Marek's disease virus (MDV) causes an acute lymphoproliferative disease in chickens, resulting in T cell lymphomas in visceral organs and peripheral nerves. Earlier studies have determined that the repeat regions of oncogenic serotype 1 MDV encode a basic leucine zipper protein, Meq, which structurally resembles the Jun͞Fos family of transcriptional activators. Meq is consistently expressed in MDV-induced tumor cells and has been suggested as the MDVassociated oncogene. To study the function of Meq, we have generated an rMd5⌬Meq virus by deleting both copies of the meq gene from the genome of a very virulent strain of MDV. Growth curves in cultured fibroblasts indicated that Meq is dispensable for in vitro virus replication. In vivo replication in lymphoid organs and feather follicular epithelium was also not impaired, suggesting that Meq is dispensable for lytic infection in chickens. Reactivation of the rMd5⌬Meq virus from peripheral blood lymphocytes was reduced, suggesting that Meq is involved but not essential for latency. Pathogenesis experiments showed that the rMd5⌬Meq virus was fully attenuated in chickens because none of the infected chickens developed Marek's disease-associated lymphomas, suggesting that Meq is involved in lymphocyte transformation. A revertant virus that restored the expression of the meq gene, showed properties similar to those of the parental virus, confirming that Meq is involved in transformation but not in lytic replication in chickens.
Chemokines induce chemotaxis, cell migration, and inflammatory responses. We report the identification of an interleukin-8 (IL-8) homolog, termed vIL-8, encoded within the genome of Marek's disease virus (MDV).The 134-amino-acid vIL-8 shares closest homology to mammalian and avian IL-8, molecules representing the prototype CXC chemokine. The gene for vIL-8 consists of three exons which map to the BamHI-L fragment within the repeats flanking the unique long region of the MDV genome. A 0.7-kb transcript encoding vIL-8 was detected in an n-butyrate-treated, MDV-transformed T-lymphoblastoid cell line, MSB-1. This induction is essentially abolished by cycloheximide and herpesvirus DNA polymerase inhibitor phosphonoacetate, indicating that vIL-8 is expressed with true late (␥ 2 ) kinetics. Baculovirus-expressed vIL-8 was found to be secreted into the medium and shown to be functional as a chemoattractant for chicken peripheral blood mononuclear cells but not for heterophils. To characterize the function of vIL-8 with respect to MDV infection in vivo, a recombinant MDV was constructed with a deletion of all three exons and a soluble-modified green fluorescent protein (smGFP) expression cassette inserted at the site of deletion. In two in vivo experiments, the vIL-8 deletion mutant (RB1BvIL-8⌬smGFP) showed a decreased level of lytic infection in comparison to its parent virus, an equal-passage-level parent virus, and to another recombinant MDV containing the insertion of a GFP expression cassette at the nonessential US2 gene. RB1BvIL-8⌬smGFP retained oncogenicity, albeit at a greatly reduced level. Nonetheless, we have been able to establish a lymphoblastoid cell line from an RB1BvIL-8⌬smGFP-induced ovarian lymphoma (MDCC-UA20) and verify the presence of a latent MDV genome lacking vIL-8. Taken together, these data describe the identification and characterization of a chemokine homolog encoded within the MDV genome that is dispensable for transformation but may affect the level of MDV in vivo lytic infection.
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 cellassociated 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⌬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.
Phosphonoacetate was an effective inhibitor of both the Marek's disease herpesvirus- and the herpesvirus of turkey-induced DNA polymerase. Using the herpesvirus of turkey-induced DNA polymerase, phosphonoacetate inhibition studies for the DNA polymerization reaction and for the deoxyribonucleoside triphosphate-pyrophosphate exchange reaction were carried out. The results demonstrated that phosphonoacetate inhibited the polymerase by interacting with it at the pyrophosphate binding site to create an alternate reaction pathway. A detailed mechanism and rate equation for the inhibition were developed. For comparison to phosphonoacetate, pyrophosphate inhibition patterns and apparent inhibition constants were determined. Twelve analogues of phosphonoacetate were tested as inhibitors of the herpesvirus of turkey-induced DNA polymerase. At the concentrations tested, only one, 2-phosphonopropionate, was an inhibitor. The apparent inhibition constant for it was about 50 times greater than the corresponding apparent inhibition constant for phosphonoacetate. DNA polymerase alpha of duck embryo fibroblasts, the host cell for the herpesviruses, was inhibited by phosphonoacetate. The apparent inhibition constants for the alpha polymerase were about 10-20 times greater than the corresponding inhibition constants for the herpesvirus-induced DNA polymerase. Duck DNA polymerase beta, Escherichia coli DNA polymerase I, and avian myeloblastosis virus reverse transcriptase were not inhibited by phosphonoacetate.
Marek's disease, a lymphoproliferative disease of chickens, is caused by an alphaherpesvirus, Marek's disease virus (MDV). This virus encodes a virokine, vIL-8, with general homology to cellular CXC chemokines such as interleukin-8 (IL-8) and Gro-␣. To study the function of vIL-8 gene, we deleted both copies of vIL-8 residing in the terminal repeat long and internal repeat long region of the viral genome and generated a mutant virus with vIL-8 deleted, rMd5/⌬vIL-8. Growth kinetics study showed that vIL-8 gene is dispensable for virus replication in cell culture. In vivo, the vIL-8 gene is involved in early cytolytic infections in lymphoid organs, as evidenced by limited viral antigen expression of rMd5/⌬vIL-8. However, the rMd5/⌬vIL-8 virus is unimpaired in virus replication in the feather follicle epithelium. vIL-8 does not appear to be important for establishment of latency, since rMd5/⌬vIL-8 and the wild-type virus have similar viremia titers at 14 days postinfection, a period when the virus titer comes primarily from reactivated latent genomes. Nevertheless, because of the impaired cytolytic infections, the overall transformation efficiency of the virus with vIL-8 deleted is much lower, as reflected by the reduced number of transformed cells at 5 weeks postinoculation and the presence of fewer gross tumors. Importantly, the revertant virus that restored the expression of vIL-8 gene also restored the wild-type phenotype, indicating the deficient phenotypes are results of vIL-8 deletion. One of the interesting differences between the MDV vIL-8 gene and its cellular counterpart is the presence of a DKR (Asp-Lys-Arg) motif instead of ELR (Glu-Leu-Arg) preceding the invariable CXC motif. To study the significance of this variation, we generated recombinant MDV, rMd5/vIL-8-ELR, carrying the ELR motif. Both in vitro and in vivo studies revealed that the DKR motif is as competent as ELR in pathogenesis of MDV.Marek's disease (MD) is a contagious, lymphoproliferative disease of domestic chickens in which mononuclear infiltration, demyelination of peripheral nerves, and T-cell lymphomas are common features (4). The etiological agent of MD is a lymphotropic, oncogenic herpesvirus, MD virus (MDV). The MDV genome is about 180 kb in length and is classified as an alphaherpesvirus on the basis of DNA sequence homology and genome structure (5, 21). Recently, the complete nucleotide sequences have been determined for all serotypes of MDV (1,16,19,32). The data showed that MDV and other alphaherpesviruses are colinear in the unique long and short regions but differ substantially in the adjacent repeats (19,30,32). MDV is grouped into three serotypes: serotype 1 consists of all pathogenic virus strains, serotype 2 comprises the naturally occurring, nononcogenic strains in chickens, and serotype 3 includes the nonpathogenic herpesvirus of turkeys (3,6,17,18). MD incidence has largely been controlled by vaccination with all three serotypes of MDV, often in bi-and multivalent combinations since the 1970s (34, 35). However, there ...
In an attempt to develop a specific diagnostic test for avian leukosis virus (ALV) subgroup J (ALV-J) strain Hc1, four monoclonal antibodies (MAbs), JE9, G2, 145, and J47, were generated that are specific for ALV-J envelope glycoprotein, gp85. Polymerase chain reaction (PCR) was used to amplify genomic pro-viral DNA of Avian Disease and Oncology Laboratory (ADOL)-Hc1 and ADOL-4817 envelope genes. Both open reading frames encoding glycoproteins gp85 and gp37 were cloned into baculoviruses. Abundant expression of gp85 and gp37 was detected in the recombinant viruses with specific antibody to Hc1 strain of the ALV-J. The expressed proteins were used for immunization of mice to produce hybridoma cell lines secreting MAbs specific to ALV-J envelope protein. A panel of MAbs was generated by fusing NS1 myeloma cells and spleen cells from mice immunized with the recombinant baculoviruses. With the use of an immunofluorescence assay, three MAbs (JE9, G2, 145) reacted with ALV-J but not with subgroups A, B, C, D, or E of ALV. MAb J47 reacted with all exogenous subgroups of ALV including A, B, C, D, and J but not with endogenous subgroup E viruses. Western blot analysis was performed with all four MAbs against recombinant baculovirus and Hc1-infected chicken embryo fibroblast (CEF) lysates. A major band with a molecular weight about 90 kD corresponding to the size of ALV-J envelope was consistently obtained. With these MAbs, we detected the Hc1 antigen in CEFs infected with several ALV-J viruses isolated in the United States and also in tissue sections from chickens infected with Hc1 strain of ALV-J. These MAbs will be useful reagents for the diagnosis of ALV-J infection because they recognize a common antigenic epitope in six isolates tested thus far.
Phosphonoformate was found to be an inhibitor of the deoxyribonucleic acid polymerase induced by the herpesvirus of turkeys. The apparent inhibition constants were 1 to 3 ,uM. Phosphonoformate was also able to block the replication in cell culture of Marek's disease herpesvirus, the herpesvirus of turkeys, and herpes simplex virus. It was as effective as phosphonoacetate. Phosphonoformate was not an effective inhibitor of a phosphonoacetate-resistant mutant of the herpesvirus of turkeys nor of its induced deoxyribonucleic acid polymerase.Phosphonoacetate is an effective inhibitor of the replication of herpesviruses (11,13,23,25). The inhibition of herpesvirus replication is through an effect on the viral-induced deoxyribonucleic acid (DNA) polymerase (9,(14)(15)(16)18). In animal model studies, the efficacy of phosphonoacetate as an antiherpesvirus drug has been clearly demonstrated (7,8,12). Its clinical use, however, may be limited because it is somewhat toxic to test animals and because it is accumulated in bone (4).Other phosphonate compounds are of interest as inhibitors of herpesvirus replication because they might exhibit an improved therapeutic ratio over phosphonoacetate either by being more effective inhibitors of virus replication or by being less toxic to animals. These compounds are also of interest at the enzymological level for the information that they might provide about the binding site on the herpesvirus-induced DNA polymerase. Consequently, using as an assay procedure the ability to inhibit the herpesvirus-induced DNA polymerase or the ability to block herpesvirus replication in cell culture or in animals, many other phosphonates have been looked at (10,13,14,23
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