Identification and characterization of a novel spliced form of the meq transcript in lymphoblastoid cell lines derived from Marek's disease tumours

Abstract

In tumour cell lines established from Marek's disease (MD) lymphomas L-meq is consistently expressed. It contains a 180 bp insertion encoding additional copies of the proline-rich repeat in the meq open reading frame and its product may contribute to the maintenance of MD virus (MDV) latency. In this study, we identified a novel spliced form of the meq transcript in MD-derived lymphoblastoid cell lines and in MDV-infected cells. This transcript, termed Dmeq, encodes an N-terminal 98 aa of the Meq protein and lacks part of the basic leucine zipper (bZIP) and transactivation domains. In MD cell lines, transcription of L-meq was significantly downregulated, while that of the Dmeq transcript was upregulated during apoptosis. These observations were also confirmed at the protein expression level. Reporter assays using meq-and interleukin-2 (IL-2)-promoter-driven luciferase vectors revealed that DMeq suppressed transactivation by L-Meq or Meq in a dose-dependent manner. Immunoprecipitation confirmed that DMeq was associated with L-Meq or Meq physically. These results suggest that DMeq could be involved in apoptosis in MD cell lines as it works as a negative regulator of L-Meq and Meq by direct interaction.

Analysis of transcriptional activities of the Meq proteins present in highly virulent Marek’s disease virus strains, RB1B and Md5

“…Therefore, the amino acid substitutions in the proline direct repeats of Meq in high-virulence MDV strains could correlate with increased transactivation activity of Meq. However, the differences in the Meq transactivation activities observed in this study were moderate compared with that observed between Meq and L-Meq [21]. Therefore, other mutations in Meq or other viral factors may also contribute to MDV virulence.…”

“…c Transient expression of Meq constructs in transfected DF-1 cells. Expression patterns of the different Meq constructs were determined by western blotting with antisera against recombinant Meq [21]. Meq expression patterns differed depending on the combination of amino acid residues at positions 283 and 320 as indicated at the bottom.…”

“…The membranes were blocked overnight at 4°C with 0.05% Tween 20 in phosphate-buffered saline (PBST) containing 3% skim milk. The membranes were then incubated at room temperature for 1 h with polyclonal rat antisera against recombinant Meq [21] or anti-phosphothreonine antibody (Cell Signaling Technology), washed three times with PBST, and incubated at room temperature for 30 min with peroxidase-conjugated goat anti-rat IgG (Cosmo Bio) or peroxidase-conjugated goat anti-rabbit-IgG (Cappel). After three washes with PBST, the membranes were incubated with 3,3 0 -diaminobenzidine tetrahydrochloride and cobalt chloride substrates to visualize the peroxidase signal.…”

“…The digested fragments were cloned into the EcoRI and NotI sites of the pCI-neo vector (Promega) to construct plasmids expressing wild type Meq from RB1B (pCI-Meq: RB1B-wt) and Md5 (pCIMeq: Md5-wt). In addition, a c-Jun expression plasmid, designated pCI-c-Jun, was constructed as previously described [21]. In brief, the chicken c-jun transcript was amplified by PCR using the primers shown in Table 1.…”

The Meq oncoprotein of Marek's disease virus interacts with p53 and inhibits its transcriptional and apoptotic activities

“…Meq is a polymorphic protein and several variants have been characterized, including L-Meq (which contains an insertion of 60 aa between residues190 and 191), S-Meq (which contains a deletion of 41 aa between residues 190 and 191), VS-Meq (which contains a deletion of 92 aa between residues 174 and 175), and ∆Meq (composed of 98 aa from the N-terminal region of Meq and a frame-shifted distinct C-terminus of 30 aa) [27-29] (Figure 4A). Meq and its variants are expressed in MD tumor cells and in MDV-infected cells, but their roles in cytolytic infection and the establishment of latency or transformation have not been elucidated fully.…”