Rapid proliferation of B cells from adenoids in response to Epstein-Barr virus infection.

EBV is a human tumor virus that is associated with different types of tumors. A unique feature of EBV is its capability to infect and immortalize human B cells both in vivo and in vitro. In cell culture, this progress is termed immortalization and infected B cells grow out to permanent, so-called lymphoblastoid cell lines. During our experiments, we observed that B lymphocytes derived from adenoids are infected efficiently by EBV and proliferate much more rapidly than any other known type of B cell. High concentrations of adhesion molecules and of CD21, the EBV receptor, present on these cells may account for this phenomenon. Adenoid B cells may therefore represent a particular subpopulation of preactivated B lymphocytes that can greatly simplify and enhance the production of lymphoblastoid cell lines for, e.g., antigen-presenting cells for gene therapeutic approaches and similar applications.

c-myc expression is activated by the immunoglobulin kappa-enhancers from a distance of at least 30 kb but not by elements located within 50 kb of the unaltered c-myc locus in vivo.

50 kb of contiguous DNA sequences covering the human c-myc coding region and approximately 20 kb of flanking upstream and downstream sequences were cloned onto a prokaryotic F-factor derived plasmid, which also contains a selectable marker and the plasmid origin of DNA replication oriP of Epstein Barr virus (EBV). Since these plasmids replicate extrachromosomally after stable transfection into EBV-positive B-cell lines, the gene regulation of c-myc can be analysed independent from chromosomal integration positions. Despite the presence of all known c-myc regulatory elements on these constructs, expression from the stably transfected c-myc gene was barely detectable in either cell line. Hypermethylation of these plasmids could be excluded as a mechanism for the lack of gene expression. Insertion of the immunoglobulin kappa-intron and 3' enhancers, however, activated c-myc transcription, when placed adjacent to or separated from the c-myc promoters by as far as 30 kb. These results indicate that transcription of c-myc in vivo requires additional and still unidentified control elements located outside this 50 kb fragment, and experimentally demonstrate long range enhancer function in vivo.

Herpesvirus vectors come of age.

Most human herpesviruses are ubiquitous and are closely associated with a number of severe acute infections and human tumors. Progress in herpesvirus genetics has made members of the herpesvirus family accessible, such that they have become more attractive as gene vectors to be used in the treatment of various diseases, including the prevention of herpesvirus-related afflictions. This review summarizes recent progress and provides a basis for development of new viral and therapeutic strategies.

Rapid method for the identification and screening of herpesviruses by DNA fingerprinting combined with blot hybridization.

Rapid characterization of herpesvirus isolates exemplified by equine herpesviruses is described. Total DNAs were isolated from virus infected small scale cell cultures. The DNA fragments obtained after restriction enzyme digestion were separated on agarose gels, transferred and immobilized on filter membranes. A radioactively labelled probe derived from the purified DNA of an EHV-1 reference strain was used for hybridization in order to detect the restriction fragments of different EHV-1 field isolates. This method allows the typing of many isolates within a short period of time.

Epstein-Barr virus maintains lymphomas via its miRNAs.

Epstein-Barr virus (EBV) has evolved exquisite controls over its host cells, human B lymphocytes, not only directing these cells during latency to proliferate and thereby expand the pool of infected cells, but also to survive and thereby persist for the lifetime of the infected individual. Although these activities ensure the virus is successful, they also make the virus oncogenic, particularly when infected people are immunosuppressed. Here we show, strikingly, that one set of EBV's microRNAs (miRNAs) both sustain Burkitt's lymphoma (BL) cells in the absence of other viral oncogenes and promote the transformation of primary B lymphocytes. BL cells were engineered to lose EBV and found to die by apoptosis and could be rescued by constitutively expressing viral miRNAs in them. Two of these EBV miRNAs were found to target caspase 3 to inhibit apoptosis at physiological concentrations.

Epstein-Barr virus vector-mediated gene transfer into human B cells: potential for antitumor vaccination.

The efficient gene transfer of immunostimulatory cytokines into autologous tumor cells or the transfer of tumor-associated antigens into professional antigen-presenting cells is a prerequisite for many immunotherapeutic approaches. In particular with B cells, the efficiency of gene uptake is one of the limiting factors in cell-based vaccine strategies, since normal and malignant human B cells are commonly refractory to transducing gene vectors. Due to its natural tropism for human B cells, Epstein-Barr virus (EBV), a human herpes virus, might be an option, which we wanted to explore. EBV efficiently infects human B cells and establishes a latent infection, while the viral genome is maintained extrachromosomally. Although these characteristics are attractive, EBV is an oncogenic virus. Here, we present a novel EBV-derived vector, which lacks three EBV genes including two viral oncogenes and an essential lytic gene, and encodes granulocyte-macrophage colony-stimulating factor (GM-CSF) as a cytokine of therapeutic interest. We could show that EBV vectors efficiently transduce different B-cell lines, primary resting B cells, and tumor cells of B-cell lineage. Vector-derived GM-CSF was expressed in sufficient amounts to support the maturation of dendritic cells and their presentation of model antigens to cognate T-cell clones in autologous settings and an allogeneic, HLA-matched assay. We conclude that the EBV vector system might offer an option for ex vivo manipulation of B cells and gene therapy of B-cell lymphomas.

Genetic design of an optimized packaging cell line for gene vectors transducing human B cells.

Viral gene vectors often rely on packaging cell lines, which provide the necessary factors in trans for the formation of virus-like particles. Previously, we reported on a first-generation packaging cell line for gene vectors, which are based on the B-lymphotropic Epstein-Barr virus (EBV), a human gamma-herpesvirus. This 293HEK-derived packaging cell line harbors a helper virus genome with a genetic modification that prevents the release of helper virions, but efficiently packages vector plasmids into virus-like particles with transducing capacity for human B cells. Here, we extended this basic approach towards a non-transforming, virus-free packaging cell line, which harbors an EBV helper virus genome with seven genetic alterations. In addition, we constructed a novel gene vector plasmid, which is devoid of a prokaryotic antibiotic resistance gene, and thus more suitable for in vivo applications in human gene therapy. We demonstrate in this paper that EBV-based gene vectors can be efficiently generated with this much-improved packaging cell line to provide helper virus-free gene vector stocks with transducing capacity for established human B-cell lines and primary B cells.

Structure and role of the terminal repeats of Epstein-Barr virus in processing and packaging of virion DNA.

The linear virion Epstein-Barr virus (EBV) DNA is terminated at both ends by a variable number of direct, tandemly arranged terminal repeats (TRs) which are approximately 500 bp in size The number of TRs at each terminus can vary. After infection of host cells, the EBV DNA circularizes via the TRs by an unknown mechanism, and replication of the viral DNA during the lytic phase of the EBV life cycle leads to large DNA concatemers which need to be cleaved into virion DNA units, eventually. This cleavage event occurs at an unknown locus within the TRs of EBV, which are the cis-acting elements essential for cleavage of the concatemers and encapsidation of the virion DNA. To investigate the mechanism of DNA processing during genome circularization and cleavage of concatemeric DNA, the genomic termini of EBV were cloned, sequenced, and analyzed by direct labeling of the virion DNA. Both termini ended with identical 11-bp elements; the right end has acquired an additional 9-bp stretch that seemed to originate from the leftmost unique sequences. The left terminus is blunt, whereas the right terminus appears to have a 3' single-base extension. In a transient packaging assay, a single terminal repeat was found to be sufficient for encapsidation of plasmid DNA, and mutagenesis of the TR element defined a region of 159 bp, including the 11-bp element, which is essential for packaging. These results indicate that the genomic termini of EBV are not generated by a simple cut of a hypothetical terminase. The mechanism for cleavage of concatemers seems to involve recombination events.

Plasmid-like replicative intermediates of the Epstein-Barr virus lytic origin of DNA replication.

During the lytic phase of herpesviruses, intermediates of viral DNA replication are found as large concatemeric molecules in the infected cells. It is not known, however, what the early events in viral DNA replication that yield these concatemers are. In an attempt to identify these early steps of DNA replication, replicative intermediates derived from the lytic origin of Epstein-Barr virus, oriLyt, were analyzed. As shown by density shift experiments with bromodeoxyuridine, oriLyt replicated semiconservatively soon after induction of the lytic cycle and oriLyt-containing DNA is amplified to yield monomeric plasmid progeny DNA (besides multimeric forms and high-molecular-weight DNA). A new class of plasmid progeny DNA which have far fewer negative supercoils than do plasmids extracted from uninduced cells is present only in cells undergoing the lytic cycle of Epstein-Barr virus. This finding is consistent with plasmid DNAs having fewer nucleosomes before extraction. The newly replicated plasmid DNAs are dependent on a functional oriLyt in cis and support an efficient marker transfer into Escherichia coli as monomeric plasmids. Multimeric forms of presumably circular progeny DNA of oriLyt, as well as detected recombination events, indicate that oriLyt-mediated DNA replication is biphasic: an early theta-like mode is followed by a complex pattern which could result from rolling-circle DNA replication.

[Immunity of the skin--infection and percutaneous immunization of rabbits with E. coli ATCC 13676]. / Immunität der Haut--Infektion und perkutane Immunisierung von Kaninchen mit E. coli ATCC 13676.

After intracutaneous infection of rabbits with a suspension of E. coli which was followed by a transient local inflammation, the local and systemic immune responses were determined using the lymphocyte stimulation test (LTT) and the hemolysis plaque assay (HPA). Lymphocytes of the lymphatic system draining the infected skin area and blood lymphocytes were used. With lymphocytes derived from the local lymph nodes, a substantial increase of specific stimulation in the LTT was detected beginning at day 3 after infection and lasting up to the termination of the experiment (3 weeks). Blood lymphocytes were stimulated at a lower level: The activity showed a peak at day 4 and an elevated level only during a 10-day period. After the intracutaneous infection with E. coli, increasing numbers of antibody-releasing lymph node cells were detected in the HPA. The antibody-secreting cells of the IgM and IgG classes clearly showed an increasing specificity for E. coli lipopolysaccharide coupled to sheep red blood cells. As with the LTT, the highest activities (values of specificity and number of plaque-forming lymphocytes) were observed at the end of the experimental period. An emulsified preparation of a heat-inactivated E. coli culture (E. coli-BKS) which had been applied locally onto the artificially altered skin evoked a similar immunological response after a 2 or 3-weeks treatment. In such animals an increased activity of lymph node cells could be registered by LTT and HPA as compared to reactions from placebo-treated control animals. However, the topical immunization with nonviable E. coli stimulated not only lymphocytes which produced antibodies directed specifically against E. coli lipopolysaccharide as demonstrated by the HPA. An increased number of lymphocytes reacted even with native sheep red blood cells. This observations is discussed in respect of a polyclonal B-cell activation by lipopolysaccharide of the E. coli-BKS.

Identification and characterization of oriLyt, a lytic origin of DNA replication of Epstein-Barr virus.

We have identified a cis-acting element of Epstein-Barr virus (EBV) that mediates viral DNA replication during the lytic phase of this virus's life cycle. This lytic origin of DNA replication, termed oriLyt, is complex in structure in that it contains multiple regions that are required for replication and additional DNA sequences that increase replication. One of the required regions of oriLyt can be functionally substituted by a transcriptional enhancing element. DNA replication mediated by oriLyt depends on EBV DNA polymerase and yields a concatemeric molecule. A vector, which contains both oriP (the EBV plasmid origin of replication) and oriLyt, can be maintained as a plasmid in latently EBV-infected cells and can be amplified 100- to 1000-fold in cells in which the lytic phase of the viral life cycle is induced.

Genetic analysis of immortalizing functions of Epstein-Barr virus in human B lymphocytes.

Epstein-Barr virus (EBV), a herpes virus, infects human B lymphocytes in vitro and efficiently immortalizes them. About 10 of the approximately 100 genes of EBV are expressed in recently immortalized B cells and although there is circumstantial evidence that at least three of these may contribute to the process of immortalization, there is no direct evidence that any particular gene is required. We have developed a genetic analysis of EBV that uses a transformation-defective strain of the virus as a helper virus in conjunction with DNA that contains all of the viral cis-acting elements required for replication, cleavage and packaging during the lytic phase of the viral life cycle. This DNA can include viral genes required for immortalization that complement the transformation-defective virus strain. The DNA can be amplified and packaged by the products of the helper virus and the packaged DNA is infectious. We have analysed two viral genes expressed in immortalized cells and find that the gene encoding EBV nuclear antigen-2 is required for immortalization, whereas the gene for the EBV nuclear antigen leader protein is not.

DNA replication of herpesviruses during the lytic phase of their life-cycles.

We have two goals in this review: the first is to relate what has been learned about DNA replication from the study primarily of herpes simplex virus type 1 (HSV-1); the second is to note briefly facets of this virus's mode of DNA replication that might serve as points of intervention for novel chemotherapeutic approaches in order to deal with primary and recurrent herpesvirus infections in man and animals. For the first goal we shall both summarize what has been learned and attempt to identify directions that may be pursued in order to further our understanding of DNA replication by herpesviruses. For the second goal we shall propose two schemes for the screening for drugs that might interfere uniquely with the DNA replication of this family of viruses.

[Plasmid-mediated pathogenicity factors of Yersinia enterocolitica from the epidemiological viewpoint]. / Plasmidvermittelte Pathogenitätsfaktoren von Y. enterocolitica unter epidemiologischen Gesichtspunkten.

In an attempt to evaluate the epidemiological significance of Y. enterocolitica strains isolated from domestic animals with latent infections, i.e. from swine (31), dogs (9), cats (2), and the environment (2) were compared, in respect of their pathogenic properties, with reference strains (27) from human patients. The results did not contradict the assumption of animals being a source of human infections. In general, these strains of the serovars 0:3 and 0:9 possessed a virulence plasmid with a molecular weight of 46 Mdal (+/- 3 Mdal), irrespective of their origin from humans or domestic animals with latent infections. There was a consistent correlation of the presence of the virulence plasmid and of positive reactions in Laird's and Cavanaugh's autoagglutination test (1980) (Tab. 1): The strains harbouring this plasmid were calcium dependent on MOX agar (18) as well. These strains were capable of colonizing the intestine of mice in a modified mouse diarrhea model (Fig. 1). In these latently infected mice low titers of species-specific antibodies were detected. It was possible to select plasmid harbouring substrains out of heterogenic strains, which had undergone conversion to plasmidless isogenic variants when applying the modified mouse diarrhea model. In a newly developed "macrophage-virulence-test", strains harbouring the virulence plasmid were able to multiply within the macrophage of mice (Fig. 4, 5) with cytopathogenic effects. In a modified Sereny-test European strains of the serovars 0:3 and 0:9 were shown to be invasive in the conjunctival epithelium of guinea pigs' eyes as an additional plasmid-associated property (Fig. 3). European strains of atypical biochemical or serological patterns (among them Y. intermedia, Y. frederiksenii) as well as some strains of the serovars 0:3 and 0:9 did not harbour the virulence plasmid. These plasmidless strains were altogether negative regarding the pathogenic properties mentioned above. Long-term in vitro subcultivation of strains correlated well with the frequency of plasmidless strains: isolates cultivated for a number of years, in contrast to newly isolated strains, hardly harboured virulence plasmis.

A transcription factor with homology to the AP-1 family links RNA transcription and DNA replication in the lytic cycle of Epstein-Barr virus.

oriLyt, the lytic origin of DNA replication of Epstein-Barr virus (EBV), ensures viral DNA amplification during the productive or lytic phase of the virus' life cycle. To understand the contribution of cis- and transacting elements involved in DNA replication of oriLyt, a detailed mutational analysis was undertaken which defined BZLF1, a viral transcriptional activator, as an essential replication factor. The BZLF1 protein belongs to the extended fos/jun family of transcription factors and binds to specific BZLF1 binding motifs within oriLyt, as well as to consensus AP-1 sites. Recombinant, chimeric transcription factors identified the transcriptional activation domain of BZLF1 as being necessary to mediate DNA replication, a function which could not be substituted by any other transcription factor tested, including jun, E2, myc or VP16.

Status of Marek's disease virus in established lymphoma cell lines: herpesvirus integration is common.

Six cell lines derived from Marek's disease lymphomas of chickens and turkeys were investigated for the status of Marek's disease virus (MDV) DNA. In the transformed T- and B-cell lines, viral DNA could be detected by conventional Southern blot hybridization, by Gardella gel electrophoresis, and by in situ hybridization of metaphase and interphase chromosomes. Integration of viral DNA into the host cell chromosome was observed in all cell lines. Two to 12 integration sites of viral DNA could be detected in metaphase chromosome spreads. The integration sites were characteristic for the individual cell lines and were preferentially located at the telomers of large- and mid-sized chromosomes or on minichromosomes. In four of six cell lines, a minor population of latently infected cells supported the lytic cycle of MDV, giving rise to linear virion DNAs. In one of these cell lines, a third species of MDV DNA could be detected with properties reminiscent of covalently closed circular DNA. The finding that MDV integrates regularly into the genomes of latently infected cells is crucial to understanding the molecular biology of herpesvirus-induced tumors in the natural host.

Molecular virology of Epstein-Barr virus.

Epstein-Barr virus (EBV) interacts with its host in three distinct ways in a highly regulated fashion: (i) EBV infects human B lymphocytes and induces proliferation of the infected cells, (ii) it enters into a latent phase in vivo that follows the proliferative phase, and (iii) it can be reactivated giving rise to the production of infectious progeny for reinfection of cells of the same type or transmission of the virus to another individual. In healthy people, these processes take place simultaneously in different anatomical and functional compartments and are linked to each other in a highly dynamic steady-state equilibrium. The development of a genetic system has paved the way for the dissection of those processes at a molecular level that can be studied in vitro, i.e. B-cell immortalization and the lytic cycle leading to production of infectious progeny. Polymerase chain reaction analyses coupled to fluorescent-activated cell sorting has on the other hand allowed a descriptive analysis of the virus-host interaction in peripheral blood cells as well as in tonsillar B cells in vivo. This paper is aimed at compiling our present knowledge on the process of B-cell immortalization in vitro as well as in vivo latency, and attempts to integrate this knowledge into the framework of the viral life cycle in vivo.