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A key aspect in the life cycle of alphaherpesviruses is their neurotropic behaviour. Sensory neurons of the trigeminal ganglion (TG) are important target cells for many alphaherpesviruses (including herpes simplex virus 1, pseudorabies virus (PRV), bovine herpesvirus 1) and constitute major sites for latent infections. The aim of this study was to develop an in vitro model that simulates the in vivo infection pattern of TG neurons by alphaherpesviruses. To this end, we developed a homologous in vitro two-chamber model using PRV and porcine TG neurons. TG of 4- to 6-week-old piglets were dissociated and cultured in the inner chamber of the in vitro model, which is separated from the outer chamber by a medium- and virus-impermeable silicon barrier. Outgrowth of axons from neuronal cell bodies in the inner chamber through the silicon barrier into the outer chamber could be observed after 2-3 weeks of cultivation. Subsequent addition of PRV to the outer chamber resulted in exclusive infection of the TG neurons by transport of virus through the axons, subsequently giving rise to productively infected TG neurons that transmitted virus to contacting neurons and non-neuronal cells in the inner chamber. Thus, we established a homologous in vitro model that mimics the natural route of alphaherpesvirus infection of TG neurons that can be used to study interactions between these viruses and this pathogenetically very important cell type.

Most large DNA viruses, like herpesviruses, encode anti-apoptotic proteins to interfere with the apoptotic cellular response to infection. Previous studies have shown that the US3 protein kinase of herpes simplex virus, in contrast to US3 of bovine herpes virus 1, is very potent in protecting cells from apoptosis induced by the virus itself or by a broad range of exogenous apoptotic stimuli. Here, we demonstrate that US3 of the swine alphaherpesvirus pseudorabies virus (PRV) suppresses PRV-induced apoptosis in swine-testicle (ST) cells at late stages in infection, and that it protects ST cells from apoptosis induced by either sorbitol or staurosporine. Interestingly, PRV US3 encodes a short and a long isoform, the latter of which contains a functional mitochondrial localization sequence. Transient transfections showed that the PRV US3 long isoform is more efficient in protecting ST cells from PRV- or staurosporine-induced apoptosis, suggesting a potential advantage for the mitochondrial localization of PRV US3 in implementing its anti-apoptotic function.

Sensory neurons of the trigeminal ganglion (TG) are of crucial importance in the pathogenesis of many alphaherpesviruses, constituting major target cells for latency and reactivation events. We showed earlier that a subpopulation of porcine TG neurons, in contrast to other porcine cell types, is highly resistant to cell death induced by infection with the porcine alphaherpesvirus pseudorabies virus (PRV). Here, we report that expression of Brn-3a, a neuron-specific transcription factor implicated in cell survival of sensory neurons, correlates with the increased resistance of TG neurons towards PRV-induced cell death. In addition, overexpression of Brn-3a in the sensory neuronal cell line ND7 markedly increased resistance of these cells to PRV-induced cell death. Hence, Brn-3a may play a hitherto uncharacterized role in protection of sensory neurons from alphaherpesvirus-induced cell death, which may have implications for different aspects of the alphaherpesvirus life cycle, including latency/reactivation events.

The multifunctional US3 protein kinase is conserved among alphaherpesviruses. Like the herpes simplex virus US3 protein kinase, the pseudorabies virus (PRV) US3 protein confers resistance against apoptosis. In the current report, we introduced a point mutation in the putative ATP binding site of the PRV US3 protein kinase. We found that, in contrast to the wild type PRV US3, the point-mutated PRV US3 does not protect cells from apoptosis induced by PRV infection or staurosporine treatment. In addition, we found that the presence of wild type PRV US3, but not of the point-mutated PRV US3, results in phosphorylation of the pro-apoptotic Bad protein in PRV-infected ST and HEp-2 cells. In PRV-infected ST cells, but not in HEp-2 cells, an additional, US3- and phosphorylation-independent alteration of Bad could be observed. These results suggest that the kinase activity of the US3 protein of PRV is crucial to protect cells from apoptotic cell death during infection, at least partly by leading to phosphorylation of the pro-apoptotic Bad protein.

α-Herpesviruses constitute closely related neurotropic viruses, including herpes simplex virus in man and pseudorabies virus (PRV) in pigs. Peripheral sensory neurons, such as trigeminal ganglion (TG) neurons, are predominant target cells for virus spread and lifelong latent infections. We report that in vitro infection of swine TG neurons with the homologous swine α-herpesvirus PRV results in the appearance of numerous synaptophysin-positive synaptic boutons (varicosities) along the axons. Nonneuronal cells that were juxtaposed to these varicosities became preferentially infected with PRV, suggesting that varicosities serve as axonal exit sites for the virus. Viral envelope glycoprotein D (gD) was found to be necessary and sufficient for the induction of varicosities. Inhibition of Cdc42 Rho GTPase and p38 mitogen-activated protein kinase signaling pathways strongly suppressed gD-induced varicosity formation. These data represent a novel aspect of the cell biology of α-herpesvirus infections of sensory neurons, demonstrating that virus attachment/entry is associated with signaling events and neuronal changes that may prepare efficient egress of progeny virus.

Trigeminal ganglion (TG) neurons are important target cells for many alphaherpesviruses and constitute a major site of virus latency and reactivation. Earlier we showed that porcine TG neurons are remarkably more resistant towards (apoptotic) cell death resulting from infection by the swine alphaherpesvirus pseudorabies virus (PRV) compared to a broad range of other primary porcine cell types and that this resistance does not depend on the strongly anti-apoptotic US3 viral protein kinase (Geenen, K., Favoreel, H.W., Nauwynck, H.J., 2005a. Higher resistance of porcine trigeminal ganglion neurons towards pseudorabies virus-induced cell death compared with other porcine cell types in vitro. J. Gen. Virol. 86, 1251-1260). Although other viral anti-apoptotic proteins may be involved in survival of TG neurons during PRV infection, an additional factor may be that TG neurons possess a cell type-dependent capacity to withstand apoptosis compared to other cell types. To investigate this, we treated uninfected porcine TG cultures, swine kidney cells, and porcine superior cervical ganglion (SCG) neurons with several apoptosis-inducing reagents (staurosporine, camptothecin and genistein). None of these reagents were able to trigger substantial apoptotic cell death in TG neurons, whereas non-neuronal TG cells, swine kidney cells, and SCG neurons showed a clear dose-dependent increase in apoptosis using either of these reagents. In conclusion, sensory TG neurons may contain a cell type-specific capacity to withstand different apoptotic assaults, including infection with an alphaherpesvirus.

Trigeminal ganglion (TG) neurons are important target cells for many alphaherpesviruses, constituting major sites for latency/reactivation events. Here, the in vitro kinetics of productive infection of the swine alphaherpesvirus pseudorabies virus (PRV) and resulting cell death in primary porcine TG neurons were determined, and these were compared with similar kinetics in many other porcine cell types. Confocal microscopy showed that all TG neurons expressed late genes such as viral glycoproteins, and that these glycoproteins were processed through the Golgi and reached the cell surface as in other cell types, albeit with a delay of ±2–6 h. However, TG neurons were much more resistant towards PRV-induced cell death compared with all other porcine cell types tested (non-neuronal TG cells, superior cervical ganglion neurons, epithelial kidney cells, arterial endothelial cells, dermal fibroblasts and cells derived from a porcine swine kidney cell line). About half of the TG neurons survived up to 96 h post-inoculation (end of experiment), whereas all other cell types almost completely succumbed within 2 days post-inoculation. In addition, infection with a strongly pro-apoptotic PRV strain that misses the anti-apoptotic US3 protein did not lead to substantial apoptosis in TG neurons, even at 72 h post-inoculation. Thus, primary porcine TG neurons can be infected with PRV in vitro, and are remarkably more resistant to PRV-induced cell death compared with other porcine cell types, suggesting a cell type-specific resistance to alphaherpesvirus-induced cell death that may have important implications for different aspects of the virus life cycle, including latency/reactivation events.