B95-8 Strain Of Epstein-Barr Virus Research Articles

Heterogeneity of the Epstein-Barr Virus (EBV) Major Internal Repeat Reveals Evolutionary Mechanisms of EBV and a Functional Defect in the Prototype EBV Strain B95-8.

ABSTRACTEpstein-Barr virus (EBV) is a ubiquitous pathogen of humans that can cause several types of lymphoma and carcinoma. Like other herpesviruses, EBV has diversified through both coevolution with its host and genetic exchange between virus strains. Sequence analysis of the EBV genome is unusually challenging because of the large number and lengths of repeat regions within the virus. Here we describe the sequence assembly and analysis of the large internal repeat 1 of EBV (IR1; also known as the BamW repeats) for more than 70 strains. The diversity of the latency protein EBV nuclear antigen leader protein (EBNA-LP) resides predominantly within the exons downstream of IR1. The integrity of the putative BWRF1 open reading frame (ORF) is retained in over 80% of strains, and deletions truncating IR1 always spare BWRF1. Conserved regions include the IR1 latency promoter (Wp) and one zone upstream of and two within BWRF1. IR1 is heterogeneous in 70% of strains, and this heterogeneity arises from sequence exchange between strains as well as from spontaneous mutation, with interstrain recombination being more common in tumor-derived viruses. This genetic exchange often incorporates regions of <1 kb, and allelic gene conversion changes the frequency of small regions within the repeat but not close to the flanks. These observations suggest that IR1—and, by extension, EBV—diversifies through both recombination and breakpoint repair, while concerted evolution of IR1 is driven by gene conversion of small regions. Finally, the prototype EBV strain B95-8 contains four nonconsensus variants within a single IR1 repeat unit, including a stop codon in the EBNA-LP gene. Repairing IR1 improves EBNA-LP levels and the quality of transformation by the B95-8 bacterial artificial chromosome (BAC).IMPORTANCE Epstein-Barr virus (EBV) infects the majority of the world population but causes illness in only a small minority of people. Nevertheless, over 1% of cancers worldwide are attributable to EBV. Recent sequencing projects investigating virus diversity to see if different strains have different disease impacts have excluded regions of repeating sequence, as they are more technically challenging. Here we analyze the sequence of the largest repeat in EBV (IR1). We first characterized the variations in protein sequences encoded across IR1. In studying variations within the repeat of each strain, we identified a mutation in the main laboratory strain of EBV that impairs virus function, and we suggest that tumor-associated viruses may be more likely to contain DNA mixed from two strains. The patterns of this mixing suggest that sequences can spread between strains (and also within the repeat) by copying sequence from another strain (or repeat unit) to repair DNA damage.

Functional analysis and molecular characterization of spontaneously outgrown human lymphoblastoid cell lines.

In vitro, the infection of human B-cells with the lymphotropic gammaherpesvirus Epstein-Barr virus (EBV) induces formation of permanently growing lymphoblastoid cell lines (LCL). In a spontaneously outgrown LCL (cell line CSIII), we detected nucleotide sequence variations of the EBV nuclear antigen 1 (EBNA1) RNA that was different from the reference sequence of EBNA1 in the prototypic EBV strain B95-8. In the present study, we molecularly and functionally characterized this virus isolate in comparison to LCL with the prototypic nucleotide sequence. Although we detected high functional similarity between CSIII and the other LCL, our data suggest that the lytic cycle might be ineffective in the CSIII LCL. DNA microarray analysis indicated that RNA binding motif, single stranded interacting protein 1 (RBMS1), which is typically expressed in latency III of EBV to prevent the lytic cycle, was the most overexpressed gene in CSIII LCL.

Establishment and characterization of gastric carcinoma cell clones expressing LMP2A of Epstein-Barr virus

Although Epstein-Barr virus (EBV) has been detected in 5-15% of gastric carcinoma (GC) cases, the mechanism by which EBV contributes to its tumorigenesis remains unclear. Only a subset of EBV latent proteins such as EBV nuclear antigen-1 and latent membrane protein 2A (LMP2A) are expressed in EBV-associated GC (EBVaGC) cases. In this study, to elucidate the role of LMP2A in the tumorigenesis of EBVaGC, we established permanent cell lines expressing LMP2A. The LMP2A gene was cloned from a naturally EBV-infected EBVaGC cell line, SNU-719 and transduced into an EBV-negative GC cell line, AGS, using a retroviral vector. The sequence of SNU-719 LMP2A showed several conserved variations compared to that of the prototype EBV strain B95-8 LMP2A. Four of seven established cell clones expressed LMP2A protein at detectable levels. These cell clones did not show enhanced cell growth compared to control cells in normal or low serum-containing medium. Furthermore, LMP2A expression had no effect on colony forming ability of the cell clones in soft agar. Our results suggest that LMP2A alone has little effect on tumorigenesis of EBVaGC.

A monoclonal antibody that recognizes Epstein-Barr virus nuclear antigen 2 (EBNA 2) amino acids 1?58 does not react with EBNA 2 in native form, consistent with the self-association of EBNA 2 through the amino-terminus

We have generated a mouse IgG1 monoclonal antibody (mAb) that recognizes amino acids 1-58 of Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA 2) of type 1 EBV strain B95-8. mAb Y101 also reacted with EBNA 2 of EBV type 2 strains MISP and Jijoye in immunoblots, whereas Jijoye EBNA 2 was not detected by the widely used mAb PE2. mAb Y101, in contrast to mAb PE2, reacted with faster migrated, hypophosphorylated proteins of type 1 EBNA 2 as intensely as slower migrated, hyperphosphorylated ones. mAb Y101 did not react in fixed-cell immunostaining or cell extract immunoprecipitation. The results implicate that the amino-terminal epitope is not exposed in a native form, consistent with the previously reported idea of self-association of EBNA 2 through the amino-terminus. mAb Y101 is the first mAb to the EBNA 2 amino-terminus and will be useful for further analyses of the structure and function of EBNA 2.

Induction of inducible nitric oxide synthase by Epstein-Barr virus B95-8-derived LMP1 in Balb/3T3 cells promotes stress-induced cell death and impairs LMP1-mediated transformation.

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) causes cellular transformation and activation of several intracellular signaling events. In this report, we show that BLMP1 (encoded by the LMP1 gene derived from the B95-8 strain of EBV) triggers the expression of inducible nitric oxide synthase (iNOS) in Balb/3T3 fibroblasts. Intriguingly, NLMP1, a natural sequence variant of LMP1 identified in EBV-positive nasopharyngeal carcinoma biopsy, does not similarly induce iNOS expression. BLMP1-induced iNOS in Balb/3T3 cells is active to produce nitric oxide (NO), and NO production can be blocked by several iNOS inhibitors. When subjected to environmental stress, Balb/3T3 cells that produce NO lose viability more rapidly than non NO-producing cells. Blockage of NO generation by iNOS inhibitors enhances the viability of NO-producing cells under stress conditions. The activities of caspase-3 and c-Jun N-terminal kinase, two important regulators mediating stress-induced apoptosis, are significantly potentiated following heat shock treatment of BLMP1-expressing/NO-producing cells, compared to parental and NLMP1-expressing cells. Furthermore, treatment with iNOS inhibitor augmented the cloning efficiency (in culture) and tumor growth (in nude mice) of BLMP1-expressing/NO-producing cells. Collectively, the results demonstrate that BLMP1 induces iNOS expression and NO production in Balb/3T3 cells, which leads to the alteration of cell functions, including sensitivity to environmental stress, capability to colonize independent of anchorage and tumorigenicity in nude mice. Our data additionally implicate that the differential iNOS induction potential of the two LMP1 forms may represent the basis of a functional difference between the two LMP1 proteins.

Epstein-Barr virus lacking latent membrane protein 2 immortalizes B cells with efficiency indistinguishable from that of wild-type virus.

Epstein-Barr virus (EBV) is a human herpesvirus that efficiently transforms and immortalizes human primary B lymphocytes. In this study, the role of latent membrane protein 2 (LMP2) in EBV growth transformation was investigated. LMP2 is a virally encoded membrane protein expressed in EBV-immortalized B cells previously shown to be nonessential for EBV transformation. However, a recent study reported that LMP2 may be an important determinant for efficient B cell transformation (Brielmeier et al., Journal of General Virology 77, 2807-2818, 1996). In this study a deletion mutation was introduced into the LMP2 gene using an E. coli mini-EBV construct containing sufficient EBV DNA to result in growth transformation of primary B cells. In an alternative approach, the introduction of the gene encoding the enhanced green fluorescent protein (EGFP) by homologous recombination into the LMP2 gene of EBV strain B95-8, generating the same LMP2 deletion mutation is reported. Careful quantification of B cell transformation using the EGFP+ LMP2- recombinant virus determined that in liquid culture medium or in culture medium containing soft agarose there was no difference in the ability of LMP2- virus to immortalize primary human B cells when compared to that of wild-type virus.

Mutations in the Epstein-Barr virus latent membrane protein-1 (BNLF-1) gene in spontaneous lymphoblastoid cell lines: effect on in vitro transformation associated parameters and tumorigenicity in SCID and nude mice

Aims-(1) To study the frequency of putative malignancy associated point mutations and a 30 base pair (bp) deletion in exon 3 of the C-terminus of the Epstein-Barr virus (EBV) encoded latent membrane protein (LMP)-1 (BNLF-1) gene in wild type EBV strains. (2) To assess the influence of these mutations on the tumorigenicity of lymphoblastoid cell lines (LCL).Methods-Eight spontaneous EBV (wild type) infected LCL were established from seven subjects. Deletions and single base mutations in the C-terminus of the BNLF-1 gene were demonstrated using bi-directional solid phase dideoxy sequencing following PCR amplification of viral DNA from the LCL. Tumorigenicity of the LCL was assessed in SCID and nude mice. Serum dependent growth and ability to form colonies in soft agarose were assessed for representative LCL.Results-All LCL showed sequence differences compared with the prototypic EBV strain B95-8. The 30 bp deletion could be detected in three of eight LCL and a 69 bp deletion (including the 30 bp deletion) was identified in an additional LCL. A range of single base mutations (including those described previously in association with EBV related neoplasias) was also seen in some of the LCL. In transformation studies, the genetic variations did not seem to influence the in vitro behaviour of the LCL. In the tumorigenicity studies, the presence of the 30 bp deletion had no influence on the behaviour of the LCL which were, as expected, tumorigenic in SCID mice but not in nude mice. In contrast, the LCL carrying the 69 bp deletion was tumorigenic in both SCID and nude mice.Conclusions-Genetic changes described previously in the C-terminus of the LMP-1 gene in various malignancy derived EBV strains are also present frequently in wild type viruses and do not simply define tumour specific EBV strains. Changes within this region may, however, still be important for the tumorigenicity of LMP-1 and thus play a role in EBV oncogenesis.

Inflammatory Pseudotumor of the Liver

We describe an "inflammatory pseudotumor" of the liver that, which on detailed investigation, proved that the spindle-cell component of this lesion is derived from follicular dendritic reticulum cells (FDRC). This contention is supported by morphologic observations and by immunophenotype. The FDRC population contain Epstein-Barr virus (EBV). It is known that FDRC express the EBV receptor CD21. In this particular case, the FDRC contained clonal EBV genomes, EBV RNA (EBER) transcripts, and expressed EBV latent membrane protein (LMP1). DNA sequencing of PCR products showed three point mutations compared with the standard LMP1 sequence of the EBV strain B95-8. The findings in this case corroborate those of other investigators concerning the possible role of EBV in the development of some inflammatory pseudotumors, including the recent production of functionally active EBV-transformed FDRC-like cell lines. This association could prove instructive in delineating the histogenesis of these tumors and further assist in making prognostic and therapeutic decisions.

CD4 mRNA Expression in CD19-Positive B Cells and Its Suppression by the Epstein-Barr Virus

We have examined for synergy between the IIIB strain of HIV-1 and Epstein-Barr virus (EBV) during infection of a homogeneous cell type. In order to obtain a cell population consisting of a homogeneous cell type, CD19-positive B lymphocytes were purified from human tonsils by flow cytometry. CD19-positive lymphocytes did not express detectable surface CD4 antigen. However, CD4 mRNA could be detected in CD19-positive lymphocytes by reverse transcription coupled to polymerase chain reaction and by dot blot hybridization using an antisense riboprobe. Transcription of CD4 mRNA in CD19-positive lymphocytes was suppressed by infection with the B95-8 strain of EBV and lost in B95-8-transformed lymphoblastoid cell lines. In contrast, the P 3HR-1K strain of EBV had no effect on the level of CD4 mRNA. HIV-1 could infect CD19-sorted B cells as measured by accumulation of reverse transcriptase and syncytia induction after coculture with SupT1 cells. HIV-1 infection of CD19-bearing lymphocytes was blocked by OKT4a antibodies. The ability of HIV-1 to replicate in CD19-positive B lymphocytes declined following preinfection with B95-8 but not with P 3HR-1K. These results as well as results with an EBNA-2 expression vector suggest that down-regulation of both CD4 mRNA and HIV-1 infection in human B cells is a function of EBV nuclear antigen EBNA-2. The fact that native CD19-positive B lymphocytes express sufficient CD4 receptor mRNA to allow HIV-1 infection strengthens the possibility that HIV-1 replication in B cells directly participates in AIDS pathogenesis. In addition, infection with EBV may modulate the ability of HIV-1 to infect and establish a latent infection in B lymphocytes in co-infected individuals.

A modified procedure for the propagation of wild type Epstein-Barr virus in cultures of marmoset blood cells

Productive infection of marmoset blood mononuclear cells by the Epstein-Barr virus was generally achieved by the co-cultivation method. By introducing 2 modifications, the success rate of infection was increased from 6% to 79%. The modifications consisted of the selection of human lymphocyte donors to serve as carriers of Epstein-Barr virus in cocultures, and the addition of cyclosporin A to culture media. So far, 10 of 10 wild type oropharyngeal EBV have been propagated successfully in cultures of marmoset blood mononuclear cells by the modified procedure. Fragment length polymorphism study failed to reveal any difference between viral genomes in human lymphocytes and that in marmoset blood mononuclear cells. Antigenic analysis of 8 wild strains showed that all were related to the B95-8 strain of Epstein-Barr virus by the neutralization test.

Nucleotide sequence of a 3.5 kilobase fragment of malignant catarrhal fever virus strain WC11

A 3.5 kilobase DNA fragment of the malignant catarrhal fever virus (MCFV), strain WC11, was mapped with a number of restriction enzymes, subcloned and sequenced. The fragment was subcloned into plasmid vector, pUC19, for direct sequencing. A complete open reading frame of 2,058 base pairs and a partial open reading frame of 630 base pairs were identified. The sequence of 3,389 nucleotides was compared to other herpesviruses. A 310 base pairs sequence in gene A was 57% homologous to a sequence in reading frame BXLF1 of Epstein-Barr virus strain B95-8.

Epstein-Barr Virus DNA Sequences in Precursor Monocyte-Macrophage Cell Lines Established from the Bone Marrow of Children with Maturation Defects of Haematopoiesis

Epstein-Barr virus (EBV) DNA sequences were detected in four established monoblast or early monocytic cell lines (CM-S, ROV-S, CV-S and AD-S) obtained from bone marrow of children suffering from maturation defects of haematopoiesis. EBV is present in these cells in a latent state. The viral DNA in these cell lines was analysed by Southern blot hybridization, using a set of cloned EBV DNA fragments from the EBV strain B95-8 as probes. A common spectrum of highly related but distinguishable EBV DNA restriction enzyme sequences was found, suggesting some genomic diversity. Propagation of the cells in long-term culture revealed a gradual decrease of EBV copies per cell in all lines with some minor changes in the restriction pattern of the EBV DNA. These findings demonstrate that human precursor monocyte cells may be susceptible to infection by EBV.

Biomolecular analysis of a defective nontransforming Epstein-Barr virus (EBV) from a patient with chronic active EBV infection.

A virus recovered from the saliva of a child with chronic active Epstein-Barr virus (EBV) infection for 8 years was shown to induce EBV early antigen (EBV-EA) in Raji cells and to be expressed into EBV-EA in fresh EBV-negative peripheral blood leukocytes. However, it did not replicate its DNA. Oropharyngeal epithelial cells scraped from recurrent mouth lesions were similarly positive for EBV-EA. DNA extracted from these cells and digested with BamHI contained a 6-kilobase-pair fragment homologous to BamHI fragment V and B1 EBV DNA probes. Furthermore, Southern blots of the BamHI and EcoRI digests of the DNA extracted from the cell lines of the patient (transformed with EBV strain B95-8) and of her mother (spontaneous) revealed, in addition to the expected BamHI G, H, H2, and B1 fragments used as probes, additional shorter ones of a presumably endogenous defective virus.

Posttranslational processing of an Epstein-Barr virus-encoded membrane protein expressed in cells transformed by Epstein-Barr virus.

The BamHI Nhet fragment of the B958 strain of Epstein-Barr virus (EBV) encodes a membrane protein (BNLF-1) that is present in cells transformed by EBV. We made a hybrid protein in which a polypeptide sequence from the carboxyl-terminal part of BNLF-1 is fused to Escherichia coli beta-galactosidase. This hybrid protein was used to immunize rabbits, and the resulting antiserum was purified by immunoaffinity chromatography. The antiserum was able to immunoprecipitate BNLF-1 from cell lysates. We found that BNLF-1 is phosphorylated at serines in EBV genome-positive B-cell lines. Pulse-chase analyses with [35S]methionine indicated that BNLF-1 is turned over in lymphoblasts with a half-life of approximately 5 h. Protein immunoblots of EBV genome-positive B-cell lines revealed both a 62,000-molecular-mass band corresponding to BNLF-1 and a myriad of lower-molecular-mass bands. We postulate that these lower-molecular-mass bands are degradation products resulting from the turnover of BNLF-1 in cells. The BNLF-1 gene was expressed in COS cells, and the protein was both phosphorylated and turned over in these cells.

Analysis of the DNAs of Epstein-Barr virus (EBV) derived from two EBV genome-positive epithelial/hybrid cell lines (NPC-KT and A2L/AH)

The authors recently reported the establishment of two Epstein-Barr virus (EBV)-producing epithelial/hybrid cell lines (A2L/AH and NPC-KT), A2L/AH cells were derived from the fusion of epithelial cell line (Ad-AH) with non-producer EBV genome-positive lymphoblastoid cells transformed with the B95-8 strain of EBV. NPC-KT cells were derived from the fusion of Ad-AH cells with primary nasopharyngeal carcinoma cells. In this study, viral DNAs derived from A2L/AH and NPC-KT cells were analyzed in comparison with viral DNA derived from B95-8 cells by use of the restriction endonuclease EcoRI and Hind III and separation of fragments in 0.4% agarose. The digestion fragments of these two viral DNAs were obviously similar, but could be distiguished each other. The molecular weight of DNA of virus from NPC-KT cells was approximately 181K base pairs (Kb), which was 12Kb larger than that of B95-8 virus DNA. Key words: NPC-KT, A2L/AH, Epstein-Barr virus, DNA A90-1177-51360

Identification of Epstein-Barr virus sequences that encode a nuclear antigen expressed in latently infected lymphocytes.

The Epstein-Barr virus (EBV) BamHI restriction endonuclease fragment K (B95-8 strain) was introduced into a polyoma virus expression vector and used to transfect murine NIH 3T3 cells. An EBV-associated nuclear antigen was detected in these cells in an indirect immunofluorescence test using anti-EBV nuclear antigen-positive human sera. These sera recognized a Mr 88,000 polypeptide in 3T3 cells transfected with the BamHI fragment K-containing polyoma virus plasmid by radioimmunoelectrophoresis. A Mr 88,000 polypeptide also was detected in a B-cell line latently infected with the B95-8 strain of EBV. Plasmids containing insertion and deletion mutations in BamHI fragment K directed the synthesis of truncated forms of the Mr 88,000 polypeptide in 3T3 cells. These data directly demonstrate that the polypeptide identified in EBV-infected lymphocyte lines by anti-EBV nuclear antigen-positive human sera is encoded by the viral genome.

DNA sequence and expression of the B95-8 Epstein-Barr virus genome.

The complete (172,282 base pairs) nucleotide sequence of the B95-8 strain of Epstein-Barr virus has been established using the dideoxynucleotide/M13 sequencing procedure. Many RNA polymerase II promoters have been mapped and the mRNAs from these promoters have been assigned to the latent or early/late productive virus cycles. Likely protein-coding regions have been identified and three of these have been shown to encode a ribonucleotide reductase, a DNA polymerase and two surface glycoproteins.

Rescue of transforming Epstein-Barr virus (EBV) from EBV-genome-positive epithelial hybrid cells transfected with subgenomic fragments of EBV DNA.

Transfection experiments using subgenomic fragments of the B95-8 strain of Epstein-Barr virus (EBV) DNA and EBV genome (HR-1)-positive epithelial/Burkitt hybrid cells (D98/HR-1) were carried out to determine whether an interaction between the transfecting virus fragment(s) and the endogenous HR-1 EBV genome could take place. Expression of EBV-specific antigens, including early antigen and virus capsid antigen, was examined in transfected cells by immunofluorescence. Attempts were also made to recover biologically active EBV from the D98/HR-1 cells after transfection with cloned fragments of B95-8 DNA. We found that D98/HR-1 cells transfected with the BamHI H or H, F, and X fragments were positive for early antigen 3 days after transfection. Spent media from transfected D98/HR-1 cells maintained for 20-30 days in culture were pooled, filtered, concentrated, and used as a potential source of virus to inoculate human umbilical cord blood lymphocytes. No evidence of transformation was observed with such preparations. However, if spent medium from D98/HR-1 cell cultures was first treated with iododeoxyuridine (to induce EBV DNA synthesis and replicative cycle) and then transfected with the BamHI H, F, and X fragments of B95-8 DNA and used to infect cord blood lymphocytes, transformation was obtained. A lymphoblastoid cell line derived in this manner, designated HI-HFX, is an EBV nuclear antigen-positive nonproducer cell line. Similar results were obtained with preparations from iododeoxyuridine-treated D98/HR-1 cells transfected with the EB 26-36 fragment of B95-8 DNA cloned in a Charon 4A vector. The EB 26-36 fragment contains the BamHI H, F, and X regions.

Limiting dilution analysis of Epstein-Barr virus-induced immunoglobulin production by human B cells.

The Epstein-Barr virus (EBV) is a herpes virus that has the capacity to infect human B cells and to induce them to secrete immunoglobulin (Ig). In the current experiments, Poisson analysis of limiting dilution cultures has been used to study the activation of human peripheral B cells by the B95-8 strain of EBV. Under the culture conditions used, 0.2-1% of peripheral blood B cells were activated by EBV to secrete IgM or IgG. In addition, when multiple replicate cultures containing limited numbers of B cells were tested for IgM and for IgG production, the precursors for IgM and IgG segregated independently; thus, individual B cell precursors matured into cells secreting IgM or IgG but not both classes of Ig. Additional experiments using limiting dilutions of EBV were undertaken to study the viral requirements for B cell activation. These studies indicated that B cell activation by EBV to produce Ig was consistent with a "one-hit" model and inconsistent with a "two-hit" model. Taken together, these results indicate that infection by one EBV virion is sufficient to induce a precursor peripheral blood B cell to secrete Ig and that only one isotype of Ig is then secreted.

The B95-8 isolate of Epstein-Barr virus arose from an isolate with a standard genome.

Blot hybridization studies revealed that the deletion which characterizes the DNA from the B95-8 strain of Epstein-Barr virus was not present in the virus from which the B95-8 strain was derived (883L). The deletion event must have occurred during establishment of the B95-8 cell line or very soon afterward, since the deletion was present in Epstein-Barr virus DNA from a cell line established with B95-8 virus soon after it became available. The presence of the deletion correlates with decreased expression of the gp220 viral envelope glycoprotein.