, the EBV DNA is completely unmethylated. In contrast, in the Burkitt's lymphoma-derived cell line Rael and in a nude mouse-passaged NPC tumor, C15, there was an extensive methylation of CpG pairs. The methylation extended into the coding regions of the two expressed genes, EBNA-1 (in both tumor types) and LMP (in C15). Two presumptive control regions were exempted from this overall methylation: the oriP that contains both an origin of DNA replication and an EBNA-1-dependent enhancer and the 5'-flanking region of the BNLF-1 open reading frame that codes for LMP. The latter was only exempted in the LMP expressing NPC. We have now investigated the relation between expression of LMP and methylation of DNA in the 5'-flanking 1 kb region of BNLF-1, coding for LMP. LMP was methylated in 3 of 12 NPC biopsies that did not express LMP but was partially or totally unmethylated in the remaining 9 that expressed the protein. The three BNLF-1 exons were highly methylated in all the tumors. The oriP region was unmethylated in all the tumors, as in the previously studied Rael cell line and nude mouse-passaged NPC. Also, the BamHI W enhancer region involved in the expression of EBNA nuclear proteins was methylated. None of the biopsies expressed EBNA-2. Our data show that the EBV genomes are highly methylated in NPC tumors. The strong reverse correlation between the methylation of the putative control region of the LMP gene and the expression of LMP suggests that methylation has a role in the regulation of this gene.     

Epstein-Barr virus (EBV) recombinants: use of positive selection markers to rescue mutants in EBV-negative B-lymphoma cells.

The objective of these experiments was to develop strategies for creation and identification of recombinant mutant Epstein-Barr viruses (EBV). EBV recombinant molecular genetics has been limited to mutations within a short DNA segment deleted from a nontransforming EBV and an underlying strategy which relies on growth transformation of primary B lymphocytes for identification of recombinants. Thus, mutations outside the deletion or mutations which affect transformation cannot be easily recovered. In these experiments we investigated whether a toxic drug resistance gene, guanine phosphoribosyltransferase or hygromycin phosphotransferase, driven by the simian virus 40 promoter can be recombined into the EBV genome and can function to identify B-lymphoma cells infected with recombinant virus. Two different strategies were used to recombine the drug resistance marker into the EBV genome. Both utilized transfection of partially permissive, EBV-infected B95-8 cells and positive selection for cells which had incorporated a functional drug resistance gene. In the first series of experiments, B95-8 clones were screened for transfected DNA that had recombined into the EBV genome. In the second series of experiments, the transfected drug resistance marker was linked to the plasmid and lytic EBV origins so that it was maintained as an episome and could recombine with the B95-8 EBV genome during virus replication. The recombinant EBV from either experiment could be recovered by infection and toxic drug selection of EBV-negative B-lymphoma cells. The EBV genome in these B-lymphoma cells is frequently an episome. Virus genes associated with latent infection of primary B lymphocytes are expressed. Expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) and the EBNA-3 genes is variable relative to that of EBNA-1, as is characteristic of some naturally infected Burkitt tumor cells. Moreover, the EBV-infected B-lymphoma cells are often partially permissive for early replicative cycle gene expression and virus replication can be induced, in contrast to previously reported in vitro infected B-lymphoma cells. These studies demonstrate that dominant selectable markers can be inserted into the EBV genome, are active in the context of the EBV genome, and can be used to recover recombinant EBV in B-lymphoma cells. This system should be particularly useful for recovering EBV genomes with mutations in essential transforming genes.