Live Cell Microscopy of Murine Polyomavirus Subnuclear Replication Centers

Area of Science:

• Virology
• Molecular Biology
• Cell Biology

Background:

• Polyomavirus (PyV) infection hijacks host proteins into viral replication centers (VRCs) for genome replication.
• Previous studies characterized VRC composition and organization using fixed-cell microscopy, but temporal dynamics remain unclear.

Purpose of the Study:

• To investigate the temporal dynamics of VRC formation and expansion during murine polyomavirus (MuPyV) infection using live cell microscopy.
• To determine the role of the early viral protein, small TAg (ST), in VRC formation and expansion.

Main Methods:

• Live cell fluorescence microscopy was employed to track VRC dynamics in a mouse fibroblast cell line expressing GFP-tagged replication protein A complex subunit (GFP-RPA32).
• GFP-RPA32 served as a marker for VRC formation and expansion.
• The impact of the small TAg (ST) protein on VRC dynamics was assessed.

Main Results:

• GFP-RPA32 correctly localized to VRCs in MuPyV-infected cells, validating its use as a VRC marker.
• VRC formation occurred at variable times post-infection, but VRC expansion proceeded at a consistent rate across cells.
• The early viral protein ST was found to be essential for VRC expansion, but not for initial VRC formation.

Conclusions:

• Viral replication centers exhibit dynamic formation and expansion during the course of polyomavirus infection.
• The early viral protein ST plays a critical role in promoting efficient viral DNA replication by driving VRC expansion.
• This study establishes a live-cell imaging approach for analyzing viral replication dynamics.