Identification of a region of simian virus 40 large T antigen required for cell transformation

Chen et al. (1990). J Virol DOI: 10.1128/jvi.64.7.3350-3357.1990 Citations: 92

Whether SV40 large T's immortalization and transformation activities shared a domain was unclear. Mutants spanning residues 105-115 were tested for both activities. Mutants lacking this region still immortalized cells but failed to grow in suspension.

Key findings

  • SV40 large T antigen mutations between residues 105 and 115 abolished full cell transformation in established rodent lines. All mutant-expressing Rat-1 lines failed anchorage-independent suspension growth; colony formation efficiency dropped from 3.6% (wild type) to 0.029% for the K1 mutant. These mutations retained viral replication competence, demonstrating that SV40 transformation and replication are separable molecular functions.

  • Transformation-defective large T mutants at residues 105–115 retained the ability to immortalize primary mouse embryo fibroblasts (MEF) and rat embryo fibroblasts (REF). Mutants K1, K8, PVU-1, and T50-L11 established MEF colonies with 71–100% survival over 17 or more doublings, comparable to wild-type large T. All immortalized lines failed suspension growth, establishing that immortalization and full transformation are genetically separable activities.

  • The 105–115 domain shared primary and predicted secondary structure similarity with conserved region 2 of adenovirus E1A, and mutations within it abolished retinoblastoma protein (p110) binding. Deletion mutants PVU-1 and T50-L11 retained immortalization capacity but were defective for focus formation, agar growth, and p110 binding, indicating the domain contributes to transformation by modulating the retinoblastoma tumor suppressor.

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Abstract

A series of replication-competent simian virus 40 (SV40) large T antigens with point and deletion mutations in the amino acid sequence between residues 105 and 115 were examined for the ability to immortalize primary cultures of mouse and rat cells. The results show that certain mutants, including one that deletes the entire region, are able to immortalize. However, consistent with previous data, the immortalized cells are not fully transformed, as judged by doubling time, sensitivity to concentrations of serum, and anchorage-independent growth. The region from 106 to 114 has structural features in common with a region involved in transformation by adenovirus E1a protein (J. Figge, T. Webster, T.F. Smith, and E. Paucha, J. Virol. 62:1814-1818, 1988) and influences the binding of the retinoblastoma gene product to large T (J.A. DeCaprio, J.W. Ludlow, J. Figge, J.-Y. Shew, C.-M. Huang, W.-H. Lee, E. Marsilio, E. Paucha, and D.M. Livingston, Cell 54:275-283, 1988). Together, these results imply that the sequence from 106 to 114 forms part of a domain that is essential for transformation of established cells, is dispensable for immortalization, and is not required for SV40 replication. The results also indicate that the ability of SV40 large T to immortalize primary cells is independent of its ability to bind to the retinoblastoma gene product.