A remarkable property of the alphaherpesvirus life cycle in the natural host is invasion and controlled spread within the peripheral nervous system (PNS) with exceedingly rare incursions into the central nervous system. The basic unit of a herpesvirus infection is the extracellular virion, a complex particle comprising several thousand protein molecules (31
). Herpes virions, in general, are approximately 200 nm in diameter with a membrane envelope containing more than 12 virus-borne membrane proteins. This host-derived membrane surrounds a tegument layer of at least 12 soluble proteins, which, in turn, surrounds an icosahedral capsid containing the 142-kb genome (36
). The assembly and movement of these distinct virion structures must be understood at the cellular level as these properties directly influence herpesvirus pathogenesis and transmission. Pseudorabies virus (PRV), an animal pathogen, has served as a model for study of directional spread of the neurotropic herpesviruses, which include the human pathogens herpes simplex virus (HSV) and varicella-zoster virus (VZV). After replication of PRV at exposed mucosal surfaces, virion components enter the axon terminals of PNS neurons, and unenveloped capsids move toward the cell body to deliver the genome to nuclei within ganglia. In natural hosts, a latent infection is typically established in these PNS neurons. Upon reactivation, capsids are assembled in the nuclei of PNS neurons, enter axons, and are moved in an anterograde direction toward axon terminals near the site of the original infection. Such directional intracellular movement is dependent upon intact microtubules (41
) and involves transport of virion components many millions of virion diameters to and from the cell body of a neuron. Directional transport within axons is likely to be regulated by different interactions of viral proteins with cellular transport machinery during retrograde and anterograde movement on microtubules. Indeed, the differential modulation of plus- and minus-end motor-based movement of capsids early and late in infection appears to affect gross directional movement in neurons (63
A recent model of herpesvirus assembly proposes that newly formed capsid proteins enter axons and are transported separately from membrane proteins as subassemblies of mature virions (53
). Genetic evidence supporting this subassembly transport model derives from studies on attenuated PRV strains utilized for tracing neural circuitry. We have reported that certain viral membrane proteins control the direction of spread between neurons within a neural circuit. For example, deletion of any one of three genes, encoding glycoprotein E (gE), gI, or Us9, from the PRV genome blocks neuronal circuit spread in the anterograde direction, from an infected presynaptic neuron to the synaptically connected postsynaptic neuron (reviewed in reference 24
). These gene products are not required for entry of PRV virions at axon terminals or spread in circuitry in the retrograde direction, from a postsynaptic to presynaptic neuron. Subsequent analysis has demonstrated that the Us9 protein is necessary for localization of newly synthesized viral glycoproteins into the axonal compartment of cultured neurons but not for that of capsid or tegument proteins (69
). In contrast, expression of the gE protein is required for efficient viral glycoprotein, capsid, and tegument localization to the axon but not for a subset of nonglycosylated viral membrane proteins (T. H. Ch'ng and L. W. Enquist, unpublished data). While these discoveries have identified some key viral components of directional spread, the mechanisms of viral protein-dependent axonal sorting remain to be elucidated.
How distinct virion components are coupled to motors, directly or via adaptors, is an important area of focus in the study of directional spread. Previous work has suggested two distinct alternatives. One possibility is that the viral tegument layer, the poorly understood proteinaceous layer surrounding the capsid, provides the physical connection between the capsid and different classes of motors. In this case, the tegument layer is the central component of directional movement. Upon entry at nerve terminals, the tegument-capsid structure is separated from the envelope. Some tegument proteins may dissociate from the capsid, which is moved from the plus end to the minus end of a microtubule, toward the nucleus in the retrograde direction. Following replication, the newly formed capsid and tegument layer are released into the cytoplasm and enter axons where this unique tegument layer, different from that present during entry, presumably binds kinesin motors for movement toward the plus end of a microtubule in the anterograde direction. Support for this proposal comes from studies on HSV type 1 (HSV-1) demonstrating that nonenveloped capsids are found in the axon following virus replication (33
), tegument structures form in the cell body of infected neurons (54
), and the tegument protein Us11 interacts with the kinesin heavy chain (20
). However, the Us11 gene is not present in many alphaherpesvirus genomes, including those of PRV and VZV (36
). In the absence of Us11 protein, other PRV and VZV tegument proteins must interact with motors for kinesin-mediated transport in this model. An alternative idea is that while tegument-capsid complexes are moved to the cell body following entry at axon terminals, newly replicated tegument-capsid structures are directed in the cytoplasm to axon-sorting compartments, where they acquire a cellular membrane. The absence of this membrane during capsid entry and its presence during egress provide the preferential interaction with different classes of motors. Indeed, enveloped capsids have been visualized in axons following virus replication (11
), and the tegument proteins VP22 and UL11 have been shown to associate with cellular membranes (1
We used two approaches to discern the subvirion structures transported in axons following virus replication and distinguish between the unenveloped versus enveloped capsid models of axonal egress. In the first approach, we constructed PRV strains expressing two fluorescent fusion proteins: the monomeric red fluorescent protein (mRFP) fused to VP26 and the green fluorescent protein (GFP) fused to VP22. The mRFP fusion protein incorporates into capsids, while the GFP fusion protein assembles in the tegument layer. Infection by the dually fluorescent virus enabled live-cell imaging and localization of subvirion structures during virus egress. Live-cell imaging of single fluorescent virion structures, including fusion proteins similar to the ones described here, have been previously reported (4
). Furthermore, dually fluorescent virus fusions have also been previously reported for HSV and have been utilized for the localization of structural proteins in the compartments of infected cells (25
). However, the high nucleotide sequence similarity of GFP variants can result in recombination and the exchange of fluorescence properties following cotransfection of epithelial cells (5
). We avoided this complication during infection by using a monomeric red fluorescent protein with limited homology to GFP (5
). A recombinant PRV strain containing the mRFP marker for tracing studies has been reported previously (2
), and mRFP was more suitable for the detection of virus spread than other red fluorescent proteins, such as DsRed.
The second approach used the dually fluorescent virus to investigate the connection between the reenvelopment pathway for alphaherpesvirus assembly and the entry and transport of virion subassemblies in axons. Specifically, we focused on the formation of tegument-capsid structures in the cytoplasm and their direction to the axonal compartment. Treatment of alphaherpesvirus-infected cells with brefeldin A (BFA) inhibits virus maturation and egress by disrupting transport within the secretory pathway (12
). More recently, release of a synchronous infection revealed that the primary block to HSV-1 assembly by BFA occurred prior to budding at the nuclear membrane (16
). Long-term BFA application (over several hours) has been used to study the compartmentalization of virion components in infected neurons. Two key studies suggest that long-term BFA treatment of rat dorsal root ganglion neurons infected with HSV-1 blocks axonal entry of viral glycoproteins and a fraction of tegument proteins but does not block capsid entry (53
). In the present study, we used long-term BFA treatment to assess the effect of secretory pathway disruption on axonal entry of fluorescent capsid and tegument fusion proteins during PRV infection. Unexpectedly, BFA treatment sufficient to block viral glycoprotein axonal entry also efficiently blocked axonal entry of fluorescent fusions to capsid (mRFP-VP26) and tegument (GFP-VP22) proteins. BFA disruption of the secretory system was reversible, enabling restoration of virus assembly transport. The data presented in this study provide a new interpretation of axonal sorting, entry, and transport of alphaherpesvirus assemblies in neurons.