Molecular mechanisms of Us9-mediated anterograde transport of alphaherpes viruses

Abstract

Alphaherpes viruses infect the nervous system of their hosts and undergo long-distance transport in neuronal axons during different steps of the life cycle. Active viral replication at the neuronal soma produces progeny virions, which must undergo anterograde transport down the axon to facilitate anterograde spread within the host. Throughout this work, the molecular mechanisms underlying anterograde transport are explored using pseudorabies virus infection as a model system. Several functional domains in the viral protein Us9, which is essential for anterograde transport, were characterized through a convergence of methodologies including live-cell imaging of fluorescent viruses as well as in vitro spread and biochemical assays. In Chapter 1, GFP-Us9 fusion proteins are employed to characterize the role of a dityrosine motif in the protein in anterograde transport of virion structural components. The dityrosine motif was required for anterograde neuron-to-cell spread in vitro as well as for axonal targeting of virion structural components. GFP-Us9 fusion proteins are then employed further in Chapter 2 to characterize the Us9 diserine motif. Interestingly, unlike the dityrosine motif, mutagenesis of the diserine motif resulted in only a modest defect in spread and was found to modulate the efficiency of anterograde transport. In Chapter 3, the requirement of Us9 for transport of viral membrane proteins is assessed for particles that do not constitute mature virions. Interestingly, we established that the viral glycoprotein M is capable of undergoing anterograde transport independently of Us9. A summation of our current understanding of the anterograde transport mechanism, known as the Married Model, is then presented in Chapter 4 through a critical analysis of experimental techniques in published works. Finally, Appendix A contains a preliminary investigation of glycoprotein E functionality with respect to Us9-Kif1A interactions and anterograde transport of virions. Together, these results expand our understanding of Us9 functionality and biochemical properties