Us9-Independent Axonal Sorting and Transport of the Pseudorabies Virus Glycoprotein gM.

Axonal sorting and transport of fully assembled pseudorabies virus (PRV) virions is dependent on the viral protein Us9. Here we identify a Us9-independent mechanism for axonal localization of viral glycoprotein M (gM). We detected gM-mCherry assemblies transporting in the anterograde direction in axons. Furthermore, unlabeled gM, but not glycoprotein B, was detected by Western blotting in isolated axons during Us9-null PRV infection. These results suggest that gM differs from other viral proteins regarding axonal transport properties.

Making the case: married versus separate models of alphaherpes virus anterograde transport in axons.

Alphaherpesvirus virions infect neurons and are transported in axons for long distance spread within the host nervous system. The assembly state of newly made herpesvirus particles during anterograde transport in axons is an essential question in alphaherpesvirus biology. The structure of the particle has remained both elusive and controversial for the past two decades, with conflicting evidence from EM, immunofluorescence, and live cell imaging studies. Two opposing models have been proposed-the Married and Separate Models. Under the Married Model, infectious virions are assembled in the neuronal cell body before sorting into axons and then traffic inside a transport vesicle. Conversely, the Separate Model postulates that vesicles containing viral membrane proteins are sorted into axons independent of capsids, with final assembly of mature virions occurring at a distant egress site. Recently, a complementary series of studies employing high-resolution EM and live cell fluorescence microscopy have provided evidence consistent with the Married Model, whereas other studies offer evidence supporting the Separate Model. In this review, we compare and discuss the published data and attempt to reconcile divergent findings and interpretations as they relate to these models.

Visualization of an alphaherpesvirus membrane protein that is essential for anterograde axonal spread of infection in neurons.

UNLABELLED: Pseudorabies virus (PRV), an alphaherpesvirus with a broad host range, replicates and spreads in chains of synaptically connected neurons. The PRV protein Us9 is a small membrane protein that is highly conserved among alphaherpesviruses and is essential for anterograde axonal spread in neurons. Specifically, the Us9 protein is required for the sorting of newly assembled PRV particles into axons. However, the molecular details underlying the function of Us9 are poorly understood. Here we constructed PRV strains that express functional green fluorescent protein (GFP)-Us9 fusion proteins in order to visualize axonal transport of viral particles in infected rat superior cervical ganglion neurons. We show that GFP-Us9-labeled structures are transported exclusively in the anterograde direction within axons. Additionally, the vast majority of anterograde-directed capsids (labeled with VP26-monomeric red fluorescent protein) and a viral membrane protein (labeled with glycoprotein M fused to mCherry) are cotransported with GFP-Us9 in the anterograde direction. In contrast, during infection with PRV strains that express nonfunctional mutant GFP-Us9 proteins, cotransport of mutant GFP-Us9 with capsids in axons is abolished. These findings show that axonal sorting of progeny viral particles is dependent upon the association of viral structures with membranes that contain functional Us9 proteins. This association is required for anterograde spread of infection in neurons. IMPORTANCE: Alphaherpesviruses, such as pseudorabies virus (PRV), are parasites of the mammalian nervous system. These viruses spread over long distances in chains of synaptically connected neurons. PRV encodes several proteins that mediate directed virion transport and spread of infection. Us9 is a highly conserved viral membrane protein that is essential for anterograde neuronal spread of infection. In the absence of Us9, newly replicated viral particles are assembled in the cell body but are not sorted into or transported within axons. Here, we constructed and characterized novel PRV strains that express functional green fluorescent protein (GFP)-Us9 fusion proteins in order to visualize its localization in living neurons during infection. This enabled us to better understand the function of Us9 in facilitating the spread of infection. We show that all viral particles moving in the anterograde direction are labeled with GFP-Us9, suggesting that the presence of Us9 determines the capacity for directed transport within axons.