Glycoproteins E and I of Marek's disease virus serotype 1 are essential for virus growth in cultured cells.
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The role of glycoprotein E (gE) and gI of Marek's disease virus serotype 1 (MDV-1) for growth in cultured cells was investigated. MDV-1 mutants lacking either gE (20DeltagE), gI (20DeltagI), or both gE and gI (20DeltagEI) were constructed by recE/T-mediated mutagenesis of a recently established infectious bacterial artificial chromosome (BAC) clone of MDV-1 (D. Schumacher, B. K. Tischer, W. Fuchs, and N. Osterrieder, J. Virol. 74:11088-11098, 2000). Deletion of either gE or gI, which form a complex in MDV-1-infected cells, resulted in the production of virus progeny that were unable to spread from cell to cell in either chicken embryo fibroblasts or quail muscle cells. This was reflected by the absence of virus plaques and the detection of only single infected cells after transfection, even after coseeding of transfected cells with uninfected cells. In contrast, growth of rescuant viruses, in which the deleted glycoprotein genes were reinserted by homologous recombination, was indistinguishable from that of parental BAC20 virus. In addition, the 20DeltagE mutant virus was able to spread from cell to cell when cotransfected into chicken embryo fibroblasts with an expression plasmid encoding MDV-1 gE, and the 20DeltagI mutant virus exhibited cell-to-cell spread capability after cotransfection with a gI expression plasmid. The 20DeltagEI mutant virus, however, was not able to spread in the presence of either a gE or gI expression plasmid, and only single infected cells were detected by indirect immunofluorescence. The results reported here demonstrate for the first time that both gE and gI are absolutely essential for cell-to-cell spread of a member of the Alphaherpesvirinae. (+info)
Improving DNA vaccine potency by linking Marek's disease virus type 1 VP22 to an antigen.
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We have previously employed an intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein to enhance DNA vaccine potency because DNA vaccines lack the intrinsic ability to amplify in cells. Recently, studies have demonstrated that the protein encoded by UL49 of Marek's disease virus type 1 (MDV-1) exhibits some degree of homology to the HSV-1 VP22 protein and features the property of intercellular transport. We therefore generated a DNA vaccine encoding MDV-1 VP22 linked to a model antigen, human papillomavirus type 16 E7. We demonstrated that compared with mice vaccinated with DNA encoding wild-type E7, mice vaccinated with MDV-1 VP22/E7 DNA exhibited a significant increase in number of gamma-interferon-secreting, E7-specific CD8(+)-T-cell precursors as well as stronger tumor prevention and treatment effects. Furthermore, our data indicated that the antitumor effect was CD8 dependent. These results suggested that the development of vaccines encoding VP22 fused to a target antigen might be a promising strategy for improving DNA vaccine potency. (+info)
Marek's disease virus VP22: subcellular localization and characterization of carboxyl terminal deletion Mutations.
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Marek's disease virus (MDV) is an alphaherpesvirus that causes T cell lymphoma and severe immunosuppression in chickens. The MDV UL49 gene, which encodes the tegument viral protein 22 (VP22), has been expressed as a green fluorescent protein (GFP) fusion protein in chicken embryonic fibroblasts to examine its subcellular localization. As with both human herpesvirus 1 and bovine herpesvirus 1VP22-GFP fusion proteins, the MDV VP22-GFP product binds to microtubules and heterochromatin. In addition, the MDV protein also binds to the centrosomes. During mitosis, VP22-GFP binds to sister chromatids, but dissociates from the centrosomes and the microtubules of the mitotic spindle. A series of VP22 carboxy terminal truncation mutants were constructed to define regions responsible for these binding properties. These mutants identified separable domains or motifs responsible for binding microtubules and heterochromatin. (+info)
Rescue of a pathogenic Marek's disease virus with overlapping cosmid DNAs: use of a pp38 mutant to validate the technology for the study of gene function.
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Marek's disease virus (MDV) genetics has lagged behind that of other herpesviruses because of the lack of tools for the introduction of site-specific mutations into the genome of highly cell-associated oncogenic strains. Overlapping cosmid clones have been successfully used for the introduction of mutations in other highly cell-associated herpesviruses. Here we describe the development of overlapping cosmid DNA clones from a very virulent oncogenic strain of MDV. Transfection of these cosmid clones into MDV-susceptible cells resulted in the generation of a recombinant MDV (rMd5) with biological properties similar to the parental strain. To demonstrate the applicability of this technology for elucidation of gene function of MDV, we have generated a mutant virus lacking an MDV unique phosphoprotein, pp38, which has previously been associated with the maintenance of transformation in MDV-induced tumor cell lines. Inoculation of Marek's disease-susceptible birds with the pp38 deletion mutant virus (rMd5 Delta pp38) revealed that pp38 is involved in early cytolytic infection in lymphocytes but not in the induction of tumors. This powerful technology will speed the characterization of MDV gene function, leading to a better understanding of the molecular mechanisms of MDV pathogenesis. In addition, because Marek's disease is a major oncogenic system, the knowledge obtained from these studies may shed light on the oncogenic mechanisms of other herpesviruses. (+info)
Generation of a permanent cell line that supports efficient growth of Marek's disease virus (MDV) by constitutive expression of MDV glycoprotein E.
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A recombinant cell line (SOgE) was established, which was derived from the permanent quail muscle cell line QM7 and constitutively expressed the glycoprotein E (gE) gene of Marek's disease virus serotype 1 (MDV-1). The SOgE cell line supported growth of virulent (RB-1B) and vaccine (CVI988, 584Ap80C) MDV-1 strains at a level comparable with that of primary chicken embryo cells (CEC). The SOgE cell line was used to produce a vaccine against Marek's disease. Chickens were immunized at 1 day old with 10(3) p.f.u. CVI988 produced on either CEC or SOgE cells. Challenge infection was performed at day 12 with hypervirulent Italian MDV-1 strain EU1. Whereas 7/7 or 6/6 animals, respectively, immunized with SOgE or QM7 cells alone developed Marek's disease, only 1/8 animals from both CVI988-immunized groups exhibited signs of disease, suggesting that SOgE cells are a valuable permanent cell culture system for MDV-1 vaccine production. (+info)
Replication-competent bacterial artificial chromosomes of Marek's disease virus: novel tools for generation of molecularly defined herpesvirus vaccines.
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Marek's disease (MD), a highly infectious disease caused by an oncogenic herpesvirus, is one of the few herpesvirus diseases against which live attenuated vaccines are used as the main strategy for control. We have constructed bacterial artificial chromosomes (BACs) of the CVI988 (Rispens) strain of the virus, the most widely used and effective vaccine against MD. Viruses derived from the BAC clones were stable after in vitro and in vivo passages and showed characteristics and growth kinetics similar to those of the parental virus. Molecular analysis of the individual BAC clones showed differences in the structure of the meq gene, indicating that the commercial vaccine contains virus populations with distinct genomic structures. We also demonstrate that, contrary to the published data, the sequence of the L-meq of the BAC clone did not show any frameshift. Virus stocks derived from one of the BAC clones (clone 10) induced 100 percent protection against infection by the virulent strain RB1B, indicating that BAC-derived viruses could be used with efficacies similar to those of the parental CVI988 vaccines. As a DNA vaccine, this BAC clone was also able to induce protection in 6 of 20 birds. Isolation of CVI988 virus from all of these six birds suggested that immunity against challenge was probably dependent on the reconstitution of the virus in vivo and that such viruses are also as immunogenic as the in vitro-grown BAC-derived or parental vaccine viruses. Although the reasons for the induction of protection only in a proportion of birds (33.3%) that received the DNA vaccine are not clear, this is most likely to be related to the suboptimal method of DNA delivery. The construction of the CVI988 BAC is a major step towards understanding the superior immunogenic features of CVI988 and provides the opportunity to exploit the power of BAC technology for generation of novel molecularly defined vaccines. (+info)
Characterization of the chromosomal binding sites and dimerization partners of the viral oncoprotein Meq in Marek's disease virus-transformed T cells.
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Marek's disease virus (MDV) is an acute transforming alphaherpesvirus that causes T-cell lymphomas in chickens. We previously reported the identification of a putative oncogene, meq, that is encoded only by the oncogenic serotype of MDV. The gene product, Meq, is a latent protein that is consistently expressed in MDV-transformed lymphoblastoid cells and tumor cells. Meq has a bZIP (basic leucine zipper) structure resembling the family of Jun/Fos. The mechanism whereby Meq transforms T cells remains poorly understood. In this study, we explored the properties of Meq as a transcriptional factor. We analyzed Meq's dimerization partners and its target genes in MSB-1, an MDV-transformed T-cell line. By using in vitro assays, we first demonstrated Meq's potential to dimerize with a variety of bZIP proteins. We then identified c-Jun as the primary dimerization partner of Meq. Both are found to be colocalized in the nucleus and corecruited to promoters with AP-1 sequences. By using chromatin immunoprecipitation (ChIP), we scanned the entire MDV genome for Meq binding sites and found three regions that were enriched with Meq binding: the MDV lytic replication origin, the promoter for Meq, and the promoter for ICP4. Transactivation assays using the above promoters showed that Meq/Meq homodimers exhibited repression activity, whereas Meq/Jun heterodimers showed activation. Finally, we were able to show by ChIP that Meq is recruited to the interleukin-2 promoter in a region encompassing an AP-1 site. Thus, in addition to providing general knowledge about the transcriptional properties of Meq, our studies revealed for the first time the ability of Meq to interact with the latent MDV and host genomes. Our data suggest, therefore, a role for Meq in viral genome regulation during latency, in addition to its putative causal role in T-cell transformation. (+info)
Marek's disease is a natural model for lymphomas overexpressing Hodgkin's disease antigen (CD30).
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Animal models are essential for elucidating the molecular mechanisms of carcinogenesis. Hodgkin's and many diverse non-Hodgkin's lymphomas overexpress the Hodgkin's disease antigen CD30 (CD30(hi)), a tumor necrosis factor receptor II family member. Here we show that chicken Marek's disease (MD) lymphoma cells are also CD30(hi) and are a unique natural model for CD30(hi) lymphoma. Chicken CD30 resembles an ancestral form, and we identify a previously undescribed potential cytoplasmic signaling domain conserved in chicken, human, and mouse CD30. Our phylogeneic analysis defines a relationship between the structures of human and mouse CD30 and confirms that mouse CD30 represents the ancestral mammalian gene structure. CD30 expression by MD virus (MDV)-transformed lymphocytes correlates with expression of the MDV Meq putative oncogene (a c-Jun homologue) in vivo. The chicken CD30 promoter has 15 predicted high-stringency Meq-binding transcription factor recognition motifs, and Meq enhances transcription from the CD30 promoter in vitro. Plasma proteomics identified a soluble form of CD30. CD30 overexpression is evolutionarily conserved and defines one class of neoplastic transformation events, regardless of etiology. We propose that CD30 is a component of a critical intracellular signaling pathway perturbed in neoplastic transformation. Specific anti-CD30 Igs occurred after infection of genetically MD-resistant chickens with oncogenic MDV, suggesting immunity to CD30 could play a role in MD lymphoma regression. (+info)