Pseudorabies virus (PRV) is a herpesvirus of swine, a member of the Alphaherpesvirinae subfamily, and the etiological agent of Aujeszky's disease. This review describes the contributions of PRV research to herpesvirus biology, neurobiology, and viral pathogenesis by focusing on (i) the molecular biology of PRV, (ii) model systems to study PRV pathogenesis and neurovirulence, (iii) PRV transsynaptic tracing of neuronal circuits, and (iv) veterinary aspects of pseudorabies disease. The structure of the enveloped infectious particle, the content of the viral DNA genome, and a step-by-step overview of the viral replication cycle are presented. PRV infection is initiated by binding to cellular receptors to allow penetration into the cell. After reaching the nucleus, the viral genome directs a regulated gene expression cascade that culminates with viral DNA replication and production of new virion constituents. Finally, progeny virions self-assemble and exit the host cells. Animal models and neuronal culture systems developed for the study of PRV pathogenesis and neurovirulence are discussed. PRV serves as a self-perpetuating transsynaptic tracer of neuronal circuitry, and we detail the original studies of PRV circuitry mapping, the biology underlying this application, and the development of the next generation of tracer viruses. The basic veterinary aspects of pseudorabies management and disease in swine are discussed. PRV infection progresses from acute infection of the respiratory epithelium to latent infection in the peripheral nervous system. Sporadic reactivation from latency can transmit PRV to new hosts. The successful management of PRV disease has relied on vaccination, prevention, and testing

The wild-type U L 31, U L 34, and U S 3 proteins localized on nuclear membranes and perinuclear virions; the U S 3 protein was also on cytoplasmic membranes and extranuclear virions. The U L 31 and U L 34 proteins were not detected in extracellular virions. U S 3 deletion caused (i) virion accumulation in nuclear membrane invaginations, (ii) delayed virus production onset, and (iii) reduced peak virus titers. These data support the herpes simplex virus type 1 deenvelopment-reenvelopment model of virion egress and suggest that the U S 3 protein plays an important, but nonessential, role in the egress pathway.Herpes simplex virus type 1 (HSV-1) virions contain a linear double-stranded DNA genome of approximately 152 kb that is packaged into an icosahedral capsid shell. An amorphous tegument layer surrounds the capsid and is, in turn, surrounded by an envelope composed of a host-derived lipid bilayer studded with viral integral membrane proteins. After the viral genome is replicated and packaged into capsids within the nucleus, assembled nucleocapsids acquire a primary lipid envelope by budding through the inner nuclear membrane (INM) into the space located between the inner and outer leaflets of the nuclear envelope (25,33). Whereas the derivation of the primary envelope from the INM is widely accepted, the route of transit of the nascent virions from the perinuclear space to the extracellular space is more controversial. An overview of the key players in herpesvirus egress and a comparison of the salient features of the two proposed envelopment models have been recently published (8,25).A single-step model of herpesvirus envelopment was proposed for the prototypical alphaherpesvirus HSV-1 (6,18,35,44). This model proposes that enveloped virions move through the endoplasmic reticulum (ER) and the Golgi apparatus in transport vesicles with concomitant modification of primary virion glycoproteins. The single-step envelopment model is supported by the observations that (i) enveloped particles within vesicles can be readily detected by electron microscopy and in fracture label studies (35, 44) and (ii) virion egress and virion-associated glycoprotein processing are both inhibited in cells treated with the ionophore monensin (18). On the other hand, neither of these observations can exclude the alternative deenvelopment-reenvelopment model. Such a model is supported by mounting ultrastructural and biochemical evidence (3,10,13,14,30,37,41,46,50) and has been proposed for HSV-1, other alphaherpesviruses such as varicella-zoster virus (VZV) and pseudorabies virus (PrV), and betaherpesviruses such as human cytomegalovirus. In this model, primary envelopment occurs by budding through the INM but the primary envelope surrounding the perinuclear virion is lost, presumably by fusion with the outer lamellae of the nuclear envelope. In a second step, reenvelopment occurs by wrapping of the nucleocapsid and its associated tegument with a lipid bilayer originating from a membranous cytoplasmic organelle bearing viral glycoprotein...

The herpes simplex virus type 1 (HSV-1) U L 34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the U L 31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with U L 34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing U Herpes simplex virus type 1 (HSV-1) nucleocapsids, like those of all herpesviruses are assembled in the nucleus and acquire a lipid bilayer envelope by budding through the inner nuclear membrane into the perinuclear space (10). Several viral proteins have been implicated in this initial budding event, including the myristylated U L 11 protein, glycoprotein K, which is necessary for envelopment in nondividing cells, and U L 34 protein (2, 21, 37). Of these, only U L 34 protein has been implicated solely in the initial envelopment step, whereas glycoprotein K and U L 11 also play roles in egress through the cytoplasm towards the extracellular space (2, 21, 37).The U L 34 sequence predicts that the protein is oriented as a type II integral membrane protein with an N-terminal cytoplasmic domain of 247 amino acids and a C-terminal transmembrane domain of 22 amino acids (32,35,37). The type II membrane topology of HSV-2 U L 34 protein has recently been addressed (39). This topology predicts that if the transmembrane domain were anchored in the outer nuclear membrane, the bulk of the protein would lie in the cytoplasm, whereas localization in the inner nuclear membrane would place the bulk of the protein within the nucleoplasm.The exact role of U L 34 protein in the envelopment process remains unclear. One possibility is that U L 34 protein interacts directly with capsids and/or tegument components and the nuclear membrane, thereby mediating wrapping of the capsid in the membrane. Alternatively, U L 34 protein may be responsible for recruiting other viral or cellular factors to the site of envelopment. Both hypotheses predict that U L 34 protein should associate with the nuclear envelope. To date, research on the localization of HSV-1 U L 34 protein and its homologues in other herpesviruses has not yielded consistent results. In baculovirus-transduced cells. HSV-1 U L 34 protein is found at the nuclear envelope and in the cytoplasm (46), whereas in HSV-1-infected cells, U L 34 protein is reportedly detectable at the cell surface (35). HSV-2 U L 34 protein has been reported to localize at the endoplasmic reticulum in transfected and in-* Corresponding author. Mailing address:

The herpes simplex virus type 1 (HSV-1) U L 31 and U L 34 proteins are dependent on each other for proper targeting to the nuclear membrane and are required for efficient envelopment of nucleocapsids at the inner nuclear membrane. In this work, we show that whereas the solubility of lamins A and C (lamin A/C) was not markedly increased, HSV induced conformational changes in the nuclear lamina of infected cells, as viewed after staining with three different lamin A/C-specific antibodies. In one case, reactivity with a monoclonal antibody that recognizes an epitope in the lamin tail domain was greatly reduced in HSV-infected cells. This apparent HSV-induced epitope masking required both U L 31 and U L 34, but these proteins were not sufficient to mask the epitope in uninfected cells, indicating that other HSV proteins are also required. In the second case, staining with a rabbit polyclonal antibody that primarily recognizes epitopes in the lamin A/C rod domain revealed that U L 34 is required for HSV-induced decreased availability of epitopes for reaction with the antibody, whereas U L 31 protein was dispensable for this effect. Still another polyclonal antibody indicated virtually no difference in lamin A/C staining in infected versus uninfected cells, indicating that the HSVinduced changes are more conformational than the result of lamin depletion at the nuclear rim. Further evidence supporting an interaction between the nuclear lamina and the U L 31/U L 34 protein complex includes the observations that (i) overexpression of the U L 31 protein in uninfected cells was sufficient to relocalize lamin A/C from the nuclear rim into nucleoplasmic aggregates, (ii) overexpression of U L 34 was sufficient to relocalize some lamin A/C into the cytoplasm, and (iii) both U L 31 and U L 34 could directly bind lamin A/C in vitro. These studies suggest that the U L 31 and U L 34 proteins modify the conformation of the nuclear lamina in infected cells, possibly by direct interaction with lamin A/C, and that other proteins are also likely involved. Given that the nuclear lamina potentially excludes nucleocapsids from envelopment sites at the inner nuclear membrane, the lamina alteration may reflect a role of the U L 31/U L 34 protein complex in perturbing the lamina to promote nucleocapsid egress from the nucleus. Alternatively, the data are compatible with a role of the lamina in targeting the U L 31/U L 34 protein complex to the nuclear membrane.The nuclear envelope consists of two leaflets, defined as the inner nuclear membrane and outer nuclear membrane. The perinuclear space, defined as the space between the two leaflets, is continuous with the lumen of the endoplasmic reticulum. A network of predominantly insoluble cellular proteins, the nuclear lamina, lines the nucleoplasmic face of the inner nuclear membrane. The nuclear lamina provides structural support for the nuclear membrane and attachment sites for chromatin. It is also required for a variety of essential cellular functions, including nuclear assembly following mi...

We describe two distinct modes of neuroinvasion and lethality after murine flank inoculation with virulent and attenuated strains of pseudorabies virus (PRV). Mice infected with virulent (e.g., PRV-Becker, PRVKaplan, or PRV-NIA3) strains self-mutilate their flank skin in response to virally induced pruritus, die rapidly with no identifiable symptoms of central nervous system (CNS) infection such as behavioral abnormalities, and have little infectious virus or viral antigen in the brain. In distinct contrast, animals infected with an attenuated PRV vaccine strain (PRV-Bartha) survive approximately three times longer than wild-type PRVinfected animals, exhibit severe CNS abnormalities, and have an abundance of infectious virus in the brain at the time of death. Interestingly, these animals have no skin lesions and do not appear pruritic at any time during infection. The severe pruritus and relatively earlier time until death induced by wild-type PRV infection may reflect the peripheral nervous system (PNS) and immune responses to infection rather than a fatal, virally induced CNS pathology. Based on previously characterized afferent (sensory) and efferent (motor) neuronal pathways that innervate the skin, we deduced that wild-type virulent strains transit through the PNS via both afferent and efferent routes, whereas PRV-Bartha travels by only efferent routes in the PNS en route to the brain.Pseudorabies virus (PRV), a swine alphaherpesvirus, is a member of the alphaherpesvirus subfamily, including human and animal pathogens such as varicella-zoster virus, herpes simplex virus type 1 (HSV-1) and HSV-2, bovine herpesvirus types 1 and 5, and equine herpesviruses types 1 and 4 (23). Although the natural hosts of PRV are adult swine, PRV is pantropic, infecting avian embryos and a wide range of mammalian species, with the notable exceptions of humans and other higher-order species of nonhuman primates.PRV routinely establishes a latent infection in PNS ganglia of adult swine and yet rarely invades the central nervous systems (CNS) of these animals (8). Viral spread between adult swine occurs primarily via direct mucosal contact. Common sequelae of wild-type PRV infection include respiratory disease, weight loss, and infertility in pregnant gilts and sows (31). In contrast, PRV infection is lethal in neonatal piglets and in nonnative hosts such as cows, dogs, rodents, and other susceptible animals. In these animals, infection induces severe, uncontrollable pruritus (itchiness), culminating in frantic selfmutilating behavior historically described as "mad itch." Death ensues within days of infection with virulent strains of PRV. The cause of death of these animals has traditionally been ascribed to fatal encephalitis.Adult swine infected with live, attenuated vaccine strains such as PRV-Bartha typically exhibit few, if any, symptoms of infection. In addition, most attenuated PRV strains are significantly less virulent in nonnative hosts such as rodents (8). Despite the attenuated phenotype, PRV-Bartha remains neuroinvas...