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

## TL;DR

The study shows that myosin-9 helps reoviruses exit cells without destroying them by using lysosomes, offering a new target for antiviral drugs.

## Contribution

The novel finding is that myosin-9 facilitates lysosome-mediated nonlytic egress of reoviruses by recruiting lysosomes and sorting mature virions.

## Key findings

- Myosin-9 promotes reovirus egress by recruiting lysosomes to viral replication factories.
- Myosin-9 sorts mature virions into modified lysosomes for nonlytic release.
- The study identifies myosin-9 as a potential therapeutic target for antiviral treatments.

## Abstract

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.

Viral egress from infected cells is an important potential target for antiviral therapeutics. Mammalian reovirus can infect a wide range of cells and tissues and has been implicated in the pathogenesis of celiac disease. Early steps in reovirus infection have been characterized in detail. However, late steps in infection, including intracellular transport and nonlytic egress, are not well understood. In a previous study, we discovered a new lysosome-mediated pathway for reovirus egress. We found that reovirus uses modified lysosomes to collect mature virions on the periphery of viral replication factories, where new viruses are formed, and mediate their transport to the cell periphery for egress. In this study, we investigated the cellular factors required for the lysosome-mediated, reovirus egress pathway. We found that myosin-9, a cytoskeleton motor protein, promotes reovirus egress, participating in two steps: (i) recruitment of lysosomes to viral factories and (ii) sorting of mature virions into the modified lysosomes. Our results enhance an understanding of how viruses remodel cellular compartments to exit host cells. Most importantly, this research uncovers a new mechanism by which viruses use myosin-9 for selecting and sorting mature viruses for egress and unveils a new target for antiviral treatments.