The pseudorabies virus (PRV) Us9 protein plays a central role in targeting viral capsids and glycoproteins to axons of dissociated sympathetic neurons. As a result, Us9 null mutants are defective in anterograde transmission of infection in vivo. However, it is unclear how Us9 promotes axonal sorting of so many viral proteins. It is known that the glycoproteins gB, gC, gD and gE are associated with lipid raft microdomains on the surface of infected swine kidney cells and monocytes, and are directed into the axon in a Us9-dependent manner. In this report, we determined that Us9 is associated with lipid rafts, and that this association is critical to Us9-mediated sorting of viral structural proteins. We used infected non-polarized and polarized PC12 cells, a rat pheochromocytoma cell line that acquires many of the characteristics of sympathetic neurons in the presence of nerve growth factor (NGF). In these cells, Us9 is highly enriched in detergent-resistant membranes (DRMs). Moreover, reducing the affinity of Us9 for lipid rafts inhibited anterograde transmission of infection from sympathetic neurons to epithelial cells in vitro. We conclude that association of Us9 with lipid rafts is key for efficient targeting of structural proteins to axons and, as a consequence, for directional spread of PRV from pre-synaptic to post-synaptic neurons and cells of the mammalian nervous system.
Alpha herpesviruses are common mammalian pathogens (e.g. herpes simplex and chickenpox virus infect humans). These viruses enter and spread in and out of the mammalian nervous system, a defining hallmark of their lifecycle and potential pathogenesis. Neurons are polarized cells, and the movement of certain cellular proteins is highly restricted to the cell body, to dendrites, or to axons. Indeed, the axon harbors only a small subset of neuronal proteins. Thus, it is remarkable that these viruses efficiently gather and move their structural components from the cell body to the axon after infection of neurons. Previously, we identified a small viral membrane protein (known as Us9) that mediates the efficient targeting of virus particles to axons of infected neurons. We now report that Us9 must localize to “lipid raft” domains, specialized regions within cellular membranes to promote axonal targeting. Reducing the affinity of Us9 for lipid rafts dramatically reduces sorting of structural proteins to axons. This is the first report, to our knowledge, to implicate lipid rafts in targeting alpha herpesvirus structural proteins to axons, an essential step for spread of infection in the mammalian nervous system.