Gavin Treadaway, University of Pittsburgh

Gavin S. Treadaway,1,2 Russell J. R. Barkley,3,4 Xayathed Somoulay,1,2 Takeshi Kobayashi,5 John S. L. Parker,3,4 Gwen M. Taylor,2,6 and Terence S. Dermody1,2,6 1 Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA 2 Institute of Infection, Inflammation, and Immunity, UPMC Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania, USA 3 Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 4 Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 5 Department of Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan 6 Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

Cell Biology of Viral Infection

Many viruses that replicate in the cytoplasm, including mammalian orthoreovirus (reovirus), form replication organelles that concentrate viral proteins, nucleic acids, and cellular components required for replication. These replication organelles are termed viral factories (VFs) in reovirus infection and initially are small puncta that mature to large, globular, dynamic structures that have characteristics of biomolecular condensates formed by liquid-liquid phase-separation (LLPS). Biomolecular condensates are membraneless compartments in cells formed by proteins with particular structural motifs, including intrinsically disordered regions and coiled-coils, and are liquid-like in material property. When transiently expressed in cells, reovirus nonstructural protein µNS forms structures like VFs, which are termed viral factory-like structures, suggesting that µNS is sufficient for nucleating VFs. Using live-cell imaging and fluorescently-tagged µNS, we observed hallmarks of LLPS for viral factory-like structures, suggesting µNS is an LLPS protein. While the structure of µNS is not known, it is predicted to include two coiled-coil domains near the C-terminus and an intrinsically disordered C-terminus. We observed loss of factory-like structure formation in cells expressing µNS mutants with alterations in coiled-coil domains relative to wild-type µNS. Additionally, expression of µNS coiled-coil mutants prior to infection with reovirus disrupted native VF formation, suggesting other components are not sufficient in promoting LLPS in the presence of a mutant µNS. Together, these data suggest that reovirus factories form by LLPS and that residues in the predicted coiled-coil domains of µNS are required for factory biogenesis.