Mutation of the YXXL endocytosis motif in the cytoplasmic tail of pseudorabies virus gE.
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The role of alphaherpesvirus membrane protein internalization during the course of viral infection remains a matter of speculation. To determine the role of internalization of the pseudorabies virus (PRV) gE and gI proteins, we constructed viral mutants encoding specific mutations in the cytoplasmic tail of the gE gene that inhibited internalization of the gE-gI complex. We used these mutants to assess the role of gE-gI endocytosis in incorporation of the proteins into the viral envelope and in gE-mediated spread or gE-promoted virulence. In addition, we report that another viral mutant, PRV 25, which encodes a gE protein defective in endocytosis, contains an additional, previously uncharacterized mutation in the gE gene. We compared PRV 25 to another viral mutant, PRV 107, that does not express the cytoplasmic tail of the gE protein. The gE protein encoded by PRV 107 is also defective in endocytosis. We conclude that efficient endocytosis of gE is not required for gE incorporation into virions, gE-mediated virulence, or spread of virus in the rat central nervous system. However, we do correlate the defect in endocytosis to a small-plaque phenotype in cultured cells. (+info)
Glycoprotein gL-independent infectivity of pseudorabies virus is mediated by a gD-gH fusion protein.
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Envelope glycoproteins gH and gL, which form a complex, are conserved throughout the family Herpesviridae. The gH-gL complex is essential for the fusion between the virion envelope and the cellular cytoplasmic membrane during penetration and is also required for direct viral cell-to-cell spread from infected to adjacent noninfected cells. It has been proposed for several herpesviruses that gL is required for proper folding, intracellular transport, and virion localization of gH. In pseudorabies virus (PrV), glycoprotein gL is necessary for infectivity but is dispensable for virion localization of gH. A virus mutant lacking gL, PrV-DeltagLbeta, is defective in entry into target cells, and direct cell-to-cell spread is drastically reduced, resulting in only single or small foci of infected cells (B. G. Klupp, W. Fuchs, E. Weiland, and T. C. Mettenleiter, J. Virol. 71:7687-7695, 1997). We used this limited cell-to-cell spreading ability of PrV-DeltagLbeta for serial passaging of cells infected with transcomplemented virus by coseeding with noninfected cells. After repeated passaging, plaque formation was restored and infectivity in the supernatant was observed. One single-plaque isolate, designated PrV-DeltagLPass, was further characterized. To identify the mutation leading to this gL-independent infectious phenotype, Southern and Western blot analyses, radioimmunoprecipitations, and DNA sequencing were performed. The results showed that rearrangement of a genomic region comprising part of the gH gene into a duplicated copy of part of the unique short region resulted in a fusion fragment predicted to encode a protein consisting of the N-terminal 271 amino acids of gD fused to the C-terminal 590 residues of gH. Western blotting and radioimmunoprecipitation with gD- and gH-specific antibodies verified the presence of a gDH fusion protein. To prove that this fusion protein mediates infectivity of PrV-DeltagLPass, cotransfection of PrV-DeltagLbeta DNA with the cloned fusion fragment was performed, and a cell line, Nde-67, carrying the fusion gene was established. After cotransfection, infectious gL-negative PrV was recovered, and propagation of PrV-DeltagLbeta on Nde-67 cells produced infectious virions. Thus, a gDH fusion polypeptide can compensate for function of the essential gL in entry and cell-to-cell spread of PrV. (+info)
Central neuronal circuit innervating the lordosis-producing muscles defined by transneuronal transport of pseudorabies virus.
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The lordosis reflex is a hormone-dependent behavior displayed by female rats during mating. This study used the transneuronal tracer pseudorabies virus (PRV) to investigate the CNS network that controls the lumbar epaxial muscles that produce this posture. After PRV was injected into lumbar epaxial muscles, the time course analysis of CNS viral infection showed progressively more PRV-labeled neurons in higher brain structures after longer survival times. In particular, the medullary reticular formation, periaqueductal gray (PAG), and ventromedial nucleus of the hypothalamus (VMN) were sequentially labeled with PRV, which supports the proposed hierarchical network of lordosis control. Closer inspection of the PRV-immunoreactive neurons in the PAG revealed a marked preponderance of spheroid neurons, rather than fusiform or triangular morphologies. Furthermore, PRV-immunoreactive neurons were concentrated in the ventrolateral column, rather than the dorsal, dorsolateral, or lateral columns of the PAG. Localization of the PRV-labeled neurons in the VMN indicated that the majority were located in the ventrolateral subdivision, although some were also in other subdivisions of the VMN. As expected, labeled cells also were found in areas traditionally associated with sympathetic outflow to blood vessels and motor pathways, including the intermediolateral nucleus of the spinal cord, the paraventricular hypothalamic nucleus, the red nucleus, and the motor cortex. These results suggest that the various brain regions along the neuraxis previously implicated in the lordosis reflex are indeed serially connected. (+info)
Distribution of B-cell epitopes on the pseudorabies virus glycoprotein B.
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In order to map antigenically important regions of glycoprotein B (gB) of pseudorabies virus (PrV), a panel of recombinant fragments of gB expressed in E. coli and truncated fragments of gB generated by cleavage of purified native gB with trypsin and cyanogen bromide was analysed by using 26 monoclonal antibodies directed against gB. Three continuous epitopes were localized in the vicinity of the N terminus of gB, between amino acids (aa) 59 and 126. One continuous epitope mapped between residues 214 and 279. The residues involved in the assembly of eight discontinuous epitopes were located between aa 540 and 734. The constituents of two discontinuous epitopes were harboured in a segment encompassing aa 540-646. The clustering of continuous epitopes at the extreme N terminus of PrV gB and the locations of residues involved in the assembly of discontinuous epitopes of PrV gB are in good agreement with data on epitope locations in gB homologues from other herpesviruses. (+info)
Retrograde, transneuronal spread of pseudorabies virus in defined neuronal circuitry of the rat brain is facilitated by gE mutations that reduce virulence.
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The pseudorabies virus (PRV) gE gene encodes a multifunctional membrane protein found in infected cell membranes and in the virion envelope. Deletion of the gE gene results in marked attenuation of the virus in almost every animal species tested that is permissive for PRV. A common inference is that gE mutants are less virulent because they have reduced ability to spread from cell to cell; e.g., gE mutants infect fewer cells and, accordingly, animals live longer. In this report, we demonstrate that this inference does not hold in a rat experimental model for virus invasion of the brain. We find that animals infected with gE mutants live longer despite extensive retrograde, transneuronal spread of virus in the rat brain. In this model of brain infection, virus is injected into the stomach musculature and virions spread to the brain in long axons of brain stem neurons that give rise to the tenth cranial nerve (the vagus). The infection then spreads from neuron to neuron in well-defined, and physically separated, areas of the brain involved in autonomic regulation of the viscera. We examined the progression of infection of five PRV strains in this circuitry: the wild-type PRV-Becker strain, the attenuated PRV-Bartha vaccine strain, and three gE mutants isogenic with the PRV-Becker strain. By 60 to 67 h after infection, all PRV-Becker-infected animals were dead. Analysis of Becker-infected rats killed prior to virus-induced death demonstrated that the virus had established an infection only in the primary vagal neurons connected directly to the stomach and synaptically linked neurons in the immediate vicinity of the caudal brain stem. There was little spread to other neurons in the vagus circuitry. In contrast, rats infected with PRV-Bartha or PRV-Becker gE mutants survived to at least 96 h and exhibited few overt signs of disease. Despite this long survival and the lack of symptoms, brains of animals sacrificed at this time revealed extensive transsynaptic infection not only of the brain stem but also of areas of the forebrain synaptically linked to neurons in the brain stem. This finding provides evidence that the gE protein plays a role in promoting symptoms of infection and death in animals that is independent of neuron-to-neuron spread during brain infection. When this early virulence function is not active, animals live longer, resulting in more extensive spread of virus in the brain. (+info)
Intracellular trafficking and localization of the pseudorabies virus Us9 type II envelope protein to host and viral membranes.
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The Us9 protein is a phosphorylated membrane protein present in the lipid envelope of pseudorabies virus (PRV) particles in a unique tail-anchored type II membrane topology. In this report, we demonstrate that the steady-state residence of the Us9 protein is in a cellular compartment in or near the trans-Golgi network (TGN). Through internalization assays with an enhanced green fluorescent protein epitope-tagged Us9 protein, we demonstrate that the maintenance of Us9 to the TGN region is a dynamic process involving retrieval of molecules from the cell surface. Deletion analysis of the cytoplasmic tail reveals that an acidic cluster containing putative phosphorylation sites is necessary for the recycling of Us9 from the plasma membrane. The absence of this cluster results in the relocalization of Us9 to the plasma membrane due to a defect in endocytosis. The acidic motif, however, does not contain signals needed to direct the incorporation of Us9 into viral envelopes. In this study, we also investigate the role of a dileucine endocytosis signal in the Us9 cytoplasmic tail in the recycling and retention of Us9 to the TGN region. Site-directed mutagenesis of the dileucine motif results in an increase in Us9 plasma membrane staining and a partial internalization defect. (+info)
The murine homolog (Mph) of human herpesvirus entry protein B (HveB) mediates entry of pseudorabies virus but not herpes simplex virus types 1 and 2.
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A mouse member of the immunoglobulin superfamily, originally designated the murine poliovirus receptor homolog (Mph), was found to be a receptor for the porcine alphaherpesvirus pseudorabies virus (PRV). This mouse protein, designated here murine herpesvirus entry protein B (mHveB), is most similar to one of three related human alphaherpesvirus receptors, the one designated HveB and also known as poliovirus receptor-related protein 2. Hamster cells resistant to PRV entry became susceptible upon expression of a cDNA encoding mHveB. Anti-mHveB antibody and a soluble protein composed of the mHveB ectodomain inhibited mHveB-dependent PRV entry. Expression of mHveB mRNA was detected in a variety of mouse cell lines, but anti-mHveB antibody inhibited PRV infection in only a subset of these cell lines, indicating that mHveB is the principal mediator of PRV entry into some mouse cell types but not others. Coexpression of mHveB with PRV gD, but not herpes simplex virus type 1 (HSV-1) gD, inhibited entry activity, suggesting that PRV gD may interact directly with mHveB as a ligand that can cause interference. By analogy with HSV-1, envelope-associated PRV gD probably also interacts directly with mHveB during viral entry. (+info)
Antibody-induced endocytosis of viral glycoproteins and major histocompatibility complex class I on pseudorabies virus-infected monocytes.
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Purified porcine monocytes, the natural carrier cells of pseudorabies virus (PrV) in the pig, were inoculated in vitro with PrV. At different time-points post-inoculation (p.i.) (from 7 to 17 h p.i.), the cells were washed and incubated with fluorescein isothiocyanate-labelled porcine PrV-specific polyclonal antibodies (IgG) at 37 degrees C. At all time-points tested p.i., 1 h of antibody incubation induced passive patching and subsequent internalization of the plasma membrane-anchored viral glycoproteins in approximately 65% of the infected monocytes. This endocytosis process is antibody-dependent, since biotinylated glycoproteins did not undergo spontaneous endocytosis. The process is fast and efficient, since only very low amounts of viral glycoproteins on the plasma membrane (7 h p.i.) and a minimal concentration of antibodies (0.04 mg IgG/ml) were needed to induce endocytosis. Experiments with PrV strains carrying deletions in the genes encoding the 11 different viral glycoproteins showed that viral glycoproteins gB and gD play a very important role in endocytosis (80% reduction with deletion mutants, P < 0.001), while the gE:gI Fc receptor complex, but not gE or gI alone, has a significant but lesser effect (45% reduction, P < 0.05). Double staining of viral glycoproteins and major histocompatibility complex class I (MHC I) showed a clear co-localization and co-endocytosis of MHC I with the viral glycoproteins, suggesting a possible role of the process in immune evasion of the virus. (+info)