Tunneling nanotubes (TNTs) are long bridge-like structures that connect eukaryotic cells and mediate intercellular communication. We found earlier that the conserved alphaherpesvirus US3 protein kinase induces long cell projections that contact distant cells and promote intercellular virus spread. In this report, we show that the US3-induced cell projections constitute TNTs. In addition, we report that US3-induced TNTs mediate intercellular transport of information (i.e. GFP) in the absence of other viral proteins. US3-induced TNTs are remarkably stable compared to most TNTs described in literature. In line with this, US3-induced TNTs were found to contain stabilized (acetylated and detyrosinated) microtubules. Transmission electron microscopy showed that virus particles are individually transported in membrane-bound vesicles in US3-induced TNTs and are released along the TNT and at the contact area between TNT and adjacent cell. Contact between US3-induced TNTs and acceptor cells is very stable, which correlated with a marked enrichment in adherens junction components beta-catenin and E-cadherin at the contact area. These data provide new structural insights in US3-induced TNTs and how they may contribute to intercellular communication and alphaherpesvirus spread. Tunneling nanotubes (TNT) represent an important, yet still poorly understood mode of long distance intercellular communication. We and others reported earlier that the conserved alphaherpesvirus US3 protein kinase induces long cellular protrusions in infected and transfected cells. Here, we show that US3-induced cell projections constitute TNTs, based on structural properties and transport of biomolecules. In addition, we report on different particular characteristics of US3-induced TNTs that aid to explain their remarkable stability compared to physiological TNTs. In addition, transmission electron microscopy assays indicate that, in infected cells, virions travel in the US3-induced TNTs in membranous transport vesicles, and leave the TNT via exocytosis. These data generate new fundamental insights in the biology of (US3-induced) TNTs, and how they may contribute to intercellular virus spread and communication.

Plasmacytoid dendritic cells (pDC) play a central role in the antiviral immune response, both in the innate response and in shaping the adaptive response, mainly because of their ability to produce massive amounts of type I interferon (TI-IFN). Here, we report that cells infected with the live attenuated Bartha vaccine strain of porcine alphaherpesvirus pseudorabies virus (PRV) trigger a dramatically increased TI-IFN response by porcine primary pDC compared to cells infected with wild-type PRV strains (Becker and Kaplan). Since Bartha is one of the relatively few examples of a highly successful alphaherpesvirus vaccine, identification of factors that may contribute to its efficacy may provide insights for the rational design of other alphaherpesvirus vaccines. The Bartha vaccine genome displays several mutations compared to the genome of wild-type PRV strains, including a large deletion in the unique short (US) region, encompassing the glycoprotein E (gE), gI, US9, and US2 genes. Using recombinant PRV Becker strains harboring the entire Bartha US deletion or single mutations in the four affected US genes, we demonstrate that the absence of the viral gE/gI complex contributes to the observed increased IFN-␣ response. Furthermore, we show that the absence of gE leads to an enhanced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in pDC, which correlates with a higher TI-IFN production by pDC. In conclusion, the PRV Bartha vaccine strain triggers strongly increased TI-IFN production by porcine pDC. Our data further indicate that the gE/gI glycoprotein complex suppresses TI-IFN production by pDC, which represents the first alphaherpesvirus factor that suppresses pDC activity.IMPORTANCE Several alphaherpesviruses, including herpes simpex virus, still lack effective vaccines. However, the highly successful Bartha vaccine has contributed substantially to eradication of the porcine alphaherpesvirus pseudorabies virus (PRV) in several countries. The impact of Bartha on the immune response is still poorly understood. Type I interferon (TI-IFN)-producing plasmacytoid dendritic cells (pDC) may play an important role in vaccine development. Here, we show that Bartha elicits a dramatically increased type I interferon (TI-IFN) response in primary porcine pDC compared to wild-type strains. In addition, we found that the gE/gI complex, which is absent in Bartha, inhibits the pDC TI-IFN response. This is the first description of an immune cell type that is differentially affected by Bartha versus wild-type PRV and is the first report describing an alphaherpesvirus protein that inhibits the TI-IFN response by pDC. These data may therefore contribute to the rational design of other alphaherpesvirus vaccines.KEYWORDS Bartha, ERK1/2, gE, herpes, immune evasion, interferon, pDC, plasmacytoid dendritic cell, pseudorabies

The nuclear factor kappa B (NF-κB) is a potent transcription factor, activation of which typically results in robust proinflammatory signaling and triggering of fast negative feedback modulators to avoid excessive inflammatory responses. Here, we report that infection of epithelial cells, including primary porcine respiratory epithelial cells, with the porcine alphaherpesvirus pseudorabies virus (PRV) results in the gradual and persistent activation of NF-κB, illustrated by proteasome-dependent degradation of the inhibitory NF-κB regulator IκB and nuclear translocation and phosphorylation of the NF-κB subunit p65. PRV-induced persistent activation of NF-κB does not result in expression of negative feedback loop genes, like the gene for IκBα or A20, and does not trigger expression of prototypical proinflammatory genes, like the gene for tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6). In addition, PRV infection inhibits TNF-α-induced canonical NF-κB activation. Hence, PRV infection triggers persistent NF-κB activation in an unorthodox way and dramatically modulates the NF-κB signaling axis, preventing typical proinflammatory gene expression and the responsiveness of cells to canonical NF-κB signaling, which may aid the virus in modulating early proinflammatory responses in the infected host. IMPORTANCE The NF-κB transcription factor is activated via different key inflammatory pathways and typically results in the fast expression of several proinflammatory genes as well as negative feedback loop genes to prevent excessive inflammation. In the current report, we describe that infection of cells with the porcine alphaherpesvirus pseudorabies virus (PRV) triggers a gradual and persistent aberrant activation of NF-κB, which does not result in expression of hallmark proinflammatory or negative feedback loop genes. In addition, although PRV-induced NF-κB activation shares some mechanistic features with canonical NF-κB activation, it also shows remarkable differences; e.g., it is largely independent of the canonical IκB kinase (IKK) and even renders infected cells resistant to canonical NF-κB activation by the inflammatory cytokine TNF-α. Aberrant PRV-induced NF-κB activation may therefore paradoxically serve as a viral immune evasion strategy and may represent an important tool to unravel currently unknown mechanisms and consequences of NF-κB activation.

The conserved alphaherpesvirus serine/threonine kinase US3 causes dramatic changes in the actin cytoskeleton, consisting of actin stress fibre breakdown and protrusion formation, associated with increased virus spread. Here, we showed that US3 expression led to RhoA phosphorylation at serine 188 (S188), one of the hallmarks of suppressed RhoA signalling, and that expression of a non-phosphorylatable RhoA variant interfered with the ability of US3 to induce actin rearrangements. Furthermore, inhibition of cellular protein kinase A (PKA) eliminated the ability of US3 to induce S188 RhoA phosphorylation, pointing to a role for PKA in US3-induced RhoA phosphorylation. Hence, the US3 kinase leads to PKA-dependent S188 RhoA phosphorylation, which contributes to US3-mediated actin rearrangements. Our data suggest that US3 efficiently usurps the antagonistic RhoA and Cdc42/Rac1/p21-activated kinase signalling branches to rearrange the actin cytoskeleton. The Alphaherpesvirinae represent the largest subfamily of the family Herpesviridae, comprising closely related pathogens of humans and animals. These include herpes simplex virus 1/2 (HSV-1/2; human herpesvirus 1/2) and varicellazoster virus (VZV; human herpesvirus 1) in humans.Pseudorabies virus (PRV) is a porcine alphaherpesvirus and is often used to study general aspects of alphaherpesvirus biology (Pomeranz et al., 2005). Infection with PRV can lead to dramatic changes in the actin cytoskeleton of the host cell, consisting of actin stress fibre breakdown and protrusion formation (Favoreel et al., 2005). These actin rearrangements are associated with increased viral cell-to-cell spread and depend on the kinase activity of the viral serine/threonine kinase US3 (Favoreel et al., 2005;Van den Broeke et al., 2009a). US3 is conserved within the alphaherpesviruses and comparable US3-induced cytoskeletal changes have been described for several other alphaherpesviruses, including HSV-2 and bovine herpesvirus 1/5 (BHV-1/5) (Brzozowska et al., 2010;Finnen et al., 2010;Ladelfa et al., 2011).

Herpesviruses are highly successful pathogens that cause lifelong persistent infections of their host. Modulation of the intracellular environment of infected cells is imperative for the success of virus infections.