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Pseudorabies virus (PRV) is a porcine alphaherpesvirus that belongs to the Herpesviridae family. We showed earlier that infection of porcine epithelial cells with PRV triggers activation of the nuclear factor kappa B (NF-kappa B) pathway, a pivotal signaling axis in the early immune response. However, PRV-induced NF-kappa B activation does not lead to NF-kappa B-dependent gene expression. Here, using electrophoretic mobility shift assays (EMSAs), we show that PRV does not disrupt the ability of NF-kappa B to interact with its kappa B target sites. Assessing basal cellular transcriptional activity in PRV-infected cells by quantitation of prespliced transcripts of constitutively expressed genes uncovered a broad suppression of cellular transcription by PRV, which also affects the inducible expression of NF-kappa B target genes. Host cell transcription inhibition was rescued when viral genome replication was blocked using phosphonoacetic acid (PAA). Remarkably, we found that host gene expression shutoff in PRV-infected cells correlated with a substantial retention of the NF-kappa B subunit p65, the TATA box binding protein, and RNA polymerase II-essential factors required for (NF-kappa B-dependent) gene transcription-in expanding PRV replication centers in the nucleus and thereby away from the host chromatin. This study reveals a potent mechanism used by the alphaherpesvirus PRV to steer the protein production capacity of infected cells to viral proteins by preventing expression of host genes, including inducible genes involved in mounting antiviral responses. IMPORTANCE 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. We reported earlier that a DNA damage response in epithelial cells infected with the alphaherpesvirus pseudorabies virus (PRV) results in activation of the hallmark proinflammatory NF-kappa B signaling axis but, remarkably, that this activation does not lead to NF-kappa B-induced (proinflammatory) gene expression. Here, we report that PRV-mediated inhibition of host gene expression stretches beyond NF-kappa B-dependent gene expression and in fact reflects a broad inhibition of host gene transcription, which correlates with a substantial recruitment of essential host transcription factors in viral replication compartments in the nucleus, away from the host chromatin. These data uncover a potent alphaherpesvirus mechanism to interfere with production of host proteins, including proteins involved in antiviral responses. 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.

Pseudorabies virus (PRV) is a member of the alphaherpesvirus subfamily and the causative agent of Aujeszky’s disease in pigs. Driven by the large economic losses associated with PRV infection, several vaccines and vaccine programs have been developed. To this day, the attenuated Bartha strain, generated by serial passaging, represents the golden standard for PRV vaccination. However, a proteomic comparison of the Bartha virion to wild-type (WT) PRV virions is lacking. Here, we present a comprehensive mass spectrometry-based proteome comparison of the attenuated Bartha strain and three commonly used WT PRV strains: Becker, Kaplan, and NIA3. We report the detection of 40 structural and 14 presumed nonstructural proteins through a combination of data-dependent and data-independent acquisition. Interstrain comparisons revealed that packaging of the capsid and most envelope proteins is largely comparable in-between all four strains, except for the envelope protein pUL56, which is less abundant in Bartha virions. However, distinct differences were noted for several tegument proteins. Most strikingly, we noted a severely reduced incorporation of the tegument proteins IE180, VP11/12, pUS3, VP22, pUL41, pUS1, and pUL40 in Bartha virions. Moreover, and likely as a consequence, we also observed that Bartha virions are on average smaller and more icosahedral compared to WT virions. Finally, we detected at least 28 host proteins that were previously described in PRV virions and noticed considerable strain-specific differences with regard to host proteins, arguing that the potential role of packaged host proteins in PRV replication and spread should be further explored. IMPORTANCE The pseudorabies virus (PRV) vaccine strain Bartha—an attenuated strain created by serial passaging—represents an exceptional success story in alphaherpesvirus vaccination. Here, we used mass spectrometry to analyze the Bartha virion composition in comparison to three established WT PRV strains. Many viral tegument proteins that are considered nonessential for viral morphogenesis were drastically less abundant in Bartha virions compared to WT virions. Interestingly, many of the proteins that are less incorporated in Bartha participate in immune evasion strategies of alphaherpesviruses. In addition, we observed a reduced size and more icosahedral morphology of the Bartha virions compared to WT PRV. Given that the Bartha vaccine strain elicits potent immune responses, our findings here suggest that differences in protein packaging may contribute to its immunogenicity. Further exploration of these observations could aid the development of efficacious vaccines against other alphaherpesvirus vaccines such as HSV-1/2 or EHV-1.

Both type I and III interferons (IFNs) play a crucial role in host antiviral response by activating the JAK/STAT (Janus kinase/signal transducer and activator of transcription) signaling pathway to trigger the expression of antiviral IFN-stimulated genes (ISGs). We report that the porcine alphaherpesvirus pseudorabies virus (PRV) triggers proteasomal degradation of the key Janus kinases Jak1 and to a lesser extent Tyk2, thereby inhibiting both type I and III IFN-induced STAT1 phosphorylation and suppressing IFN-induced expression of ISGs. UV-inactivated PRV did not interfere with IFN signaling. In addition, deletion of the EP0 gene from the PRV genome or inhibition of viral genome replication did not affect PRV-induced inhibition of IFN signaling. To our knowledge, this is the first report describing Janus kinase degradation by alphaherpesviruses. These findings thus reveal a novel alphaherpesvirus evasion mechanism of type I and type III IFNs. IMPORTANCE Type I and III interferons (IFNs) trigger signaling via Janus kinases that phosphorylate and activate signal transducer and activator of transcription (STAT) transcription factors, leading to the expression of antiviral interferon-stimulated genes (ISGs) that result in an antiviral state of host cells. Viruses have evolved various mechanisms to evade this response. Our results indicate that an alphaherpesvirus, the porcine pseudorabies virus (PRV), inhibits both type I and III IFN signaling pathways by triggering proteasome-dependent degradation of the key Janus kinases Jak1 and Tyk2 and consequent inhibition of STAT1 phosphorylation and suppression of ISG expression. Moreover, we found that this inhibition is not caused by incoming virions and does not depend on expression of the viral EP0 protein or viral true late proteins. These data for the first time address alphaherpesvirus evasion of type III IFN-mediated signaling and reveal a previously uncharacterized alphaherpesvirus mechanism of IFN evasion via proteasomal degradation of Janus kinases.

Corneal graft rejection is a major problem in chronic herpetic keratitis (HK) patients with latent infection. A new class of antiviral agents targeting latent and active forms of herpes simplex virus type 1 (HSV-1) is importantly required. Meganucleases are sequence-specific homing endonucleases capable of inducing DNA double-strand breaks. A proof-of-concept experiment has shown that tailor-made meganucleases are efficient against HSV-1 in vitro. To take this work a step forward, we hypothesized that the pre-treatment of human corneas in eye banks using meganuclease-encoding vectors will allow HK patients to receive a medicated cornea to resist the recurrence of the infection and the common graft rejection problem. However, this strategy requires efficient gene delivery to human corneal endothelium. Using recombinant adeno-associated virus, serotype 2/1 (rAAV2/1), efficient gene delivery of a reporter gene was demonstrated in human corneas ex vivo. The optimum viral dose was 3.7 × 10(11) VG with an exposure time of 1 day, followed by 6 days incubation in de-swelling medium. In addition, 12 days incubation can result in transgene expression in excess of 70%. Using similar transduction conditions, meganuclease transgene expression was detected in 39.4% of the endothelial cells after 2 weeks in culture. Reduction of the total viral load in the media and the endothelial cells of corneas infected with HSV-1 was shown. Collectively, this work provides information about the optimum conditions to deliver genetic material to the cornea, and demonstrates for the first time the expression of meganuclease in human corneas ex vivo and its antiviral activity. In conclusion, we demonstrate that the treatment of human corneas in eye banks before transplantation is a new approach to address the unmet clinical needs in corneal diseases.