Submitted to: Avian Diseases
Publication Type: Peer reviewed journal
Publication Acceptance Date: 2/6/2004
Publication Date: 9/1/2004
Citation: Prigge, J.T., Majerciak, V., Hunt, H.D., Dienglewicz, R.L., Parcells, M.S. 2004. Construction and characterization of Marek's disease viruses having green fluorescent protein expression tied directly or indirectly to phosphoprotein 38 expression. Avian Diseases. 48:471-87. Interpretive Summary: Marek's disease (MD) is a cancer-like disease of chickens induced by a herpesvirus known as MD virus (MDV). The role of a region of the MDV genome producing a phosphoprotein called pp38 in induction of cancer was investigated. When a reporter gene called enhanced green fluorescent protein (EGFP) was directly linked to pp38 the virus became attenuated and did not induce cancer in chickens. However when the EGFP was separated from pp38, the virus induced cancer. This result suggests that either the pp38 directly linked to EGFP is incapable of inducing cancer or that this direct linkage disrupted a small gene of unknown function called LORF12, which is involved in cancer induction. This information regarding induction of cancer by MDV should be of interest to scientists in academia, industry and government.
Technical Abstract: Marek's Disease (MD) is caused by Marek's Disease Virus (MDV), a highly cell-associated alphaherpesvirus. MD is primarily characterized by lymphocyte infiltration of the nerves, and the development of lymphomas in visceral organs, muscle, and skin. MDV encodes a highly-expressed phosphoprotein family (pp38/pp24) that were initially identified in MDV-induced tumors, but are now known to be linked primarily to MDV lytic infection. To examine the function of pp38 in vivo, we constructed two recombinant viruses, one having enhanced green fluorescent protein (eGFP) fused in-frame to the pp38 ORF (RB1Bpp38/eGFP) and the other having soluble-modified GFP (smGFP) downstream but out-of-frame with pp38 (RB1Bpp38/smGFP). While constructing RB1Bpp38/eGFP, an open reading frame (ORF) located downstream of pp38 (LORF12) was partially deleted, but in RB1Bpp38/smGFP, LORF12 was left intact. This report describes the cell culture and in vivo characterization of RB1Bpp38/eGFP and RB1Bpp38/smGFP. Structural analysis showed that the virus stocks of RB1Bpp38/eGFP and RB1Bpp38/smGFP had incorporated the GFP cassette and were free of contaminating parent virus (RB1B). Expression analysis showed that the transcription of genes in RB1Bpp38/eGFP- and RB1Bpp38/smGFP-infected CEF were similar to RB1B-infected CEF, with the notable exception of deletion of a LORF12-specific transcript in RB1Bpp38/eGFP-infected cells. In chicken embryo fibroblasts (CEF), RB1Bpp38/eGFP and RB1Bpp38/smGFP showed comparable one-step growth kinetics to parent virus (RB1B). RB1Bpp38/eGFP and RB1Bpp38/smGFP showed quite distinct growth characteristics in vivo. RB1Bpp38/eGFP was highly attenuated, while RB1Bpp38/smGFP exhibited pathogenesis similar to parent virus and retained oncogenicity. Our results suggest that the RB1Bpp38/eGFP phenotype could be attributed to an interference with pp38 function via GFP direct fusion, or due to the deletion of LORF12 (a.k.a. Mys, for mystery protein). The construction of these viruses and the establishment of cell lines from RB1Bpp38/smGFP will provide useful tools for the study of MDV lytic infection, reactivation from latency and the small open reading frame LORF12 adjacent to pp38.