Homodimerization of Marek's disease virus-encoded Meq protein is not sufficient for transformation of lymphocytes in chickens

Abstract

Marek's disease virus (MDV), the etiologic agent of Marek's disease, is a potent oncogenic herpesvirus. MDV is highly contagious and elicits a rapid onset of malignant T-cell lymphomas in chickens within several weeks after infection. MDV genome codes an oncoprotein, Meq, which shares resemblance with the Jun/Fos family of bZIP transcription factors. Similar to Jun, the leucine zipper region of Meq allows the formation of homo- and heterodimers. Meq homo- and heterodimers have different DNA binding affinities and transcriptional activity; therefore, they may differentially regulate transcription of viral and cellular genes. In this study we investigated the role of Meq homodimers in the pathogenicity of MDV by generating a chimeric meq gene, which contains the leucine zipper region of the yeast transcription factor GCN4 (meqGCN). A recombinant virus (rMd5-MeqGCN) containing the chimeric meqGCN gene in place of parental meq was generated with overlapping cosmid clones of Md5, a very virulent MDV strain. The rMd5-MeqGCN virus replicated in vitro and in vivo but was unable to transform T cells in infected chickens. These data provide the first in vivo evidence that Meq homodimers are not sufficient for MDV-induced transformation.

FIG. 1.

Cosmid clones used to recover recombinant viruses. (A) Organization of the serotype 1 MDV genome. (B) Schematic representation of the overlapping cosmid clones used to reconstitute recombinant viruses rMd5 and rMeqGCN, derived from a very virulent strain of MDV (Md5). (C) Location of EcoQ fragment and Meq gene in cosmids SN5 and A6. (D) Location of KpnI site described in Materials and Methods, located within the EcoQ fragment at nucleotide 385 of the meq gene.

FIG. 2.

Schematic representation of the meq gene. The DNA binding basic regions (BR1 and BR2) and transactivation domains are depicted. The leucine zipper (LZ) sequence of parental meq and GCN4 used to replace parental meq LZ in rMd5-MeqGCN are shown. Asterisks indicate the conserved leucine sites.

FIG. 3.

Homo- and heterodimerization of Meq and MeqGCN. (a) Coimmunoprecipitation analysis of tagged Meq and MeqGCN proteins. The indicated plasmids were cotransfected in DF-1 cells. Twenty percent of the total cell lysates that were used for coimmunoprecipitation were also included as controls. Meq coprecipitates only with Meq (left panel), and MeqGCN coprecipitates only with MeqGCN (right panel), demonstrating homodimerization. Membranes were reprobed with anti-Flag antibody as a control. (b) MeqGCN had impaired ability to form heterodimer with c-Jun or c-Fos. The indicated plasmids were cotransfected in DF-1 cells, immunoprecipitated with either anti-Fos or anti-Jun antibody, and blotted with anti-Meq antibody. Ten percent of the total cell lysates that were used for immunoprecipitation were also included in the same gel as a control. Both c-Fos and c-Jun are effectively precipitated Meq but only weakly precipitated MeqGCN.

FIG. 4.

EMSAs of Meq and MeqGCN protein were performed to test the DNA binding capacity of MeqGCN. Purified baculovirus-expressed proteins, Meq and MeqGCN, were incubated with radiolabeled oligonucleotide probes, followed by gel retardation analysis. Two different probes were used: a consensus AP-1 oligonucleotide and an ACACA probe derived from the MDV origin of replication (MDV Ori). Band shifts are observed by Meq and MeqGCN (black arrow), the intensity of which decreases in the presence of unlabeled competitor. NS, nonspecific bands.

FIG. 5.

Luciferase assays demonstrate that MeqGCN has functional transactivation and repression activities. DF-1 cells were transfected with meq promoter, pp14 promoter, pp38 promoter, or 3X-Ori luciferase reporter plasmids and pcDNA (empty vector), pcDNA-Meq, or pcDNA-MeqGCN. Both Meq and MeqGCN activate the meq promoter and repress the pp38/14 bidirectional promoters and 3X-Ori, indicating MeqGCN maintains transactivation/repressive functions. Luciferase values are expressed as the fold difference relative to the pcDNA vector. Error bars indicate the standard deviation.

FIG. 6.

In vitro soft-agar assay demonstrating that MeqGCN has reduced transformation potential. (a) The top row shows immunofluorescence analysis of Meq expression from pools of selected DF-1 cells transfected with pcDNA, pcDNA-Meq, and pcDNA-MeqGCN. In the bottom panels, a soft-agar assay was performed using pcDNA, pcDNA-Meq, and pcDNA-MeqGCN selected DF-1 cells to assess anchorage-independent growth. (b) Number of colonies >100 μm observed in cells expressing pcDNA, pcDNA-Meq, or pcDNA-MeqGCN. The average numbers of colonies counted from three random fields are shown. Error bars indicate the standard deviation.

FIG. 7.

Southern blot analysis of rMd5, rMd5-MeqGCN, and rMd5ΔMeq. (A) DNA was digested with EcoRI and probed with total viral MDV DNA. The restriction profile of rMd5-MeqGCN is similar to rMd5, indicating that no gross genome rearrangements occurred. The arrow indicates the location of the EcoQ fragment. Due to the meq deletion in the EcoQ fragment of rMd5ΔMeq, this fragment migrates faster. (B) DNA was digested with PstI and probed with EcoQ fragment. The introduced LZ mutations in rMd5-MeqGCN resulted in the loss of a PstI site, and therefore a single band is observed, in contrast to the two bands for rMd5. Likewise, rMd5ΔMeq does not have a PstI site and, as a consequence of the meq deletion, results in a faster-migrating single band.

FIG. 8.

Immunofluorescence analysis of DEF cells infected with recombinant viruses. (a) rMd5ΔMeq-infected DEF (magnification, ×100); (b) rMd5-MeqGCN-infected DEF (magnification, ×100); (c) rMd5-infected DEF (magnification, ×100); (d) rMd5-MeqGCN-infected DEF (magnification, ×1,000). Meq expression is observed in the nucleus of rMd5- and rMd5-MeqGCN-infected DEF but not rMd5ΔMeq-infected DEF.

FIG. 9.

In vitro growth properties of rMd5 and rMd5-MeqGCN. DEFs were infected with the indicated viruses and harvested on days 2, 4, and 6 after infection, and titers were determined on fresh DEF. Day 0 indicates the titer of the virus in the inoculum. The experiment was performed in duplicate, and the titer (logarithm of the mean number of PFU per dish) is indicated.

FIG. 10.

Infection of lymphoid tissue and feather follicles. Immunohistochemistry of bursa and feather follicle at days 6 and 21 postinfection, respectively, with anti-pp38 monoclonal antibody (H19). Positive cells are indicated by double staining; counterstaining was performed with hematoxylin.

FIG. 11.

Lymphoid organ/body weight ratios of rMd5-, rMd5-MeqGCN-, and rMd5ΔMeq-infected chickens day 14 postinfection. Three birds from each group were euthanized, lymphoid organs were collected, and chickens and lymphoid organs were weighed. Statistical analysis was performed by using the Kruskal-Wallis test. Significance was set at P < 0.05. Although no significant differences were observed, less lymphoid organ atrophy was observed in chickens infected with rMd5-MeqGCN compared to birds infected with rMd5.

FIG. 12.

Detection of MDV pp38 protein in latently infected cells at 21 days postinfection. IFA was performed with anti-pp38 monoclonal antibody (H19) on peripheral blood mononuclear cells collected from rMd5- and rMd5-MeqGCN-infected chickens. Texas Red-conjugated secondary antibodies were used to detect pp38 (white arrows), and nuclei were stained with DAPI (4′,6′-diamidino-2-phenylindole).

FIG. 13.

Mortality in chickens inoculated with rMd5, rMd5-MeqGCN, and rMd5ΔMeq. Chickens were inoculated with 2,000 PFU of the indicated viruses at 1 day of age and maintained in isolation for 8 weeks, and weekly mortality was recorded. Uninoculated chickens served as negative controls. Chickens that died during the experiment were evaluated for MDV-specific gross lesions.