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Overexpression of simian virus 40 small-T antigen blocks centrosome function and mitotic progression in human
S Gaillard1, K M Fahrbach, R Parkati
1Department of Microbiology-Immunology and the Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois 60611-3010, USA.
Abstract:
Recombinant adenoviruses that express high levels of the simian virus 40 (SV40) small-t (ST) antigen have been used to study the requirement for ST to drive cell cycle proliferation of confluent human diploid fibroblasts. This occurs when either large-T (LT) antigen or serum is added to provide a second signal. While cells readily completed S phase in these experiments, they were found to accumulate with 4N DNA content. Cellular and nuclear morphology, as well as the biochemical status of cyclin B complexes, showed that these cells entered mitosis but were blocked prior to mitotic metaphase. The defect appears to reflect an inability of cells overexpressing ST to form organized centrosomes that duplicate and separate normally during the cell cycle and, therefore, the absence of a mitotic spindle. The ability of ST to bind protein phosphatase 2A was required for this pattern, suggesting that altered phosphorylation of key centrosomal components may occur when ST is overexpressed. Although the possible significance of ST effects on the centrosome cycle is not fully understood, these findings suggest that ST could influence chromosomal instability patterns that are a hallmark of SV40-transformed cells and LT expression.
Insights
Simian virus 40 (SV40) small-t antigen disrupts cell division by preventing proper centrosome formation, leading to mitotic arrest. This defect, linked to protein phosphatase 2A binding, may contribute to chromosomal instability.
Area of Science:
- Cell Biology
- Virology
- Molecular Biology
Background:
- Simian virus 40 (SV40) small-t (ST) antigen is implicated in viral oncogenesis.
- The role of ST antigen in driving cell cycle progression, particularly in quiescent cells, requires further elucidation.
- Understanding ST antigen's impact on cell cycle regulation is crucial for comprehending viral transformation.
Purpose of the Study:
- To investigate the requirement of SV40 ST antigen for driving cell cycle proliferation in human diploid fibroblasts.
- To determine the specific cell cycle phase affected by ST antigen overexpression.
- To explore the underlying molecular mechanisms, including centrosome function and protein interactions, responsible for ST-induced cell cycle defects.
Main Methods:
- Utilized recombinant adenoviruses to express high levels of SV40 ST antigen in confluent human diploid fibroblasts.
- Applied serum or SV40 large-T (LT) antigen as a second signal to induce cell cycle progression.
- Assessed cell cycle progression, DNA content (4N), cellular and nuclear morphology, cyclin B complex status, centrosome organization, and mitotic spindle formation.
Main Results:
- Cells expressing high levels of SV40 ST antigen, upon receiving a second signal, completed S phase but accumulated with 4N DNA content.
- These cells entered mitosis but were arrested prior to metaphase, exhibiting defects in centrosome duplication and separation, and lacking a mitotic spindle.
- The observed phenotype required the ability of ST antigen to bind protein phosphatase 2A, suggesting altered phosphorylation of centrosomal proteins.
Conclusions:
- SV40 ST antigen overexpression disrupts normal centrosome cycle progression, leading to mitotic arrest in human diploid fibroblasts.
- The interaction of ST antigen with protein phosphatase 2A is critical for inducing these centrosome-related defects.
- These findings suggest a potential mechanism by which ST antigen contributes to chromosomal instability observed in SV40-transformed cells.