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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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Pseudorabies virus encodes a membrane protein (Us9) that is essential for the axonal sorting of virus particles within neurons and anterograde spread in the mammalian nervous system. Enhanced green fluorescent protein (GFP)-tagged Us9 mimicked the trafficking properties of the wild-type protein in nonneuronal cells. We constructed a pseudorabies virus strain that expressed Us9-GFP and tested its spread capabilities in the rat visual system and in primary neuronal cultures. We report that Us9-EGFP does not promote anterograde spread of infection and may disrupt packing of viral membrane proteins in lipid rafts, an essential step for Us9-mediated axonal sorting.

Alpha-herpesviruses, including herpes simplex virus and pseudorabies virus (PRV), infect the peripheral nervous system (PNS) of their hosts. Here, we describe an in vitro method for studying neuron-to-cell spread of infection as well as viral transport in axons. The method centers on a novel microfluidic chamber system that directs growth of axons into a fluidically isolated environment. The system uses substantially smaller amounts of virus inoculum and media than previous chamber systems and yet offers the flexibility of applying multiple virology and cell biology assays including live-cell optical imaging. Using PRV infection of cultured PNS neurons, we demonstrate that the microfluidic chamber recapitulates all known facets of neuron-to-cell spread demonstrated in animals and other compartmented cell systems.

Pseudorabies virus (PRV) mutants lacking the Us9 gene cannot spread from presynaptic to postsynaptic neurons in the rat visual system, although retrograde spread remains unaffected. We sought to recapitulate these findings in vitro using the isolator chamber system developed in our lab for analysis of the transneuronal spread of infection. The wild-type PRV Becker strain spreads efficiently to postsynaptic neurons in vitro, whereas the Us9-null strain does not. As determined by indirect immunofluorescence, the axons of Us9-null infected neurons do not contain the glycoproteins gB and gE, suggesting that their axonal sorting is dependent on Us9. Importantly, we failed to detect viral capsids in the axons of Us9-null infected neurons. We confirmed this observation by using three different techniques: by direct fluorescence of green fluorescent protein-tagged capsids; by transmission electron microscopy; and by live-cell imaging in cultured, sympathetic neurons. This finding has broad impact on two competing models for how virus particles are trafficked inside axons during anterograde transport and redefines a role for Us9 in viral sorting and transport.

The neurotropic alphaherpesviruses invade and spread in the nervous system in a directional manner between synaptically connected neurons. Until now, this property has been studied only in living animals and has not been accessible to in vitro analysis. In this study, we describe an in vitro system in which cultured peripheral nervous system neurons are separated from their neuron targets by an isolator chamber ring. Using pseudorabies virus (PRV), an alphaherpesvirus capable of transneuronal spread in neural circuits of many animals, we have recapitulated in vitro all known genetic requirements for retrograde and anterograde transneuronal spread as determined previously in vivo. We show that in vitro transneuronal spread requires intact axons and the presence of the viral proteins gE, gI, and Us9. We also show that transneuronal spread is dependent on the viral glycoprotein gB, which is required for membrane fusion, but not on gD, which is required for extracellular spread. We demonstrate ultrastructural differences between anterograde- and retrograde-traveling virions. Finally, we show live imaging of dynamic fluorescent virion components in axons and postsynaptic target neurons.

The pseudorabies virus (PRV) Us3 gene is conserved among the alphaherpesviruses and encodes a serine/threonine protein kinase that is not required for growth in standard cell lines. In this report, we used a compartmented culture system to investigate the role of PRV Us3 in viral replication in neurons, in spread from neurons to PK15 cells, and in axon-mediated spread of infection. We also examined the role of Us3 in neuroinvasion and virulence in rodents. Us3 null mutants produce about 10-fold less infectious virus from neurons than wild-type virus and have no discernible phenotypes for axonal targeting of viral components in cultured peripheral nervous system neurons. After eye infection in rodents, Us3 null mutants were slightly attenuated for virulence, with a delayed onset of symptoms compared to the wild type or a Us3 null revertant. While initially delayed, the symptoms increased in severity until they approximated those of the wild-type virus. Us3 null mutants were neuroinvasive, spreading in both efferent and afferent circuits innervating eye tissues.

The molecular mechanisms responsible for long-distance, directional spread of alphaherpesvirus infections via axons of infected neurons are poorly understood. We describe the use of red and green fluorescent protein (GFP) fusions to capsid and tegument components, respectively, to visualize purified, single extracellular virions and axonal assemblies after pseudorabies virus (PRV) infection of cultured neurons. We observed heterogeneity in GFP fluorescence when GFP was fused to the tegument component VP22 in both single extracellular virions and discrete puncta in infected axons. This heterogeneity was observed in the presence or absence of a capsid structure detected by a fusion of monomeric red fluorescent protein to VP26. The similarity of the heterogeneous distribution of these fluorescent protein fusions in both purified virions and in axons suggested that tegument-capsid assembly and axonal targeting of viral components are linked. One possibility was that the assembly of extracellular and axonal particles containing the dually fluorescent fusion proteins occurred by the same process in the cell body. We tested this hypothesis by treating infected cultured neurons with brefeldin A, a potent inhibitor of herpesvirus maturation and secretion. Brefeldin A treatment disrupted the neuronal secretory pathway, affected fluorescent capsid and tegument transport in the cell body, and blocked subsequent entry into axons of capsid and tegument proteins. Electron microscopy demonstrated that in the absence of brefeldin A treatment, enveloped capsids entered axons, but in the presence of the inhibitor, unenveloped capsids accumulated in the cell body. These results support an assembly process in which PRV capsids acquire a membrane in the cell body prior to axonal entry and subsequent transport.

The core structures of many viruses move within cells by association with host cytoskeletal motor proteins; however, the mechanisms by which intracellular viral particles are transported toward sites of replication or the cell periphery at distinct stages of infection remain to be understood. The regulation of herpesvirus directional transport in sensory neurons was examined by tracking individual viral capsids within axons at multiple frames per s. After entry into axons, capsids underwent bidirectional and saltatory movement to the cell body independently of endosomes. A comparison of entry transport to a previous analysis of capsid axonal transport during egress revealed that capsid targeting in and out of cells occurs by modulation of plus-end, but not minus-end, motion. Entry transport was unperturbed by the presence of egressing virus from a prior infection, indicating that transport direction is not modulated globally by viral gene expression, but rather directly by a component of the viral particle.

Pseudorabies virus, an alpha-herpesvirus, is capable of infecting the nervous system and spreading between synaptically connected neurons in diverse hosts. At least three viral membrane proteins (gE, gI, and Us9) are necessary for the spread of infection from presynaptic to postsynaptic neurons (anterograde spread) in infected rodents. To understand how these proteins effect anterograde spread between neurons, we analyzed the subcellular localization of viral proteins after infection of cultured rat sympathetic neurons with wild-type or mutant viruses. After Us9-null mutant infections but not gE-null mutant infections, only a subset of the viral structural proteins had entered axons. Surprisingly, capsid and tegument proteins but not viral membrane proteins were detected in axons. The spread of Us9 missense mutants in the rodent nervous system correlated with the amount of viral membrane proteins localized to axons. We conclude that the Us9 membrane protein controls axonal localization of diverse viral membrane proteins but not that of capsid or tegument proteins. The data support a model where virion subassemblies but not complete virions are transported in the axon. Our results provide new insight into the process of virion assembly and exit from neurons that leads to directional spread of herpesviruses in the nervous system.

The alpha herpesviruses, a subfamily of the herpesviruses, are neurotropic pathogens found associated with most mammalian species. The prototypic member of this subfamily is herpes simplex virus type 1, the causative agent of recurrent cold sores in humans. The mild nature of this disease is a testament to the complex and highly regulated life cycle of the alpha herpesviruses. The cellular mechanisms used by these viruses to disseminate infection in the nervous system are beginning to be understood. Here, we overview the life cycle of alpha herpesviruses with an emphasis on assembly and transport of viral particles in neurons.

Alpha herpesviruses infect the vertebrate nervous system resulting in either mild recurrent lesions in mucosal epithelia or fatal encephalitis. Movement of virions within the nervous system is a critical factor in the outcome of infection; however, the dynamics of individual virion transport have never been assessed. Here we visualized and tracked individual viral capsids as they moved in axons away from infected neuronal cell bodies in culture. The observed movement was compatible with fast axonal flow mediated by multiple microtubule motors. Capsids accumulated at axon terminals, suggesting that spread from infected neurons required cell contact.

Pseudorabies virus (PRV), a neurotropic swine alpha herpesvirus, has been used extensively for transneuronal analysis ofmultisynaptic circuitry after peripheral injection. In the present analysis, we examined the influence of viral concentration and neuronal architecture on the invasiveness, replication, and transynaptic passage of an attenuated strain of PRV (PRV-Bartha) injected into rat striatum. Different concentrations of PRV-Bartha were injected into the striatum at a constant rate of infusion (10 nl/minute), and animals were killed 50 hours later. Viral concentration was manipulated by either altering the volume of the inoculum (100, 50, 20 nl) or by diluting the inoculum within a constant volume of 100 nl. Immunohistochemical localization of infected neurons revealed dramatic differences in the progression of infection that were dependent directly on the concentration of injected virus. In every case, the pattern of infection was consistent with preferential uptake of virions by axon terminals and retrograde transynaptic passage of virus from the injection site. The known topographically organized corticostriatal projections permitted a precise definition of the zone of viral uptake. This analysis demonstrated that the "effective zone of viral uptake" (i.e., the zone within which viral uptake led to productive replication of virus) varied in relation to the concentration of injected virus, with the highest concentration of PRV invading terminals within a 500 microm radius of the canula. Concentration-dependent changes in the progression of retrograde transynaptic infection also were observed. The highest concentration of virus produced the most extensive infection. The distribution of infected neurons in these cases included those with known afferent projections to striatum as well as those that became infected by retrograde transynaptic infection. Lesser concentrations of PRV-Bartha produced an increasingly restricted infection of the same circuitry within the same postinoculation interval. It is noteworthy that neurons known to elaborate dense striatal terminal fields were less sensitive to reduction in viral concentration than those giving rise to terminal fields of lesser density. Collectively, the data indicate that the onset of viral replication after intracerebral injection of PRV is directly dependent on virus concentration and terminal field density at the site of virus injection.