Mutations in the rel-homology domain alter the biochemical properties of v-rel and render it transformation defective in chicken embryo fibroblasts.

Sequential deletions of approximately 100 base pairs were made in the rel-homology domain of the viral rel protein. Each deletion mutant was cloned into a replication-competent viral vector and assayed in chicken embryo fibroblasts (CEFs). The deleted v-rel proteins were analysed for localization, complex formation and ability to induce transformation. In vitro-translated mutant proteins were assayed for binding to a NF-kappa B consensus sequence. All the deletion mutants between nucleotides 37 and 798 in v-rel were transformation defective. Each of the mutants localized predominantly in the cytoplasm, whereas wild-type v-rel localizes predominantly in the nucleus of CEFs. Any disruption of the rel-homology domain reduced binding of the mutant v-rel proteins to the cellular protein, p36, while the requirements for binding to p68c-rel, p115 and p124 appeared to be more complicated. The binding of these three proteins to v-rel appeared to be linked and mediated through c-rel, suggesting that v-rel disrupts normal c-rel function. None of the deletion mutants in this region were able to bind to the NF-kappa B site. However, mutants which lie outside the rel-homology domain retained the ability to transform CEFs, localize to the nucleus, complex with p36, p115, p124 and p68c-rel and bind to the NK-kappa B site. These results suggest that transformation by v-rel requires an intact rel-homology domain and that the biochemical properties of v-rel are linked and dependent upon higher order protein structure for full function.