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, 76 (10), 4928-39

Mutational Analysis of the v-Rel Dimerization Interface Reveals a Critical Role for v-Rel Homodimers in Transformation

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Mutational Analysis of the v-Rel Dimerization Interface Reveals a Critical Role for v-Rel Homodimers in Transformation

Andrew S Liss et al. J Virol.

Abstract

The v-rel oncogene encoded by reticuloendotheliosis virus strain T is the acutely transforming member of the Rel/NF-kappaB family of transcription factors. In v-Rel-transformed cells, v-Rel exists as homodimers or heterodimers with the endogenous Rel/NF-kappaB proteins c-Rel, NF-kappaB1, NF-kappaB2, and RelA. To examine the contribution of these complexes to v-Rel-mediated transformation, mutations were introduced into the dimerization interface of v-Rel to generate v-Rel mutants with selective dimerization properties. Nine mutants are described in this study that are defective in homodimer and/or heterodimer formation with specific Rel/NF-kappaB family members. Viruses expressing mutants that failed to homodimerize but were able to form heterodimeric complexes were unable to transform splenic lymphocytes in vitro, indicating that the dimerization of v-Rel with endogenously expressed Rel/NF-kappaB proteins is not in itself sufficient for transformation. In addition, two partially transforming mutants were identified that exhibited an impaired ability to form homodimers. Sequence analysis of the proviral DNA from cells transformed by these mutants revealed the presence of multiple secondary mutations in sequences responsible for dimerization and DNA binding. Two of these mutations either enhanced or restored the ability of these proteins to bind DNA as a homodimer. Viruses expressing these proteins transformed cells at levels comparable to or slightly less than v-Rel, suggesting that a threshold level of DNA binding by v-Rel homodimers is required for transformation.

Figures

FIG. 1.
FIG. 1.
Expression of v-Rel dimerization mutants in CEF cultures. (A) Western blot analysis of CEFs expressing v-Rel dimerization mutants. Protein (40 μg) in lysates from cells expressing CSV, v-Rel, or v-Rel dimerization mutants was resolved by SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blotting with the anti-Rel monoclonal antibody HY87. The migration of v-Rel and c-Rel is indicated. (B) Northern blot analysis of CEFs expressing v-Rel dimerization mutants. Total RNA (10 μg) from the cultures expressing CSV, v-Rel, or v-Rel dimerization mutants in panel A was analyzed for the expression of viral RNA. The hybridization of the viral genomic RNA with a v-rel probe is shown in the top panel. The lower panel shows the methylene blue staining of the 26S rRNA after transfer to the membrane. (C) Half-life analysis of v-Rel mutants in CEF cultures. CEF cultures expressing v-Rel (top panel), M1 (middle panel), or M3 (lower panel) were treated with cycloheximide and whole-cell extracts prepared at various times after treatment. The expression of v-Rel and v-Rel dimerization mutants was evaluated by Western blot analysis. The location of v-Rel, M1, M3, or c-Rel is indicated on the left side of each panel. The length (in hours) of cycloheximide treatment is indicated on the top.
FIG. 2.
FIG. 2.
Mutations in proviral DNA from cells transformed by v-Rel dimerization mutants. Genomic DNA was isolated from four cell lines (D10-1, D10-2, D11-1, and D11-4) transformed by viruses expressing M1 and three cell lines (D4-1, D4-2, and D4-3) transformed by viruses expressing M3. Proviral DNA was amplified with virus-specific primers and cloned into pGEM-T. Three separate clones of the PCR products from each cell line were sequenced over the entire length of v-rel. The amino acids from 1 to 360 of v-Rel and the location of amino acids involved in dimerization (⧫), DNA binding (•), and intramolecular interactions (▪) of chicken c-Rel are indicated. Only PCR products containing mutations that altered the amino acid sequence of the protein are shown. The gray letters represent the original amino acid differences found in M1 and M3 relative to v-Rel. The black letters represent secondary amino acid changes resulting from mutations in the proviral DNA.
FIG. 3.
FIG. 3.
Association of v-Rel dimerization mutants with Rel/NF-κB proteins. v-Rel proteins were cotranslated in an in vitro system with truncated v-Rel proteins (v-RelΔ), c-Rel (aa 1 to 284), NF-κB1 (p50), and NF-κB2 (p52) in the presence of [35S]methionine. All cotranslations contained approximately equal amounts of each protein. Translated products were directly analyzed by SDS-PAGE (first four lanes) or were subjected to immunoprecipitation with an antiserum specific to the C terminus of v-Rel and then resolved by SDS-PAGE (last four lanes). Proteins were visualized by phosphorimager analysis. The identity of the v-Rel protein studied is indicated to the right of each panel. The Rel/NF-κB proteins present in each reaction are indicated on the top of each panel.
FIG. 4.
FIG. 4.
DNA binding of v-Rel dimerization mutants. v-Rel proteins were translated alone or cotranslated with c-Rel (aa 1 to 284), NF-κB1 (p50), and NF-κB2 (p52) and used in EMSAs with a 32P-labeled palindromic κB site probe (GGGGAATTCCCC). The Rel/NF-κB proteins in each reaction are indicated on the top of each panel. Reactions with translation mixtures containing v-Rel (A), M1 and M1M (B), and M3 and M3R (C) are indicated above each panel. The locations of heterodimeric complexes are indicated by asterisks. All cotranslations contained approximately equal amounts of each protein. The DNA-binding activity of v-Rel, M1, M1M, M3, and M3R homodimers when ca. 15-fold more protein is used are shown in panel D.
FIG. 5.
FIG. 5.
Transformation potential of v-Rel dimerization mutants M1M and M3R. (A) Expression of v-Rel dimerization mutants in CEFs. Proteins in lysates (40 μg) from CEFs expressing CSV, v-Rel, M3R, and M1M were resolved by SDS-PAGE and analyzed by Western blotting with monoclonal antibody HY87 (top panel). The location of v-Rel and c-Rel is indicated. Northern blot analysis of CEFs expressing v-Rel dimerization mutants is shown in the middle panel. Total RNA (10 μg) from the CEFs expressing v-Rel or v-Rel dimerization mutants described above was analyzed for the expression of viral RNA. The hybridization of the viral genomic RNA is shown. The lower panel shows the methylene blue staining of the 26S rRNA after transfer to the membrane. (B) Transformation potential of v-Rel, M3R, and M1M. Splenic lymphocytes were infected with 5 × 105 infectious units of a REV-based retrovirus expressing v-Rel, M3R, or M1M. Infected lymphocytes were plated in soft agar, and colonies were scored microscopically after 10 days. The relative transforming potential of each protein is indicated.

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