Related Experiment Videos
Identification of a novel antiapoptotic functional domain in simian virus 40 large T antigen
S D Conzen1, C A Snay, C N Cole
1Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03756, USA.
Abstract:
The ability of DNA tumor virus proteins to trigger apoptosis in mammalian cells is well established. For example, transgenic expression of a simian virus 40 (SV40) T-antigen N-terminal fragment (N-termTag) is known to induce apoptosis in choroid plexus epithelial cells. SV40 T-antigen-induced apoptosis has generally been considered to be a p53-dependent event because cell death in the brain is greatly diminished in a p53-/- background strain and is abrogated by expression of wild-type (p53-binding) SV40 T antigen. We now show that while N-termTags triggered apoptosis in rat embryo fibroblasts cultured in low serum, expression of full-length T antigens unable to bind p53 [mut(p53-)Tags] protected against apoptosis without causing transformation. One domain essential for blocking apoptosis by T antigen was mapped to amino acids 525 to 541. This domain has >60% homology with a domain of adenovirus type 5 E1B 19K required to prevent E1A-induced apoptosis. In the context of both wild-type T antigen and mut(p53-)Tags, mutation of two conserved amino acids in this region eliminated T antigen's antiapoptotic activity in REF-52 cells. These data suggest that SV40 T antigen contains a novel functional domain involved in preventing apoptosis independently of inactivation of p53.
Insights
Simian virus 40 T-antigen can prevent cell death independently of p53. A specific domain (amino acids 525-541) is crucial for this novel anti-apoptotic function, distinct from p53 inactivation.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- DNA tumor viruses, like simian virus 40 (SV40), are known to induce apoptosis (programmed cell death) in mammalian cells.
- SV40 T-antigen is a key viral protein involved in cell cycle regulation and apoptosis.
- SV40 T-antigen-induced apoptosis has been largely attributed to its interaction with the tumor suppressor protein p53.
Purpose of the Study:
- To investigate the role of SV40 T-antigen in apoptosis, particularly its dependence on p53.
- To identify specific domains within SV40 T-antigen responsible for modulating apoptosis.
- To explore potential p53-independent mechanisms by which SV40 T-antigen affects apoptosis.
Main Methods:
- Transgenic expression of SV40 T-antigen fragments and mutants in rat embryo fibroblasts (REF-52 cells).
- Culturing cells in low serum conditions to induce apoptosis.
- Assessing apoptosis induction and protection by different T-antigen variants.
- Mapping functional domains by site-directed mutagenesis of conserved amino acids.
Main Results:
- While SV40 T-antigen N-terminal fragments induced apoptosis, full-length T-antigens unable to bind p53 protected cells from apoptosis.
- A specific domain within SV40 T-antigen (amino acids 525-541) was identified as essential for blocking apoptosis.
- This domain shares homology with a similar domain in adenovirus E1B 19K protein, known for anti-apoptotic activity.
- Mutating conserved amino acids within this domain abolished the anti-apoptotic function of T-antigen, even in p53-binding-deficient mutants.
Conclusions:
- SV40 T-antigen possesses a novel functional domain capable of preventing apoptosis independently of p53 inactivation.
- This p53-independent anti-apoptotic activity is mediated by amino acids 525-541, highlighting a new mechanism of viral oncogenesis.
- These findings challenge the prevailing view of SV40 T-antigen's apoptotic effects being solely p53-dependent.