Abstract
Virus host evasion genes are ready-made tools for gene manipulation and therapy. In this work we have assessed the impact in vivo of the evasion gene A238L of the African Swine Fever Virus, a gene which inhibits transcription mediated by both NF-κB and NFAT. The A238L gene has been selectively expressed in mouse T lymphocytes using tissue specific promoter, enhancer and locus control region sequences for CD2. The resulting two independently derived transgenic mice expressed the transgene and developed a metastasic, angiogenic and transplantable CD4+CD8+CD69– lymphoma. The CD4+CD8+CD69– cells also grew vigorously in vitro. The absence of CD69 from the tumour cells suggests that they were derived from T cells at a stage prior to positive selection. In contrast, transgenic mice similarly expressing a mutant A238L, solely inhibiting transcription mediated by NF-κB, were indistinguishable from wild type mice. Expression of Rag1, Rag2, TCRβ-V8.2, CD25, FoxP3, Bcl3, Bcl2 l14, Myc, IL-2, NFAT1 and Itk, by purified CD4+CD8+CD69– thymocytes from A238L transgenic mice was consistent with the phenotype. Similarly evaluated expression profiles of CD4+CD8+ CD69– thymocytes from the mutant A238L transgenic mice were comparable to those of wild type mice. These features, together with the demonstration of (mono-)oligoclonality, suggest a transgene-NFAT-dependent transformation yielding a lymphoma with a phenotype reminiscent of some acute lymphoblastic lymphomas.
Highlights
Viruses have evolved multiple strategies to manipulate and evade host cell biology and immune responses [1,2,3,4,5,6,7,8]
Evaluation of the Number of Copies of the Transgene The number of copies of the transgene incorporated in the genome of the two A238L founders, as assessed by the Light Cycler technology, was 10 and 30, and the transgene sequence was identical to the published NCBI A238L sequence
In all the F1 and F2 mice from both founders of each genotype that were analysed, expression of the transgene was confirmed at both protein and RNA levels with transgenic thymus, spleen, lymph nodes, in the organs where there was infiltration of the transgenic cells, and in the thymic cell lines established from the A238L transgenic mice (Figure 1C)
Summary
Viruses have evolved multiple strategies to manipulate and evade host cell biology and immune responses [1,2,3,4,5,6,7,8]. One domain of A238L, with homology to IkBa, interacts with p65 of the NF-kB family of transcription factors, thereby inhibiting its activation [9,10]. A number of mice lacking expression of NF-kB or NFAT have been constructed in order to define the roles of these transcription factors. Manipulation of such key intracellular signalling molecules through transgenic expression of a viral host modification gene, provides an alternative strategy to explore the role of such proteins. Expression of the A238L virus transgene was restricted to T cells, as the development and function of these lymphocytes is very well understood, thereby providing an excellent system to explore the impact of the transgene. The mutA238L transgenic mice were indistinguishable from wild type mice, suggesting that NFAT is essential for the neoplastic transformation in the A238L, T cell restricted transgenic mice
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