Murine polyomavirus DNA transitions through spatially distinct nuclear replication subdomains during infection

Abstract

The replication of small DNA viruses requires both host DNA replication and repair factors that are often recruited to subnuclear domains termed viral replication centers (VRCs). Aside from serving as a spatial focus for viral replication, little is known about these dynamic areas in the nucleus. We investigated the organization and function of VRCs during murine polyomavirus (MuPyV) infection using 3D structured illumination microscopy (3D-SIM). We localized MuPyV replication center components, such as the viral large T-antigen (LT) and the cellular replication protein A (RPA), to spatially distinct subdomains within VRCs. We found that viral DNA (vDNA) trafficked sequentially through these subdomains post-synthesis, suggesting their distinct functional roles in vDNA processing. Additionally, we observed disruption of VRC organization and vDNA trafficking during mutant MuPyV infections or inhibition of DNA synthesis. These results reveal a dynamic organization of VRC components that coordinates virus replication.

Live Cell Microscopy of Murine Polyomavirus Subnuclear Replication Centers

“…Virus 808A has a mutation in the MT splice acceptor that prevents the expression of MT (expression of LT and ST are unaffected) [ 21 , 22 ]. Infections were carried out as described previously [ 7 ]. Briefly, cells were grown to 40% confluency and then cultured overnight in DMEM/A-A/βME without serum.…”

“…1.5) and infected as described above. Cells were pre-extracted and fixed as described previously [ 6 , 7 ]. After fixation, cells were blocked overnight at 4 °C in 10% BCS/PBS (block solution), incubated with primary antibody diluted in block solution at 37 °C for 1 h, and then rinsed three times with 4 °C block solution.…”

“…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 ].…”

“…VRCs are minimally defined as nuclear domains where LT and vDNA colocalize, although many cellular proteins are associated with VRCs [ 5 , 6 , 7 ]. For example, immunofluorescence microscopy of fixed, infected cells has shown that proteins involved with cellular DNA replication and repair are enriched at VRCs [ 5 , 6 , 7 , 23 ]. As with other DNA viruses, PyVs engage with the cellular DNA damage response (DDR) during infection.…”

“…In particular, the activity of double-strand break repair and homologous recombination repair proteins are critical for PyV infection, possibly to resolve the Cairns intermediate that results from bidirectional replication of the viral genome [ 4 , 6 , 24 , 25 , 26 ]. We previously used super-resolution fluorescence microscopy of fixed cells to define vDNA replication- and repair-associated subdomains within MuPyV replication centers that are spatially and functionally distinct [ 7 ]. LT localized to the replication-associated subdomain, whereas DDR signaling proteins, such as phosphorylated ataxia-telangiectasia mutated kinase (pATM S1981 ), localized to the repair-associated subdomain.…”