Marek's disease virus-encoded Meq gene is involved in transformation of lymphocytes but is dispensable for replication

Abstract

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 Jun/Fos family of transcriptional activators. Meq is consistently expressed in MDV-induced tumor cells and has been suggested as the MDV-associated oncogene. To study the function of Meq, we have generated an rMd5DeltaMeq 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 rMd5DeltaMeq virus from peripheral blood lymphocytes was reduced, suggesting that Meq is involved but not essential for latency. Pathogenesis experiments showed that the rMd5DeltaMeq 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.

Fig. 1.

Organization of the serotype 1 MDV genome. (A) The MDV genome consists of a unique long (U_L) region flanked by inverted repeats, terminal repeat long (TR_L), internal repeat long (IR_L), and a unique short region (U_S) also flanked by inverted repeats, internal repeat short (IR_S) and terminal repeat short (TR_S). (B) Schematic representation of the overlapping cosmid clones generated to reconstitute an infectious virus from a very virulent (vv) strain of MDV (Md5). The restriction enzymes used to generate the cosmid clones and their positions are indicated. (C) Location of TaqI restriction sites in cosmids SN5 and A6 used to delete the meq gene.

Fig. 2.

Immunofluorescence analysis of DEF cells infected with recombinant viruses. Parental, rMd5, and revertant rMd5ΔMeqR express Meq protein, whereas the deletion mutant virus rMd5ΔMeq does not. The presence of rMd5ΔMeq virus was confirmed by staining of MDV-specific pp38 protein. Note the intranuclear expression of Meq and cytoplasmic expression of pp38.

Fig. 3.

Southern blot analysis of rMd5 and rMd5ΔMeq. DNA digested with EcoRI and probed with total viral MDV DNA (lanes 1 and 2) or an EcoQ (spanning meq gene)-specific probe (lanes 3 and 4) confirmed the deletion of a 1,085-bp fragment corresponding to the Meq gene (lane 4) and the absence of gross rearrangement in the viral genome (lanes 1 and 2). The band corresponding to the 5,063-bp fragment is explained in Results.

Fig. 4.

Immunohistochemical analysis of lymphoid organs at 6 days (bursa, spleen, and thymus) and FFE at 14 days after inoculation of rMd5, rMd5Δmeq, and control chickens. Frozen sections were stained with pp38-specific mAb.

Fig. 5.

Incidence of mortality in chickens inoculated with rMd5, rMd5ΔMeq, and rMd5ΔMeqR. Chickens were inoculated with 1,500 pfu of the indicated viruses at 1 day of age and maintained in isolation for 8 weeks. Uninoculated chickens served as negative controls. Weekly mortality was recorded. Chickens that died during the experiment were evaluated for MDV-specific gross lesions. One chicken from mock-infected and rMd5ΔMeq groups died on day 8 due to nonspecific causes.