Investigating the Role of SMPD4 in Anterograde Sorting of Pseudorabies Virus in Neurons

Abstract

Pseudorabies virus (PRV) is an alpha herpesvirus of swine causing disease characterized by encephalomyelitis and inflammation of the respiratory tract. Efforts to studying PRV have led to discoveries uncovering the molecular mechanisms of herpesvirus pathogenesis and neuronal invasion. Consequently, PRV can be a powerful tool in exploring the neuroanatomical pathways of the brain, because infection spreads among synaptically connected neurons. PRV infection requires the viral Us9 protein to spread form pre-synaptically connected neurons to post-synaptically connected neurons. Us9 is a membrane protein found in lipid rafts and is required for sorting newly assembled virions into axons. Various studies suggest that sphingolipids, significant lipid raft constituents, and sphingomyelinases are necessary for viral intracellular transport. The host protein, SMPD4, is a neutral sphingomyelinase involved in ceramide production and biogenesis of exosomes. Previous work in this laboratory demonstrated that SMPD4 protein interacts with Us9. SMPD4 was the second most enriched host protein associated with GFP-Us9 in an immunoprecipitation and mass-spectrometry experiment. In this thesis, we studied SMPD4 through a series of immunofluorescence and western blot experiments. We used the neuronal cell line (Neuro2a) to study the function of SMPD4 and Us9 interaction. These cells can be differentiated into central nervous system (CNS) neurons. However, PRV infection in these cells was variable and the production of infection virus was low. Various environmental conditions were explored to optimize PRV infection of these cells. While natural mouse laminin coating induces the growth of two or more neurite-like projections per cell by 25%, it did not affect the percent of Neuro2a cells infected. The infectious cycle is not synchronous in Neuro2a cells, which may reflect the lack of synchrony of the cell cycle. In addition, infected cells appeared to round up and die, perhaps indicating an apoptotic response to infection. We detected SMPD4 using immunofluorescence microscopy. The protein appeared as individual puncta throughout the cell in uninfected and infected Neuro2a cells. Furthermore, we could observe colocalization between SMPD4 and viral capsids.