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Live Cell Microscopy of Murine Polyomavirus Subnuclear Replication Centers
Douglas K Peters1, Kimberly D Erickson1, Robert L Garcea1,2
1BioFrontiers Institute, University of Colorado Boulder, Boulder, CO 80309, USA.
Abstract:
During polyomavirus (PyV) infection, host proteins localize to subnuclear domains, termed viral replication centers (VRCs), to mediate viral genome replication. Although the protein composition and spatial organization of VRCs have been described using high-resolution immunofluorescence microscopy, little is known about the temporal dynamics of VRC formation over the course of infection. We used live cell fluorescence microscopy to analyze VRC formation during murine PyV (MuPyV) infection of a mouse fibroblast cell line that constitutively expresses a GFP-tagged replication protein A complex subunit (GFP-RPA32). The RPA complex forms a heterotrimer (RPA70/32/14) that regulates cellular DNA replication and repair and is a known VRC component. We validated previous observations that GFP-RPA32 relocalized to sites of cellular DNA damage in uninfected cells and to VRCs in MuPyV-infected cells. We then used GFP-RPA32 as a marker of VRC formation and expansion during live cell microscopy of infected cells. VRC formation occurred at variable times post-infection, but the rate of VRC expansion was similar between cells. Additionally, we found that the early viral protein, small TAg (ST), was required for VRC expansion but not VRC formation, consistent with the role of ST in promoting efficient vDNA replication. These results demonstrate the dynamic nature of VRCs over the course of infection and establish an approach for analyzing viral replication in live cells.
Insights
Viral replication centers (VRCs) form during polyomavirus infection. Live cell microscopy revealed VRCs expand similarly in cells, with early viral protein small TAg (ST) essential for this expansion.
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.
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