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.
Highlights
Viruses are obligate intracellular pathogens that have developed diverse mechanisms to utilize host cell factors to permit and enhance infection
The replication protein A (RPA) complex is a known component of viral replication centers (VRCs) and localizes to both replication- and repair-associated VRC subdomains identified by super-resolution microscopy [7]
GFP-RPA32 fusion proteins have been used in previous studies in human cells [44], we validated the appropriate localization of GFP-RPA32 in mouse fibroblasts
Summary
Viruses are obligate intracellular pathogens that have developed diverse mechanisms to utilize host cell factors to permit and enhance infection. Must these cellular factors be successfully co-opted, but their activities must be regulated spatially and temporally to ensure productive and efficient infection [1]. DNA viruses, including polyomaviruses (PyVs), adenoviruses, papillomaviruses, and herpesviruses, form specialized nuclear compartments during infection that spatially organize replication, transcription, and viral assembly [2,3,4]. We investigated the spatiotemporal dynamics of compartments formed during murine PyV (MuPyV) infection, termed viral replication centers (VRCs) [5,6,7]. The TAgs are Viruses 2020, 12, 1123; doi:10.3390/v12101123 www.mdpi.com/journal/viruses
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