Georg W. Bornkamm

Geographical prevalence of two types of Epstein-Barr virus

The Jijoye EBV strain is characterized by a substitution of 1.8 kb in the C-terminal part of the EBNA 2 gene compared to B95-8 or M-ABA virus. This made it possible to construct hybridization probes specific for M-ABA (type A) and Jijoye viruses (type B), which have been used to type the EBV genomes in 38 spontaneously established cell lines. Type A is more prevalent being found in 31 of 38 cases; type B virus was found in five cell lines (Jijoye, LY 67, QIMR-GOR, BL 16, and BL 29); and two cell lines, Daudi and EB-3, contained neither the M-ABA- nor the Jijoye-specific sequences. EBV type B appears to be less ubiquitous, since all type B isolates, including AG 876 virus, originated from Central Africa, La Réunion, and New Guinea. All the other cell lines, carrying EBV type A, were established from patients from Central Africa (4), North Africa (7), New Guinea (1), and Asia (6) and from white individuals (13). The restricted geographical localization of EBV type B in parts of the southern hemisphere and its similarity to herpesvirus papio (T. Dambaugh, K. Hennessy, L. Chamnankit, and E. Kieff (1984) Proc. Natl. Acad. Sci. USA 81, 7632-7636) could suggest that such viruses may have evolved by recombination of EBV with a related Old World monkey virus, alternatively, evolution of virus variants within the human species also being conceivable.

A putative transforming gene of Jijoye virus differs from that of Epstein-Barr virus prototypes

The P3HR-1 strain of Epstein-Barr virus (EBV), a nontransforming clonal derivative of Jijoye (EBV), is characterized by a deletion of 6.6 kb involving part of the BamHI-W repeats and the adjacent region including the NotI repeats. In the transforming parental Jijoye virus this region differs from the corresponding regions in B95-8 or M-ABA virus. The HindIII-B fragments which carry this region from both Jijoye and prototype M-ABA (EBV) viruses have been cloned and subclones have been constructed which contain the left-hand part of HindIII-B from the HindIII to the BglII site (BglII-delta C fragment). By restriction enzyme analysis the inserts were found to be of equal size (6.3 kb) but to differ in their restriction enzyme pattern. Heteroduplexes formed under stringent conditions in the presence of T4 gene 32 protein revealed a substitution loop of 1750 +/- 200 nucleotides. Heteroduplex formation under nonstringent conditions showed that the substituted sequences are partially homologous to each other, with the regions of nonhomology confined to three distinct areas of 100 to 200 nucleotides. The partial homology observed between both regions indicates that they have evolved from a common ancestor. By hybridization of a Jijoye virus subclone containing only sequences of the substituted region to Northern blots a 2.8-kb polyadenylated transcript was detected indicating that the substituted region is expressed in Jijoye cells.

Two deletions in the Epstein-Barr virus genome of the Burkitt lymphoma nonproducer line Raji.

The Epstein-Barr virus genome carried in the Burkitt lymphoma nonproducer cell line Raji was characterized by partial denaturation mapping and by hybridization of cloned viral fragments to filters containing separated Raji DNA fragments. Partial denaturation mapping revealed that the EBV DNA population of Raji cells is homogeneous and that two deletions are observed in distant parts of the genome compared to linear DNA isolated from virus particles of different strains. These deletions were characterized by blot analysis. One deletion of 3.15 kb lies within HindIII-E; the second is 2.4 kb and is located close to the right terminus of linear viral DNA. The two deletions were observed in several cell lines derived from the Raji line. These deletions might contribute to the inability of Raji cells to produce EBV either spontaneously or upon induction.

Characterization of an EBV-like virus from African green monkey lymphoblasts

Abstract A lymphoblastoid cell line (AGM-2206) has been established from the peripheral blood of an African green monkey. Approximately 1% of these cells showed fluorescence for EBV capsid antigens (VCA), when examined with human EBV-VCA-positive sera by indirect immunofluorescence. Titration of human and monkey sera on AGM-2206 and P3HR-1 cells revealed a partial cross-reactivity between capsid antigens of the AGM virus and human EBV. In addition, cross-reactivity within the EA complex is suggested by reactivity of about 20% of monkey sera with human EBV-EA. Neither human nor monkey sera reacted with an EBNA-like antigen in AGM lymphoblasts. Cleavage patterns of AGM-EBV DNA with Eco RI and Hin dIII restriction endonucleases showed differences when compared to human EBV DNA isolated from B 95-8, P3HR-1, and QIMR-WIL cells. Hybridization of labeled EBV DNA from B 95-8 cells to fragments of AGM-EBV DNA blotted onto nitrocellulose filters resulted in some hybridization to a number of AGM-EBV DNA bands.

C-myc and immunoglobulin kappa light chain constant genes are on the 8q+ chromosome of three Burkitt lymphoma lines with t(2;8) translocations

We have determined the localization of c‐myc and the immunoglobulin kappa light chain genes on the 8q+/2p‐ chromosomes of the three Burkitt lymphoma lines BL21, LY66 and LY91 with t(2;8) translocation by in situ hybridization. BL21 is characterized by a complex translocation in which a piece of chromosome 9 appears to be located between the fragments of chromosome 8 and 2 on the 8q+ chromosome. Our data indicate that in all three cell lines the c‐myc gene is located on the 8q+ chromosome proximal to the breakpoint in band 8q24. In all cell lines examined the cluster of kappa variable genes has remained on the 2p‐ chromosome. In LY91 cells the major part of the joining region remained on 2p‐, while the joining region has moved to 8q+ in the cell lines BL21 and LY66. In all three cell lines the constant kappa light chain gene was found on the 8q+ chromosome. The fact that an essentially identical pattern was found in the cell line BL21, with the complex translocation, suggests that the insertion of the piece of chromosome 9 into the 8q+ chromosome might be a secondary event. Our present data fit into the concept that in all Burkitt lymphoma lines investigated so far, including cases with t(8;14) and the variant translocations t(2;8) and t(8;22), the c‐myc gene becomes situated at the 5′ side of an immunoglobulin constant gene. This may have implications for the generation of somatic mutations in the coding and non‐coding part of the c‐myc gene.

Nucleic acid hybridization for the detection of viral genomes.

Single-stranded nucleic acids can form stable duplexes if they encounter complementary sequences under appropriate conditions. This reaction is called hybridization and has been extensively used during the past 15 years to explore the structure and expression of cellular and viral genes. Particularly with the advance of molecular cloning, hybridization with specific probes has made it possible to isolate and study individual genes from genomic libraries and has thus contributed significantly to our understanding of eukaryotic gene organization.

No homology detectable between Marek's disease virus (MDV) DNA and herpesvirus of the Turkey (HVT) DNA

The relation of four different strains of MDV and two strains of HVT was analyzed by gel electrophoresis of viral DNA digested by various restriction endonucleases and by filter hybridization of viral DNA with complementary RNA.The four MDV strains showed fragment patterns completely different from those of HVT upon digestion of the viral DNA with Bam H I, Eco R I, Hind III, Hpa I, and Xho and separation of fragments on agarose gels.The cleavage patterns of the four MDV strains showed great similarities among each other as well as some differences between the individual strains. In the cleavage patterns of HVT a similar close relationship was observed between the two HVT strains with slight divergence between both.Filter hybridizations of viral DNA with labelled complementary RNA prepared from the DNA of the GA strain of MDV or from the DNA of the PH-THV1 strain of HVT revealed no cross-hybridization between the MDV and the HVT strains.cRNA prepared from the DNA of an MDV strain hybridized only to restriction enzyme fragments of the MDV strains transferred to nitrocellulose filters, but not to fragments of HVT DNA, and vice versa.

Establishment of spontaneously outgrowing lymphoblastoid cell lines with Cyclosporin A

To establish spontaneously outgrowing EBV (Epstein-Barr virus) transformed lymphoid cell lines from healthy EBV-seropositive adults different culture conditions were investigated. The effect of T-cell depletion was compared with that of adding Cyclosporin A (CSA) to unseparated mononuclear blood cells, either at the onset of cultivation or after 3 weeks. The highest frequency of spontaneous outgrowth of EBV-transformed cells was obtained in unseparated cultures when the addition of CSA was delayed. The presence of unimpaired T cells at the onset of the culture period appeared to have a favourable effect on the spontaneous outgrowth of EBV-carrying lymphoblastoid cell lines.

EBNA2 and c-myc in B cell immortalization by Epstein-Barr virus and in the pathogenesis of Burkitt's lymphoma.

Epstein-Barr virus (EBV) is a ubiquitous human herpes virus with the unique ability to immortalize primary resting human B lymphocytes. The virus is the causative agent of infectious mononucleosis and is associated with a number of human malignancies including Burkitt’s Lymphoma, Nasopharyngeal Carcinoma, Hodgkin’s Disease, and Immunoblastic Lymphoma arising in immunocompromized individuals (for review, see Rickinson and KiefF, 1995). The pathogenicity of the virus is closely linked to its immortalizing capacity. Studying the role of EBV in B cell immortalization may thus help to better understand the role of the virus in disease pathogenesis. In EBV immortalized cells only a limited set of viral gene products is expressed including six nuclear (EBNA1, EBNA2, EBNA3A, -3B, -3C and EBNA-LP) and two membrane proteins (LMP1 and LMP2) (for review, see Kieff, 1995). For several reasons EBNA2 plays a pivotal role in B cell immortalization: (i) deletion of the EBNA2 gene leads to the loss of the immortalizing capacity of the virus, (ii) EBNA2, together with EBNA-LP, is the first viral gene expressed in infected B cells, and (iii) EBNA2 is a transcriptional regulator acting as a master switch regulating the expression of a number of cellular genes (e.g. CD21 and CD23) and viral genes involved in B cell immortalization. Amongst the viral genes regulated by EBNA2 are the LMP1 and LMP2 genes and the large transcription unit giving rise to EBNA-LP and EBNA2 as well as the family of EBNA3 proteins. EBNA2 exerts its function as a transcriptional regulator without binding to DNA directly. It is thus of primary importance to elucidate the molecular mechanism of action of EBNA2.

Chromosomentranslokationen und Epstein-Barr-Virus beim Burkitt-Lymphom

Das Burkitt-Lymphom ist ein Tumor mit besonderen epidemiologi-schen Eigenheiten. Es ist ein sehr haufiger Tumor im Kindesalter in tropischen Teilen Afrikas und kommt mit viel niedrigerer Inzidenz uberall auf der Welt vor. Chromosomentranslokationen, die den langen Arm von Chromosom 8 und einen der drei Immunglobulingen-loci auf Chromsom 2, 14 oder 22 betreffen, werden regelmasig beim Burkitt-Lymphom beobachtet, unabhangig von der geographischen Herkunft. Das haufige Auftreten des Burkitt-Lymphoms in Zentral-afrika ist mit zwei Faktoren korreliert: holo- oder hyperendemischer Malaria und Prasenz des Epstein-Barr-Virus-Genoms in den Tumorzellen. Wir stellen hier ein neues Konzept zur Pathogenese des Burkitt-Lymphoms vor: Stimulierung des B-Zellsystems durch Malaria stellt danach den ersten Schritt in der Entwicklung des Tumors dar. Durch die vermehrte Neubildung von B-Zellen soil die Wahrscheinlichkeit fur das Auftreten der Translokation steigen. Die Chromoso-mentranslokation und Aktivierung des Proto-Onkogens c-myc erlaubt den Zellen, auf Wachstumsfaktoren zu antworten, ohne das Proliferation direkt induziert wird. Nach unserem Modell vermittelt die Infektion mit Epstein-Barr-Virus der Zelle einen zusatzlichen Wachstums-vorteil und treibt sie weiter in Richtung maligne Zellen.