Pseudorabies virus Us9 directs axonal sorting of viral capsids

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 axonal sorting activity of pseudorabies virus Us9 protein depends on the state of neuronal maturation

Alpha-herpesviruses establish a life-long infection in the nervous system of the affected host; while this infection is restricted to peripheral neurons in a healthy host, the reactivated virus can spread within the neuronal circuitry, such as to the brain, in compromised individuals and lead to adverse health outcomes. Pseudorabies virus (PRV), an alpha-herpesvirus, requires the viral protein Us9 to sort virus particles into axons and facilitate neuronal spread. Us9 sorts virus particles by mediating the interaction of virus particles with neuronal transport machinery. Here, we report that Us9-mediated regulation of axonal sorting also depends on the state of neuronal maturation. Specifically, the development of dendrites and axons is accompanied with proteomic changes that influence neuronal processes. Immature superior cervical ganglionic neurons (SCGs) have rudimentary neurites that lack markers of mature axons. Immature SCGs can be infected by PRV, but they show markedly reduced Us9-dependent regulation of sorting, and increased Us9-independent transport of particles into neurites. Mature SCGs have relatively higher abundances of proteins characteristic of vesicle-transport machinery. We also identify Us9-associated neuronal proteins that can contribute to axonal sorting and subsequent anterograde spread of virus particles in axons. We show that SMPD4/nsMase3, a sphingomyelinase abundant in lipid-rafts, associates with Us9 and is a negative regulator of PRV sorting into axons and neuronal spread, a potential antiviral function.     

Fusion of enhanced green fluorescent protein to the pseudorabies virus axonal sorting protein Us9 blocks anterograde spread of infection in mammalian neurons

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.     

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.     

Role of Us9 Phosphorylation in Axonal Sorting and Anterograde Transport of Pseudorabies Virus

Alphaherpes viruses, such as pseudorabies virus (PRV), undergo anterograde transport in neuronal axons to facilitate anterograde spread within hosts. Axonal sorting and anterograde transport of virions is dependent on the viral membrane protein Us9, which interacts with the host motor protein Kif1A to direct transport. Us9-Kif1A interactions are necessary but not sufficient for these processes, indicating that additional cofactors or post-translational modifications are needed. In this study, we characterized two conserved serine phosphorylation sites (S51 and S53) in the PRV Us9 protein that are necessary for anterograde spread in vivo. We assessed the subcellular localization of phospho-Us9 subspecies during infection of neurons and found that the phospho-form is detectable on the majority, but not all, of axonal vesicles containing Us9 protein. In biochemical assays, phospho-Us9 was enriched in lipid raft membrane microdomains, though Us9 phosphorylation did not require prior lipid raft association. During infections of chambered neuronal cultures, we observed only a modest reduction in anterograde spread capacity for diserine mutant Us9, and no defect for monoserine mutants. Conversely, mutation of the kinase recognition sequence residues adjacent to the phosphorylation sites completely abrogated anterograde spread. In live-cell imaging analyses, anterograde transport of virions was reduced during infection with a recombinant PRV strain expressing GFP-tagged diserine mutant Us9. Phosphorylation was not required for Us9-Kif1A interaction, suggesting that Us9-Kif1A binding is a distinct step from the activation and/or stabilization of the transport complex. Taken together, our findings indicate that, while not essential, Us9 phosphorylation enhances Us9-Kif1A-based transport of virions in axons to modulate the overall efficiency of long-distance anterograde spread of infection.     

Efficient axonal localization of alphaherpesvirus structural proteins in cultured sympathetic neurons requires viral glycoprotein E

Pseudorabies virus (PRV) glycoprotein E (gE) is a type I viral membrane protein that facilitates the anterograde spread of viral infection from the peripheral nervous system to the brain. In animal models, a gE-null mutant infection spreads inefficiently from presynaptic neurons to postsynaptic neurons (anterograde spread of infection). However, the retrograde spread of infection from post- to presynaptic neurons remains unaffected. Here we show that gE is required for wild-type localization of viral structural proteins in axons of infected neurons. During a gE-null PRV infection, a subset of viral glycoproteins, capsids, and tegument proteins enter and localize to the axon inefficiently. This defect is most obvious in the distal axon and growth cones. However, axonal entry and localization of other viral membrane proteins and endogenous cellular proteins remains unaffected. Neurons infected with gE-null mutants produce wild-type levels of viral structural proteins and infectious virions in the cell body. Our results indicate that reduced axonal targeting of viral structural proteins is a compelling explanation for the lack of anterograde spread in neural circuits following infection by a gE-null mutant.     

Copatching and lipid raft association of different viral glycoproteins expressed on the surfaces of pseudorabies virus-infected cells

Pseudorabies virus (PRV) is a swine alphaherpesvirus that is closely related to human herpes simplex virus (HSV). Both PRV and HSV express a variety of viral envelope glycoproteins in the plasma membranes of infected cells. Here we show that at least four major PRV glycoproteins (gB, gC, gD, and gE) in the plasma membrane of infected swine kidney cells and monocytes seem to be linked, since monospecific antibody-induced patching of any one of these proteins results in copatching of the others. Further, for all four PRV glycoproteins, monospecific antibody-induced patches were enriched in GM1, a typical marker of lipid raft microdomains, but were excluded for transferrin receptor, a nonraft marker, suggesting that these viral proteins may associate with lipid rafts. However, only gB and, to a lesser extent, gE were found in lipid raft fractions by using detergent floatation assays, indicating that gC and gD do not show strong lipid raft association. Addition of methyl-beta-cyclodextrin (MCD), a cholesterol-depleting agent that is commonly used to disrupt lipid rafts, only slightly reduced copatching efficiency between the different viral proteins, indicating that other factors, perhaps tegument-glycoprotein interactions, may be important for the observed copatching events. On the other hand, MCD strongly reduced polarization of the antibody-induced viral glycoprotein patches to a cap structure, a gE-dependent process that has been described for specific PRV- and HSV-infected cells. Therefore, we hypothesize that efficient gE-mediated capping of antibody-antigen patches may require the lipid raft-associated signal transduction machinery.     

Role of pseudorabies virus Us9, a type II membrane protein, in infection of tissue culture cells and the rat nervous system

The protein product of the pseudorabies virus (PRV) Us9 gene is a phosphorylated, type II membrane protein that is inserted into virion envelopes and accumulates in the trans-Golgi network. It is among a linked group of three envelope protein genes in the unique short region of the PRV genome which are absent from the attenuated Bartha strain. We found that two different Us9 null mutants exhibited no obvious phenotype after infection of PK15 cells in culture. Unlike those of gE and gI null mutants, the plaque size of Us9 null mutants on Madin-Darby bovine kidney cells was indistinguishable from that of wild-type virus. However, both of the Us9 null mutants exhibited a defect in anterograde spread in the visual and cortical circuitry of the rat. The visual system defect was characterized by restricted infection of a functionally distinct subset of visual projections involved in the temporal organization of behavior, whereas decreased anterograde spread of virus to the cortical projection targets was characteristic of animals receiving direct injections of virus into the cortex. Spread of virus through retrograde pathways in the brain was not compromised by a Us9 deletion. The virulence of the Us9 null mutants, as measured by time to death and appearance of symptoms of infection, also was reduced after their injection into the eye, but not after cortical injection. Through sequence analysis, construction of revertants, measurement of gE and gI protein synthesis in the Us9 null mutants, and mixed-infection studies of rats, we conclude that the restricted-spread phenotype after infection of the rat nervous system reflects the loss of Us9 and is not an indirect effect of the Us9 mutations on expression of glycoproteins gE and gI. Therefore, at least three viral envelope proteins, Us9, gE, and gI, function together to promote efficient anterograde transneuronal infection by PRV in the rat central nervous system.     

In vitro analysis of transneuronal spread of an alphaherpesvirus infection in peripheral nervous system neurons

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.     

Comparison of the Pseudorabies Virus Us9 Protein with Homologs from Other Veterinary and Human Alphaherpesviruses

Pseudorabies virus (PRV) Us9 is a small, tail-anchored (TA) membrane protein that is essential for axonal sorting of viral structural proteins and is highly conserved among other members of the alphaherpesvirus subfamily. We cloned the Us9 homologs from two human pathogens, varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1), as well as two veterinary pathogens, equine herpesvirus type 1 (EHV-1) and bovine herpesvirus type 1 (BHV-1), and fused them to enhanced green fluorescent protein to examine their subcellular localization and membrane topology. Akin to PRV Us9, all of the Us9 homologs localized to the trans-Golgi network and had a type II membrane topology (typical of TA proteins). Furthermore, we examined whether any of the Us9 homologs could compensate for the loss of PRV Us9 in anterograde, neuron-to-cell spread of infection in a compartmented chamber system. EHV-1 and BHV-1 Us9 were able to fully compensate for the loss of PRV Us9, whereas VZV and HSV-1 Us9 proteins were unable to functionally replace PRV Us9 when they were expressed in a PRV background.Alphaherpesviruses are classified by their variable host range, short reproductive cycle, and ability to establish latency in the peripheral nervous system (PNS) (36, 37). Commonly studied pathogens of this subfamily include herpes simplex virus (HSV) and varicella-zoster virus (VZV), as well as the veterinary pathogens pseudorabies virus (PRV), equine herpesvirus (EHV), and bovine herpesvirus (BHV). Initial infection begins with the virus entering the host mucosal surfaces and spreading between cells of the mucosal epithelium. Invariably, virus enters the PNS through the infection of peripheral nerves that innervate this region. The virus establishes a latent infection in PNS neurons that can be reactivated and that persists for the life of the host (36). In most natural infections, virus replication in the PNS never spreads to the central nervous system (CNS). However, on rare occasions, invasion of the CNS does occur, resulting in devastating encephalitis (46). Trafficking of virus particles from infected epithelial cells into the axon and subsequent transport to neuronal cell bodies is known as retrograde spread of infection. Trafficking of virus particles that are assembled in the neuronal cell body and subsequently sorted into axons for transport to epithelial cells at the initial site of infection (upon reactivation from latency) is known as anterograde spread of infection.Though the natural host of PRV is swine, the virus infects a wide variety of animals, including rodents, cats, dogs, rabbits, cattle, and chicken embryos, but not higher primates (1, 30, 47). In contrast to the well-contained spread of PRV within its natural host, infection of other mammals is usually lethal. Instead of stopping in the PNS, infection continues on to second-order and third-order neurons in the CNS (reviewed in reference 35). This facet of PRV infection makes it a useful tracer of neuronal connections (18). Work in our lab has identified three PRV proteins, Us9 and the gE/gI heterodimer, which are critical for efficient anterograde spread of infection in vivo (i.e., spread from presynaptic to postsynaptic neurons) (6, 45). The molecular mechanism by which these proteins function has been further elucidated in vitro using primary neuronal cultures of superior cervical ganglion (SCG) harvested from embryonic rat pups. PRV Us9 and, to a lesser extent, gE/gI are required for efficient axonal targeting of viral structural proteins, a necessary step for subsequent anterograde, transneuronal spread (10, 11, 27, 28, 42).PRV Us9 is a type II, tail-anchored (TA) membrane protein that is highly enriched in lipid raft microdomains and resides predominantly in or near the trans-Golgi network (TGN) inside infected cells (5-7, 27). It has homologs in most of the alphaherpesviruses, including VZV (16), HSV-1 (22), HSV-2 (17), EHV-1 (21, 40), EHV-4 (41), BHV-1 (25), and BHV-5 (14). Though several studies have examined individually the Us9 proteins encoded by VZV (16), HSV-1 (4, 22, 34, 39), BHV-1 (13), and BHV-5 (14), several gaps in our understanding of Us9 biology remain, namely, whether all of the PRV Us9 homologs are type II membrane proteins, if the proteins localize to similar subcellular compartments within different cell types, and if they can functionally substitute for the loss of PRV Us9 in axonal sorting and anterograde spread of infection. The aim of this study is to examine PRV Us9 in parallel assays with its homologs from VZV, HSV-1, EHV-1, and BHV-1 to identify potential similarities and differences between these highly conserved alphaherpesvirus proteins.     

The Us9 Gene Product of Pseudorabies Virus, an Alphaherpesvirus, Is a Phosphorylated, Tail-Anchored Type II Membrane Protein

The Us9 gene is highly conserved among the alphaherpesviruses sequenced to date, yet its function remains unknown. In this report, we demonstrate that the pseudorabies virus (PRV) Us9 protein is present in infected cell lysates as several phosphorylated polypeptides ranging from 17 to 20 kDa. Synthesis is first detected at 6 h postinfection and is sensitive to the DNA synthesis inhibitor phosphonoacetic acid. Unlike the herpes simplex virus type 1 Us9 homolog, which was reported to be associated with nucleocapsids in the nuclei of infected cells (M. C. Frame, D. J. McGeoch, F. J. Rixon, A. C. Orr, and H. S. Marsden, Virology 150:321–332, 1986), PRV Us9 localizes to the secretory pathway (predominately to the Golgi apparatus) and not to the nucleus. By fusing the enhanced green fluorescent protein (EGFP) reporter molecule to the carboxy terminus of Us9, we demonstrated that Us9 not only is capable of targeting a Us9-EGFP fusion protein to the Golgi compartment but also is able to direct efficient incorporation of such chimeric molecules into infectious viral particles. Moreover, through protease digestion experiments with Us9-EGFP-containing viral particles, we demonstrated that the Us9 protein is inserted into the viral envelope as a type II, tail-anchored membrane protein.     

Directional transneuronal infection by pseudorabies virus is dependent on an acidic internalization motif in the Us9 cytoplasmic tail

The Us9 gene is conserved among most alphaherpesviruses. In pseudorabies virus (PRV), the Us9 protein is a 98-amino-acid, type II membrane protein found in the virion envelope. It localizes to the trans-Golgi network (TGN) region in infected and transfected cells and is maintained in this compartment by endocytosis from the plasma membrane. Viruses with Us9 deleted have no observable defects in tissue culture yet have reduced virulence and restricted spread to retinorecipient neurons in the rodent brain. In this report, we demonstrate that Us9-promoted transneuronal spread in vivo is dependent on a conserved acidic motif previously shown to be essential for the maintenance of Us9 in the TGN region and recycling from the plasma membrane. Mutant viruses with the acidic motif deleted have an anterograde spread defect indistinguishable from that of Us9 null viruses. Transneuronal spread, however, is not dependent on a dileucine endocytosis motif in the Us9 cytoplasmic tail. Through alanine scanning mutagenesis of the acidic motif, we have identified two conserved tyrosine residues that are essential for Us9-mediated spread as well as two serine residues, comprising putative consensus casein kinase II sites, that modulate the rate of PRV transneuronal spread in vivo.     

Different Patterns of Neuronal Infection after Intracerebral Injection of Two Strains of Pseudorabies Virus

Pseudorabies virus (PRV), a swine neurotropic alphaherpesvirus, is known to invade the central nervous system (CNS) of a variety of animal species through peripherally projecting axons, replicate in the parent neurons, and then pass transsynaptically to infect other neurons of a circuit. Studies of the human pathogen herpes simplex virus type 1 have reported differences in the direction of transport of two strains of this virus after direct injection into the primate motor cortex. In the present study we examined the direction of transport of virulent and attenuated strains of PRV, utilizing injections into the rat prefrontal cortex to evaluate specific movement of virus through CNS circuitry. The data demonstrate strain-dependent patterns of infection consistent with bidirectional (anterograde and retrograde) transport of virulent virus and unidirectional (retrograde) transport of attenuated PRV from the site of injection. The distribution of infected neurons and the extent of transsynaptic passage also suggest that a release defect in the attenuated strain reduces the apparent rate of viral transport through neuronal circuitry. Finally, injection of different concentrations of virus influenced the onset of replication within a neural circuit. Taken together, these data suggest that viral envelope glycoproteins and virus concentration at the site of injection are important determinants of the rate and direction of viral transport through a multisynaptic circuit in the CNS.     

Role of pseudorabies virus Us3 protein kinase during neuronal infection

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.     

Pseudorabies virus membrane proteins gI and gE facilitate anterograde spread of infection in projection-specific neurons in the rat

The membrane proteins gI and gE of Pseudorabies virus (PRV) are required for viral invasion and spread through some neural pathways of the rodent central nervous system. Following infection of the rat retina with wild-type PRV, virus replicates in retinal ganglion neurons and anterogradely spreads to infect all visual centers in the brain. By contrast, gI and gE null mutants do not infect a specific subset of the visual centers, e.g., the superior colliculus and the dorsal lateral geniculate nucleus. In previous experiments, we suggested that the defect was not due to inability to infect projection-specific retinal ganglion cells, because mixed infection of a gE deletion mutant and a gI deletion mutant restored the wild-type phenotype (i.e., genetic complementation occurred). In the present study, we provide direct evidence that gE and gI function to promote the spread of infection after entry into primary neurons. We used stereotaxic central nervous system injection of a fluorescent retrograde tracer into the superior colliculus and subsequent inoculation of a PRV gI-gE double null mutant into the eye of the same animal to demonstrate that viral antigen and fluorescent tracer colocalize in retinal ganglion cells. Furthermore, we demonstrate that direct injection of a PRV gI-gE double null mutant into the superior colliculus resulted in robust infection followed by retrograde transport to the eye and replication in retinal ganglion neuron cell bodies. These experiments provide additional proof that the retinal ganglion cells projecting to the superior colliculus are susceptible and permissive to gE and gI mutant viruses. Our studies confirm that gI and gE specifically facilitate anterograde spread of infection by affecting intracellular processes in the primary infected neuron such as anterograde transport in axons or egress from axon terminals.     

The gE and gI homologs from two alphaherpesviruses have conserved and divergent neuroinvasive properties.

The membrane glycoproteins gE and gI are encoded by pseudorabies virus (PRV), a neurotropic, broad-host-range alphaherpesvirus of swine. PRV gE and gI are required for anterograde spread to a restricted set of retinorecipient neurons in the brain after infection of the rat retina. A related alphaherpesvirus, encoding gE and gI homologs, is called bovine herpesvirus 1.1 (BHV-1.1). BHV-1.1 is a respiratory pathogen of highly restricted host range and, in contrast to PRV, is unable to propagate in or cause disease in rodents. We have shown previously that the BHV-1.1 gE and gI proteins are capable of complementing the virulence functions of PRV gE and gI in a rodent model (A. C. Knapp and L. W. Enquist, J. Virol. 71:2731-2739, 1997). We examined the ability of the BHV-1.1 gE and gI homologs to direct circuit-specific invasion of the rat central nervous system by PRV. Both complete open reading frames were cloned into a PRV mutant lacking the PRV gE and gI genes. Recombinant viruses were analyzed for the ability to invade the rat brain after infection of the retina. Surprisingly, in a portion of the animals tested, the BHV-1.1 gE and gI proteins functioned autonomously to promote spread of PRV to a subset of retinorecipient regions of the brain. First, the presence of BHV-1.1 gI alone, but not PRV gI alone, promoted viral invasion of the optic tectum. Second, expression of BHV-1.1 gE alone facilitated PRV infection of a subset of neurons in the hippocampus not normally infected by PRV. When both BHV-1.1 proteins were expressed in a coinfection, all retinorecipient regions of the rat brain were infected. Therefore, depending on the viral source, homologs of gE and gI differentially affect spread between synaptically connected neurons in the rat.     

Anterograde Transport of Herpes Simplex Virus Proteins in Axons of Peripheral Human Fetal Neurons: an Immunoelectron Microscopy Study

Herpes simplex virus (HSV) reactivates from latency in the neurons of dorsal root ganglia (DRG) and is subsequently transported anterogradely along the axon to be shed at the skin or mucosa. Although we have previously shown that only unenveloped nucleocapsids are present in axons during anterograde transport, the mode of transport of tegument proteins and glycoproteins is not known. We used a two-chamber culture model with human fetal DRG cultivated in an inner chamber, allowing axons to grow out and penetrate an agarose barrier and interact with autologous epidermal cells in the outer chamber. After HSV infection of the DRG, anterograde transport of viral components could be examined in the axons in the outer chamber at different time points by electron and immunoelectron microscopy (IEM). In the axons, unenveloped nucleocapsids or focal collections of gold immunolabel for nucleocapsid (VP5) and/or tegument (VP16) were detected. VP5 and VP16 usually colocalized in both scanning and transmission IEM. In contrast, immunolabel for glycoproteins gB, gC, and gD was diffusely distributed in axons and was rarely associated with VP5 or VP16. In longitudinal sections of axons, immunolabel for glycoprotein was arrayed along the membranes of axonal vesicles. These findings provide evidence that in DRG axons, virus nucleocapsids coated with tegument proteins are transported separately from glycoproteins and suggest that final assembly of enveloped virus occurs at the axon terminus.     

Bovine herpesvirus 5 (BHV-5) Us9 is essential for BHV-5 neuropathogenesis

Bovine herpesvirus 5 (BHV-5) is a neurovirulent alphaherpesvirus that causes fatal encephalitis in calves. In a rabbit model, the virus invades the central nervous system (CNS) anterogradely from the olfactory mucosa following intranasal infection. In addition to glycoproteins E and I (gE and gI, respectively), Us9 and its homologue in alphaherpesviruses are necessary for the viral anterograde spread from the presynaptic to postsynaptic neurons. The BHV-5 Us9 gene sequence was determined, and the predicted amino acid sequence of BHV-5 Us9 was compared with the corresponding Us9 sequences of BHV-1.1. Alignment results showed that they share 77% identity and 83% similarity. BHV-5 Us9 peptide-specific antibody recognized a doublet of 17- and 19-kDa protein bands in BHV-5-infected cell lysates and in purified virions. To determine the role of the BHV-5 Us9 gene in BHV-5 neuropathogenesis, a BHV-5 Us9 deletion recombinant was generated and its neurovirulence and neuroinvasive properties were compared with those of a Us9 rescue mutant of BHV-5 in a rabbit model. Following intranasal infection, the Us9 rescue mutant of BHV-5 displayed a wild-type level of neurovirulence and neural spread in the olfactory pathway, but the Us9 deletion mutant of BHV-5 was virtually avirulent and failed to invade the CNS. In the olfactory mucosa containing the olfactory receptor neurons, the Us9 deletion mutant virus replicated with an efficiency similar to that of the Us9 rescue mutant of BHV-5. However, the Us9 deletion mutant virus was not transported to the bulb. Confocal microscopy of the olfactory epithelium detected similar amounts of virus-specific antigens in the cell bodies of olfactory receptor neuron for both the viruses, but only the Us9 rescue mutant viral proteins were detected in the processes of the olfactory receptor neurons. When injected directly into the bulb, both viruses were equally neurovirulent, and they were transported retrogradely to areas connected to the bulb. Taken together, these results indicate that Us9 is essential for the anterograde spread of the virus from the olfactory mucosa to the bulb.     

In vivo egress of an alphaherpesvirus from axons

Many alphaherpesviruses establish a latent infection in the peripheral nervous systems of their hosts. This life cycle requires the virus to move long distances in axons toward the neuron's cell body during infection and away from the cell body during reactivation. While the events underlying entry of the virion into neurons during infection are understood in principle, no such consensus exists regarding viral egress from neurons after reactivation. In this study, we challenged two different models of viral egress from neurons by using pseudorabies virus (PRV) infection of the rat retina: does PRV egress solely from axon terminals, or can the virus egress from axon shafts as well as axon terminals? We took advantage of PRV gD mutants that are not infectious as extracellular particles but are capable of spreading by cell-cell contact. We observed that both wild-type virus and a PRV gD null mutant are capable of spreading from axons to closely apposed nonneuronal cells within the rat optic nerve after intravitreal infection. However, infection does not spread from these infected nonneuronal cells. We suggest that viral egress can occur sporadically along the length of infected axons and is not confined solely to axon terminals. Moreover, it is likely that extracellular particles are not involved in nonneuronal cell infections. Taking these together with previous data, we suggest a model of viral egress from neurons that unifies previous apparently contradictory data.     

Integrity of membrane lipid rafts is necessary for the ordered assembly and release of infectious Newcastle disease virus particles

Membrane lipid raft domains are thought to be sites of assembly for many enveloped viruses. The roles of both classical lipid rafts and lipid rafts associated with the membrane cytoskeleton in the assembly of Newcastle disease virus (NDV) were investigated. The lipid raft-associated proteins caveolin-1, flotillin-2, and actin were incorporated into virions, while the non-lipid raft-associated transferrin receptor was excluded. Kinetic analyses of the distribution of viral proteins in lipid rafts, as defined by detergent-resistant membranes (DRMs), in non-lipid raft membranes, and in virions showed an accumulation of HN, F, and NP viral proteins in lipid rafts early after synthesis. Subsequently, these proteins exited the DRMs and were recovered quantitatively in purified virions, while levels of these proteins in detergent-soluble cell fractions remained relatively constant. Cholesterol depletion of infected cells drastically altered the association of viral proteins with DRMs and resulted in an enhanced release of virus particles with reduced infectivity. Decreased infectivity was not due to effects on subsequent virus entry, since the extraction of cholesterol from intact virus did not significantly reduce infectivity. Particles released from cholesterol-depleted cells had very heterogeneous densities and altered ratios of NP and glycoproteins, demonstrating structural abnormalities which potentially contributed to their lowered infectivity. Taken together, these results indicate that lipid rafts, including cytoskeleton-associated lipid rafts, are sites of NDV assembly and that these domains are important for ordered assembly and release of infectious Newcastle disease virus particles.