Myosin-9 is required for lysosome-mediated nonlytic reovirus egress

Mammalian orthoreoviruses (reoviruses) are nonenveloped, double-stranded RNA viruses that assemble progeny particles in cytoplasmic viral factories (VFs) and exit some types of cells using a nonlytic release mechanism. In human brain microvascular endothelial cells (HBMECs), progeny reovirus virions are selectively sorted from VFs into sorting organelles (SOs), which are derived from lysosomes. Smaller membranous carriers (MCs) bud from SOs and transport progeny virions to the plasma membrane where they are released nonlytically by fusion of MCs with the plasma membrane. To discover cellular factors required for lysosomal modification and nonlytic egress, we used mass spectrometry to identify proteins associated with lysosomes purified from uninfected and reovirus-infected HBMECs as well as virions purified from HBMECs and L929 cells, which differ in the pathways used by reovirus for egress. Network analysis of the proteomic results from HBMECs yielded an enrichment of cytoskeletal proteins centered on myosin-9. Using siRNA gene-silencing of myosin-9, pharmacological inhibition of myosin-9, super-resolution light microscopy, electron microscopy, and three-dimensional electron tomography, we found that myosin-9 acts at late stages of reovirus replication to promote viral egress. Myosin-9 associates with actin filaments attached to mature virions and mediates nonlytic egress of viral progeny from HBMECs. Our findings provide insights into the role of myosin-9 in the intracellular lysosome-mediated reovirus egress pathway and illuminate a new potential therapeutic target for viruses that use this nonlytic egress pathway.