Studying spread of pseudorabies virus infection in dissociated and compartmented neuronal culture systems

Pseudorabies virus (PRV) is an alphaherpesvirus that is able to infect the nervous system of the host and spread between synaptically connected neurons. Several highly conserved viral proteins have been identified as crucial mediators of long distance transneuronal spread of infection. This thesis represents the study and characterization of glycoprotein E (gE), a viral protein that is required for the directional spread of infection from presynaptic to postsynaptic neurons (anterograde spread of infection). Our studies revealed that gE is required for the efficient entry and localization of various virus structural proteins, including viral capsids, glycoproteins and tegument proteins, in axons. Strikingly, in the absence of gE, viral proteins fail to accumulate efficiently in distal axonal structures such as varicosities and growth cones. We conclude that gE, like the viral membrane protein Us9, must be playing a role in either sorting, recruiting, targeting or transporting viral cargo into axons. Domain analysis of gE in cultured neurons as well as animal infection studies indicate that both the gE cytoplasmic tail and gE ectodomain are necessary for wild type sorting of viral proteins into axons.; In order to study the directional spread of infection between neurons and their targets cells, we also developed an in vitro compartmented culture system known as the trichamber system. In the trichamber system, neuron cell bodies are physical separated from the axons in different compartments which allows us to study axonal transmission of infection to and from the neuron cell bodies. All our results from the trichamber system are consistent with the animal infection studies. We show that PRV spread from neurons to target cells is gD-independent and requires intact axons. Furthermore, we demonstrate that the vaccine strain PRV Bartha is completely defective in neuron-to-cell spread while viral mutants lacking either gE, gI or Us9 expression have a kinetic defect in spread to target cells. In addition, the trichamber system also revealed that PRV Bartha has a slight defect in axon-mediated infection of cell bodies.