Gwen Taylor, University of Pittsburgh

Gavin Treadaway - University of Pittsburgh, USA Isabel Fernandez de Castro - National Center for Biotechnology, CNB-CSIC, Madrid, Spain Cristina Risco - National Center for Biotechnology, CNB-CSIC, Madrid, Spain Terence Dermody - University of Pittsburgh, USA

Cell Biology of Viral Infection

Infection of polarized cells by mammalian orthoreovirus (reovirus) leads to nonlytic egress, a process that requires vesicular compartments called sorting organelles (SOs) and membranous carriers (MCs). SOs are modified lysosomes that are recruited to the periphery of viral factories (VFs) where they collect mature virions. MCs bud from SOs and transport newly assembled virions to the plasma membrane for nonlytic egress. Mature reovirus virions are attached to actin filaments inside VFs, SOs, MCs, and the extracellular environment, suggesting that these filaments are involved in transport of mature virions through the nonlytic egress pathway. Host factors required for the nonlytic reovirus egress mechanism are uncharacterized. We hypothesized that egress mediators associate with viral outer-capsid proteins, including μ1. To identify host proteins that interact with μ1 during reovirus infection, we used the BioID proximity-based labeling approach. Several proteins were identified that function in formation of cytoskeletal networks and vesicular transport, including dynamin 2 (DNM2), a GTPase involved in membrane remodeling processes, including endocytosis, vesicle trafficking, and exocytosis. Using siRNA-mediated gene silencing, we observed significantly diminished virus release following infection of DNM2 siRNA-treated cells relative to control siRNA-treated cells. Live-cell immunofluorescence microscopy revealed that DNM2 localizes to VFs during reovirus infection and that DNM2-positive puncta traffic from VFs to the cell surface. These data suggest that DNM2 functions in sorting mature reovirus virions through the nonlytic egress pathway. The function of DNM2 in nonlytic egress will be further characterized using doxycycline-inducible shRNA-mediated gene silencing of DNM2 and super-resolution light microscopy and electron microscopy.