Enhancer-mediated role for polyomavirus middle T/small T in DNA replication

Area of Science:

• Virology
• Molecular Biology
• Cellular Biology

Background:

• Polyomavirus middle T/small T antigens play a role in viral DNA synthesis.
• Middle T/small T-deficient mutants (hr-t mutants) exhibit replication defects.

Purpose of the Study:

• To investigate the role of polyomavirus middle T/small T antigens in viral DNA replication.
• To understand the mechanisms by which hr-t mutants are affected and how these defects can be alleviated.

Main Methods:

• Replication studies of hr-t mutants in NIH 3T3 cells.
• Analysis of viral genome accumulation and early transcript levels.
• Investigating the effects of phorbol ester and enhancer duplications on replication.
• Mixed infection competition experiments.

Main Results:

• hr-t mutants showed a 16- to 100-fold defect in genome accumulation in low-serum conditions.
• Replication defects were partially rescued by 12-O-tetradecanoylphorbol-13-acetate and alpha core enhancer duplication.
• The alpha core duplication provided a replication advantage to hr-t mutants, especially in early replication stages.
• Middle T/small T presence eliminated the advantage of the duplication-bearing genome.

Conclusions:

• Middle T/small T antigens are essential for efficient polyomavirus DNA replication.
• Factors binding the alpha core domain are likely limiting in NIH 3T3 cells and crucial for replication.
• Middle T/small T likely stimulates replication by activating these limiting factors.
• Enhancer duplications in hr-t mutants partially compensate for the lack of middle T/small T function.