Pseudorabies virus inhibits progesterone-induced inactivation of TRPML1 to facilitate viral entry.

Viral infection is a significant risk factor for fertility issues. Here, we demonstrated that infection by neurotropic alphaherpesviruses, such as pseudorabies virus (PRV), could impair female fertility by disrupting the hypothalamus-pituitary-ovary axis (HPOA), reducing progesterone (P4) levels, and consequently lowering pregnancy rates. Our study revealed that PRV exploited the transient receptor potential mucolipin 1 (TRPML1) and its lipid activator, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), to facilitate viral entry through lysosomal cholesterol and Ca2+. P4 antagonized this process by inducing lysosomal storage disorders and promoting the proteasomal degradation of TRPML1 via murine double minute 2 (MDM2)-mediated polyubiquitination. Overall, the study identifies a novel mechanism by which PRV hijacks the lysosomal pathway to evade P4-mediated antiviral defense and impair female fertility. This mechanism may be common among alphaherpesviruses and could contribute significantly to their impact on female reproductive health, providing new insights for the development of antiviral therapies.

Pseudorabies virus upregulates low-density lipoprotein receptors to facilitate viral entry.

Pseudorabies virus (PRV) is the causative agent of Aujeszky's disease in pigs. The low-density lipoprotein receptor (LDLR) is a transcriptional target of the sterol-regulatory element-binding proteins (SREBPs) and participates in the uptake of LDL-derived cholesterol. However, the involvement of LDLR in PRV infection has not been well characterized. We observed an increased expression level of LDLR mRNA in PRV-infected 3D4/21, PK-15, HeLa, RAW264.7, and L929 cells. The LDLR protein level was also upregulated by PRV infection in PK-15 cells and in murine lung and brain. The treatment of cells with the SREBP inhibitor, fatostatin, or with SREBP2-specific small interfering RNA prevented the PRV-induced upregulation of LDLR expression as well as viral protein expression and progeny virus production. This suggested that PRV activated SREBPs to induce LDLR expression. Furthermore, interference in LDLR expression affected PRV proliferation, while LDLR overexpression promoted it. This indicated that LDLR was involved in PRV infection. The study also demonstrated that LDLR participated in PRV invasions. The overexpression of LDLR or inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which binds to LDLR and targets it for lysosomal degradation, significantly enhanced PRV attachment and entry. Mechanistically, LDLR interacted with PRV on the plasma membrane, and pretreatment of cells with LDLR antibodies was able to neutralize viral entry. An in vivo study indicated that the treatment of mice with the PCSK9 inhibitor SBC-115076 promoted PRV proliferation. The data from the study indicate that PRV hijacks LDLR for viral entry through the activation of SREBPs.IMPORTANCEPseudorabies virus (PRV) is a herpesvirus that primarily manifests as fever, pruritus, and encephalomyelitis in various domestic and wild animals. Owing to its lifelong latent infection characteristics, PRV outbreaks have led to significant financial setbacks in the global pig industry. There is evidence that PRV variant strains can infect humans, thereby crossing the species barrier. Therefore, gaining deeper insights into PRV pathogenesis and developing updated strategies to contain its spread are critical. This study posits that the low-density lipoprotein receptor (LDLR) could be a co-receptor for PRV infection. Hence, strategies targeting LDLR may provide a promising avenue for the development of effective PRV vaccines and therapeutic interventions.

Neuronal expression of herpes simplex virus-1 VP16 protein induces pseudorabies virus escape from silencing and reactivation.

Alpha herpesvirus (α-HV) particles enter their hosts from mucosal surfaces and efficiently maintain fast transport in peripheral nervous system (PNS) axons to establish infections in the peripheral ganglia. The path from axons to distant neuronal nuclei is challenging to dissect due to the difficulty of monitoring early events in a dispersed neuron culture model. We have established well-controlled, reproducible, and reactivateable latent infections in compartmented rodent neurons by infecting physically isolated axons with a small number of viral particles. This system not only recapitulates the physiological infection route but also facilitates independent treatment of isolated cell bodies or axons. Consequently, this system enables study not only of the stimuli that promote reactivation but also the factors that regulate the initial switch from productive to latent infection. Adeno-associated virus (AAV)-mediated expression of herpes simplex-1 (HSV-1) VP16 alone in neuronal cell bodies enabled the escape from silencing of incoming pseudorabies virus (PRV) genomes. Furthermore, the expression of HSV VP16 alone reactivated a latent PRV infection in this system. Surprisingly, the expression of PRV VP16 protein supported neither PRV escape from silencing nor reactivation. We compared transcription transactivation activity of both VP16 proteins in primary neurons by RNA sequencing and found that these homolog viral proteins produce different gene expression profiles. AAV-transduced HSV VP16 specifically induced the expression of proto-oncogenes including c-Jun and Pim2. In addition, HSV VP16 induces phosphorylation of c-Jun in neurons, and when this activity is inhibited, escape of PRV silencing is dramatically reduced.IMPORTANCEDuring latency, alpha herpesvirus genomes are silenced yet retain the capacity to reactivate. Currently, host and viral protein interactions that determine the establishment of latency, induce escape from genome silencing or reactivation are not completely understood. By using a compartmented neuronal culture model of latency, we investigated the effect of the viral transcriptional activator, VP16 on pseudorabies virus (PRV) escape from genome silencing. This model recapitulates the physiological infection route and enables the study of the stimuli that regulate the initial switch from a latent to productive infection. We investigated the neuronal transcriptional activation profiles of two homolog VP16 proteins (encoded by HSV-1 or PRV) and found distinct gene activation signatures leading to diverse infection outcomes. This study contributes to understanding of how alpha herpesvirus proteins modulate neuronal gene expression leading to the initiation of a productive or a latent infection.

TMEM41B Is an Interferon-Stimulated Gene That Promotes Pseudorabies Virus Replication.

Pseudorabies virus (PRV) is a double-stranded DNA virus that causes Aujeszky's disease and is responsible for economic loss worldwide. Transmembrane protein 41B (TMEM41B) is a novel endoplasmic reticulum (ER)-localized regulator of autophagosome biogenesis and lipid mobilization; however, the role of TMEM41B in regulating PRV replication remains undocumented. In this study, PRV infection was found to upregulate TMEM41B mRNA and protein levels both in vitro and in vivo. For the first time, we found that TMEM41B could be induced by interferon (IFN), suggesting that TMEM41B is an IFN-stimulated gene (ISG). While TMEM41B knockdown suppressed PRV proliferation, TMEM41B overexpression promoted PRV proliferation. We next studied the specific stages of the virus life cycle and found that TMEM41B knockdown affected PRV entry. Mechanistically, we demonstrated that the knockdown of TMEM41B blocked PRV-stimulated expression of the key enzymes involved in lipid synthesis. Additionally, TMEM41B knockdown played a role in the dynamics of lipid-regulated PRV entry-dependent clathrin-coated pits (CCPs). Lipid replenishment restored the CCP dynamic and PRV entry in TMEM41B knockdown cells. Together, our results indicate that TMEM41B plays a role in PRV infection via regulating lipid homeostasis. IMPORTANCE PRV belongs to the alphaherpesvirus subfamily and can establish and maintain a lifelong latent infection in pigs. As such, an intermittent active cycle presents great challenges to the prevention and control of PRV disease and is responsible for serious economic losses to the pig breeding industry. Studies have shown that lipids play a crucial role in PRV proliferation. Thus, the manipulation of lipid metabolism may represent a new perspective for the prevention and treatment of PRV. In this study, we report that the ER transmembrane protein TMEM41B is a novel ISG involved in PRV infection by regulating lipid synthesis. Therefore, our findings indicate that targeting TMEM41B may be a promising approach for the development of PRV vaccines and therapeutics.

Pseudorabies virus UL38 attenuates the cGAS-STING signaling pathway by recruiting Tollip to promote STING for autophagy degradation.

Natural immunity is the first defense line of the host immune system, which plays a significant role in combating foreign pathogenic microorganisms. The IFN-ß (interferon-beta) signaling pathway, being a typical example of innate immunity, plays a vital function. This study aimed to elucidate the function of pseudorabies virus (PRV) UL38 protein (unique long region 38) in suppressing the activation of the IFN-ß signaling pathway. The findings from our study indicate that the PRV UL38 protein effectively hampers the activation of IFN-ß by poly (dA: dT) (poly(deoxyadenylic-deoxythymidylic)) and 2'3'-cGAMP (2'-3'-cyclic GMP-AMP). Furthermore, UL38 exhibits spatial co-localization with STING (stimulator of interferon genes) and effectively hinders STING dimerization. Subsequently, STING was downgraded to suppress the production of IFN-ß and ISGs (interferon stimulated genes). Immunoprecipitation analysis revealed that the interaction between UL38 and STING, which subsequently initiated the degradation of STING via selective autophagy mediated by TOLLIP (toll interacting protein). To summarize, this research elucidates the function of UL38 in counteracting the cGAS (cGAMP synthase)-STING-induced IFN-ß pathway. The PRV UL38 protein may attenuate the activation of IFN-ß as a means of regulating the virus's persistence in the host.

Establishment of pseudorabies virus latency and reactivation model in mice dorsal root ganglia culture.

Pseudorabies virus (PRV) belongs to the alpha herpesvirus family and is responsible for Aujeszky's disease in pigs. Similar to other alpha herpesviruses, PRV establishes a lifelong latent infection in trigeminal ganglion. These latently infected pigs serve as a reservoir for recurrent infections when reactivation is triggered, making the eradication of PRV a challenging task. However, the molecular mechanism underlying PRV latency and reactivation in neurons is still poorly understood due to limitations in the in vitro model. To establish a pseudorabies virus latency and reactivation model in primary neuron cultures, we isolated dorsal root ganglion (DRG) from newborn Kunming mice using a method named epineurium-pulling for DRG collection (EPDC) and cultured primary neurons in vitro. A dual-colour recombinant PRV BAC mRuby-VP16 was constructed and 0.5 multiplicity of infection (MOI) was found as an appropriate dose in the presence of aciclovir to establish latency. Reactivation was induced using UV-inactivated herpesviruses or a series of chemical inhibitors. Interestingly, we found that not only UV-PRV, but also UV-HSV-1 and UV-BHoV-5 were able to induce rapid PRV reactivation. The efficiency of reactivation for LY294002, forskolin, etoposide, dexamethasone, and acetylcholine was found to be dependent on their concentration. In conclusion, we developed a valuable model of PRV latency and reactivation, which provides a basis for future mechanism research.

A Pseudorabies Virus Serine/Threonine Kinase, US3, Promotes Retrograde Transport in Axons via Akt/mToRC1.

Infection of peripheral axons by alpha herpesviruses (AHVs) is a critical stage in establishing a lifelong infection in the host. Upon entering the cytoplasm of axons, AHV nucleocapsids and associated inner-tegument proteins must engage the cellular retrograde transport machinery to promote the long-distance movement of virion components to the nucleus. The current model outlining this process is incomplete, and further investigation is required to discover all viral and cellular determinants involved as well as the temporality of the events. Using a modified trichamber system, we have discovered a novel role of the pseudorabies virus (PRV) serine/threonine kinase US3 in promoting efficient retrograde transport of nucleocapsids. We discovered that transporting nucleocapsids move at similar velocities in both the presence and absence of a functional US3 kinase; however, fewer nucleocapsids are moving when US3 is absent, and they move for shorter periods of time before stopping, suggesting that US3 is required for efficient nucleocapsid engagement with the retrograde transport machinery. This led to fewer nucleocapsids reaching the cell bodies to produce a productive infection 12 h later. Furthermore, US3 was responsible for the induction of local translation in axons as early as 1 h postinfection (hpi) through the stimulation of a phosphatidylinositol 3-kinase (PI3K)/Akt-mToRC1 pathway. These data describe a novel role for US3 in the induction of local translation in axons during AHV infection, a critical step in transport of nucleocapsids to the cell body. IMPORTANCE Neurons are highly polarized cells with axons that can reach centimeters in length. Communication between axons at the periphery and the distant cell body is a relatively slow process involving the active transport of chemical messengers. There is a need for axons to respond rapidly to extracellular stimuli. Translation of repressed mRNAs present within the axon occurs to enable rapid, localized responses independently of the cell body. AHVs have evolved a way to hijack local translation in the axons to promote their transport to the nucleus. We have determined the cellular mechanism and viral components involved in the induction of axonal translation. The US3 serine/threonine kinase of PRV activates Akt-mToRC1 signaling pathways early during infection to promote axonal translation. When US3 is not present, the number of moving nucleocapsids and their processivity are reduced, suggesting that US3 activity is required for efficient engagement of nucleocapsids with the retrograde transport machinery.

An ESCRT/VPS4 Envelopment Trap To Examine the Mechanism of Alphaherpesvirus Assembly and Transport in Neurons.

The assembly and egress of alphaherpesviruses, including herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), within neurons is poorly understood. A key unresolved question is the structure of the viral particle that moves by anterograde transport along the axon, and two alternative mechanisms have been described. In the "married" model, capsids acquire their envelopes in the cell body and then traffic along axons as enveloped virions within a bounding organelle. In the "separate" model, nonenveloped capsids travel from the cell body into and along the axon, eventually encountering their envelopment organelles at a distal site, such as the nerve cell terminal. Here, we describe an "envelopment trap" to test these models using the dominant negative terminal endosomal sorting complex required for transport (ESCRT) component VPS4-EQ. Green fluorescent protein (GFP)-tagged VPS4-EQ was used to arrest HSV-1 or PRV capsid envelopment, inhibit downstream trafficking, and GFP-label envelopment intermediates. We found that GFP-VPS4-EQ inhibited trafficking of HSV-1 capsids into and along the neurites and axons of mouse CAD cells and rat embryonic primary cortical neurons, consistent with egress via the married pathway. In contrast, transport of HSV-1 capsids was unaffected in the neurites of human SK-N-SH neuroblastoma cells, consistent with the separate mechanism. Unexpectedly, PRV (generally thought to utilize the married pathway) also appeared to employ the separate mechanism in SK-N-SH cells. We propose that apparent differences in the methods of HSV-1 and PRV egress are more likely a reflection of the host neuron in which transport is studied rather than true biological differences between the viruses themselves. IMPORTANCE Alphaherpesviruses, including herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), are pathogens of the nervous system. They replicate in the nerve cell body and then travel great distances along axons to reach nerve termini and spread to adjacent epithelial cells; however, key aspects of how these viruses travel along axons remain controversial. Here, we test two alternative mechanisms for transport, the married and separate models, by blocking envelope assembly, a critical step in viral egress. When we arrest formation of the viral envelope using a mutated component of the cellular ESCRT apparatus, we find that entry of viral particles into axons is blocked in some types of neurons but not others. This approach allows us to determine whether envelope assembly occurs prior to entry of viruses into axons or afterwards and, thus, to distinguish between the alternative models for viral transport.

Pseudorabies Virus Infection Results in a Broad Inhibition of Host Gene Transcription.

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 κB (NF-κB) pathway, a pivotal signaling axis in the early immune response. However, PRV-induced NF-κB activation does not lead to NF-κB-dependent gene expression. Here, using electrophoretic mobility shift assays (EMSAs), we show that PRV does not disrupt the ability of NF-κB to interact with its κB 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-κB 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-κB subunit p65, the TATA box binding protein, and RNA polymerase II-essential factors required for (NF-κB-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-κB signaling axis but, remarkably, that this activation does not lead to NF-κB-induced (proinflammatory) gene expression. Here, we report that PRV-mediated inhibition of host gene expression stretches beyond NF-κB-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.

Alphaherpesvirus-induced activation of plasmacytoid dendritic cells depends on the viral glycoprotein gD and is inhibited by non-infectious light particles.

Plasmacytoid dendritic cells (pDC) are important innate immune cells during the onset of viral infections as they are specialized in the production of massive amounts of antiviral type I interferon (IFN). Alphaherpesviruses such as herpes simplex virus (HSV) or pseudorabies virus (PRV) are double stranded DNA viruses and potent stimulators of pDC. Detailed information on how PRV activates porcine pDC is lacking. Using PRV and porcine primary pDC, we report here that PRV virions, so-called heavy (H-)particles, trigger IFNα production by pDC, whereas light (L-) particles that lack viral DNA and capsid do not. Activation of pDC requires endosomal acidification and, importantly, depends on the PRV gD envelope glycoprotein and O-glycosylations. Intriguingly, both for PRV and HSV-1, we found that L-particles suppress H-particle-mediated activation of pDC, a process which again depends on viral gD. This is the first report describing that gD plays a critical role in alphaherpesvirus-induced pDC activation and that L-particles directly interfere with alphaherpesvirus-induced IFNα production by pDC.

Pseudorabies virus kinase UL13 phosphorylates H2AX to foster viral replication.

The DNA damage response (DDR) pathway is critical for maintaining genomic integrity and sustaining organismal development. Viruses can either utilize or circumvent the DDR to facilitate their replication. Pseudorabies virus (PRV) infection was shown to induce apoptosis via stimulating DDR. However, the underlying mechanisms have not been fully explored to date. This study showed that PRV infection robustly activates the ATM and DNA-PK signaling pathways shortly after infection. However, inhibition of ATM, but not DNA-PK, could dampen PRV replication in cells. Importantly, we found that PRV-encoded serine/threonine kinase UL13 interacts with and subsequently phosphorylates H2AX. Furthermore, we found that UL13 deletion largely attenuates PRV neuroinvasiveness and virulence in vivo. In addtion, we showed that UL13 contributes to H2AX phosphorylation upon PRV infection both in vitro and in vivo, but does not affect ATM phosphorylation. Finally, we showed that knockdown of H2AX reduces PRV replication, while this reduction can be further enhanced by deletion of UL13. Taken together, we conclude that PRV-encoded kinase UL13 regulates DNA damage marker γH2AX and UL13-mediated H2AX phosphorylation plays a pivotal role in efficient PRV replication and progeny production.

RhoA suppresses pseudorabies virus replication in vitro.

The porcine pseudorabies virus (PRV) is one of the most devastating pathogens and brings great economic losses to the swine industry worldwide. Viruses are intracellular parasites that have evolved numerous strategies to subvert and utilize different host processes for their life cycle. Among the different systems of the host cell, the cytoskeleton is one of the most important which not only facilitate viral invasion and spread into neighboring cells, but also help viruses to evade the host immune system. RhoA is a key regulator of cytoskeleton system that may participate in virus infection. In this study, we characterized the function of RhoA in the PRV replication by chemical drugs treatment, gene knockdown and gene over-expression strategy. Inhibition of RhoA by specific inhibitor and gene knockdown promoted PRV proliferation. On the contrary, overexpression of RhoA or activation of RhoA by chemical drug inhibited PRV infection. Besides, our data demonstrated that PRV infection induced the disruption of actin stress fiber, which was consistent with previous report. In turn, the actin specific inhibitor cytochalasin D markedly disrupted the normal fibrous structure of intracellular actin cytoskeleton and decreased the PRV replication, suggesting that actin cytoskeleton polymerization contributed to PRV replication in vitro. In summary, our data displayed that RhoA was a host restriction factor that inhibited PRV replication, which may deepen our understanding the pathogenesis of PRV and provide further insight into the prevention of PRV infection and the development of anti-viral drugs.

Guanylate-binding protein 1 inhibits nuclear delivery of pseudorabies virus by disrupting structure of actin filaments.

The alphaherpesvirus pseudorabies virus (PRV) is the causative agent of pseudorabies, responsible for severe economic losses to the swine industry worldwide. The interferon-inducible GTPase guanylate-binding protein 1 (GBP1) exhibits antiviral immunity. Our findings show that there is a robust upregulation in the expression of porcine GBP1 during PRV infection. GBP1 knockout promotes PRV infection, while GBP1 overexpression restricts it. Importantly, we found that GBP1 impeded the normal structure of actin filaments in a GTPase-dependent manner, preventing PRV virions from reaching the nucleus. We also discovered that viral US3 protein bound GBP1 to interfere with its GTPase activity. Finally, the interaction between US3 and GBP1 requires US3 serine/threonine kinase activity sites and the GTPase domain (aa 1 to 308) of GBP1. Taken together, this study offers fresh perspectives on how PRV manipulates the host's antiviral immune system.

Peroxiredoxin 1 Interacts with TBK1/IKKε and Negatively Regulates Pseudorabies Virus Propagation by Promoting Innate Immunity.

Peroxiredoxin 1 (PRDX1) is a cellular antioxidant enzyme that is crucial for diverse fundamental biological processes, such as autophagy, inflammation, and carcinogenesis. However, molecular mechanisms underpinning its diverse roles are not well understood. Here, we report that PRDX1 positively regulates interferon (IFN) induction and that pseudorabies virus (PRV) targets PRDX1 to evade IFN induction. PRV UL13 encodes a serine/threonine kinase important for PRV infection, although its biological function remains obscure. We identified PRDX1 as a UL13-interacting protein. Virological and biochemical assays demonstrate that PRDX1 promotes IFN induction by interacting with TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε). Conversely, UL13 accelerates PRDX1 degradation via the ubiquitin-proteosome pathway in a kinase-dependent manner. In doing so, PRV inhibits IFN induction during productive infection, which requires PRDX1 expression. This study uncovers an essential role of PRDX1 in the innate immune response and reveals a new viral immune evasion strategy to counteract cellular defenses. IMPORTANCE PRV interacts with numerous cellular proteins during productive infection. Here, we demonstrated the interaction of viral protein UL13 with the antioxidant enzyme PRDX1, which functions in multiple signal transduction pathways. We found that PRDX1 participates in the type I IFN pathway by interacting with TBK1 and IKKε, thereby negatively regulating PRV propagation. However, UL13 ubiquitinates PRDX1, which routes PRDX1 into proteasomes for degradation and effectively reduces its expression. These results illuminate the fundamental role that PRDX1 plays in the IFN pathway, and they identify a potential target for the control of PRV infection.

CRISPR/Cas9-Constructed Pseudorabies Virus Mutants Reveal the Importance of UL13 in Alphaherpesvirus Escape from Genome Silencing.

Latent and recurrent productive infection of long-living cells, such as neurons, enables alphaherpesviruses to persist in their host populations. Still, the viral factors involved in these events remain largely obscure. Using a complementation assay in compartmented primary peripheral nervous system (PNS) neuronal cultures, we previously reported that productive replication of axonally delivered genomes is facilitated by pseudorabies virus (PRV) tegument proteins. Here, we sought to unravel the role of tegument protein UL13 in this escape from silencing. We first constructed four new PRV mutants in the virulent Becker strain using CRISPR/Cas9-mediated gene replacement: (i) PRV Becker defective for UL13 expression (PRV ΔUL13), (ii) PRV where UL13 is fused to eGFP (PRV UL13-eGFP), and two control viruses (iii and iv) PRV where VP16 is fused with mTurquoise at either the N terminus (PRV mTurq-VP16) or the C terminus (PRV VP16-mTurq). Live-cell imaging of PRV capsids showed efficient retrograde transport after axonal infection with PRV UL13-eGFP, although we did not detect dual-color particles. However, immunofluorescence staining of particles in mid-axons indicated that UL13 might be cotransported with PRV capsids in PNS axons. Superinfecting nerve cell bodies with UV-inactivated PRV ΔUL13 failed to efficiently promote escape from genome silencing compared to UV-PRV wild type and UV-PRV UL13-eGFP superinfection. However, UL13 does not act directly in the escape from genome silencing, as adeno-associated virus (AAV)-mediated UL13 expression in neuronal cell bodies was not sufficient to provoke escape from genome silencing. Based on this, we suggest that UL13 may contribute to initiation of productive infection through phosphorylation of other tegument proteins.IMPORTANCE Alphaherpesviruses have mastered various strategies to persist in an immunocompetent host, including the induction of latency and reactivation in peripheral nervous system (PNS) ganglia. We recently discovered that the molecular mechanism underlying escape from latency by the alphaherpesvirus pseudorabies virus (PRV) relies on a structural viral tegument protein. This study aimed at unravelling the role of tegument protein UL13 in PRV escape from latency. First, we confirmed the use of CRISPR/Cas9-mediated gene replacement as a versatile tool to modify the PRV genome. Next, we used our new set of viral mutants and AAV vectors to conclude the indirect role of UL13 in PRV escape from latency in primary neurons, along with its spatial localization during retrograde capsid transport in axons. Based on these findings, we speculate that UL13 phosphorylates one or more tegument proteins, thereby priming these putative proteins to induce escape from genome silencing.

Inhibition of PARP1 Dampens Pseudorabies Virus Infection through DNA Damage-Induced Antiviral Innate Immunity.

Pseudorabies virus (PRV) is the causative pathogen of Aujeszky's disease in pigs. Although vaccination is currently applied to prevent the morbidity of PRV infection, new applications are urgently needed to control this infectious disease. Poly(ADP-ribose) polymerase 1 (PARP1) functions in DNA damage repair. We report here that pharmacological and genetic inhibition of PARP1 significantly influenced PRV replication. Moreover, we demonstrate that inhibition of PARP1 induced DNA damage response and antiviral innate immunity. Mechanistically, PARP1 inhibition-induced DNA damage response resulted in the release of double-stranded DNA (dsDNA) into the cytosol, where dsDNA interacted with cyclic GMP-AMP (cGAMP) synthase (cGAS). cGAS subsequently catalyzed cGAMP production to activate the STING/TBK1/IRF3 innate immune signaling pathway. Furthermore, challenge of mice with PARP1 inhibitor stimulated antiviral innate immunity and protected mice from PRV infection in vivo. Our results demonstrate that PARP1 inhibitors may be used as a new strategy to prevent Aujeszky's disease in pigs. IMPORTANCE Aujeszky's disease is a notifiable infectious disease of pigs and causes economic losses worldwide in the pig industry. The causative pathogen is PRV, which is a member of the subfamily Alphaherpesvirinae of the family Herpesviridae. PRV has a wide range of hosts, such as ruminants, carnivores, and rodents. More seriously, recent reports suggest that PRV can cause human endophthalmitis and encephalitis, which indicates that PRV may be a potential zoonotic pathogen. Although vaccination is currently the major strategy used to control the disease, new applications are also urgently needed for the pig industry and public health. We report here that inhibition of PARP1 induces DNA damage-induced antiviral innate immunity through the cGAS-STING signaling pathway. Therefore, PARP1 is a therapeutic target for PRV infection as well as alphaherpesvirus infection.

Coinfection of Porcine Circovirus 2 and Pseudorabies Virus Enhances Immunosuppression and Inflammation through NF-κB, JAK/STAT, MAPK, and NLRP3 Pathways.

Porcine circovirus 2 (PCV2) and pseudorabies virus (PRV) are economically important pathogens in swine. PCV2 and PRV coinfection can cause more severe neurological and respiratory symptoms and higher mortality of piglets. However, the exact mechanism involved in the coinfection of PRV and PCV2 and its pathogenesis remain unknown. Here, porcine kidney cells (PK-15) were infected with PCV2 and/or PRV, and then the activation of immune and inflammatory pathways was evaluated to clarify the influence of the coinfection on immune and inflammatory responses. We found that the coinfection of PCV2 and PRV can promote the activation of nuclear factor-κB (NF-κB), c-Jun N-terminal protein kinases (JNK), p38, and nod-like receptor protein 3 (NLRP3) pathways, thus enhancing the expression of interferon-γ (IFN-γ), interferon-λ1 (IFN-λ1), interferon-stimulated gene (ISG15), interleukin 6 (IL6), and interleukin 1ß (IL1ß). Meanwhile, PCV2 and PRV also inhibit the expression and signal transduction of IFN-ß, tumor necrosis factor α (TNFα), and the Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway. In addition, PCV2 and PRV infection can also weaken extracellular-signal-regulated kinase (ERK) activity. These results indicate that the regulations of cellular antiviral immune responses and inflammatory responses mediated by NF-κB, JAK/STAT, mitogen-activated protein kinase (MAPK), and NLRP3 pathways, contribute to immune escape of PCV2 and PRV and host antiviral responses.

The Antiviral Effect of Panax Notoginseng Polysaccharides by Inhibiting PRV Adsorption and Replication In Vitro.

Porcine pseudorabies (PR) is an important infectious disease caused by pseudorabies virus (PRV), which poses a major threat to food safety and security. Vaccine immunization has become the main means to prevent and control the disease. However, since 2011, a new PRV variant has caused huge economic losses to the Chinese pig industry. Panax notoginseng polysaccharides have immunomodulatory activity and other functions, but the antiviral effect has not been reported. We studied the anti-PRV activity of Panax notoginseng polysaccharides in vitro. A less cytopathic effect was observed by increasing the concentration of Panax notoginseng polysaccharides. Western blot, TCID50, plaque assay, and IFA revealed that Panax notoginseng polysaccharides could significantly inhibit the infectivity of PRV XJ5 on PK15 cells. In addition, we also found that Panax notoginseng polysaccharides blocked the adsorption and replication of PRV to PK15 cells in a dose-dependent manner. These results show that Panax notoginseng polysaccharides play an antiviral effect mainly by inhibiting virus adsorption and replication in vitro. Therefore, Panax notoginseng polysaccharides may be a potential anti-PRV agent.

Roles of the Different Isoforms of the Pseudorabies Virus Protein Kinase pUS3 in Nuclear Egress.

Protein kinases homologous to the US3 gene product (pUS3) of herpes simplex virus (HSV) are conserved throughout the alphaherpesviruses but are absent from betaherpesviruses and gammaherpesviruses. pUS3 homologs are multifunctional and are involved in many processes, including modification of the cytoskeleton, inhibition of apoptosis, and immune evasion. pUS3 also plays a role in efficient nuclear egress of alphaherpesvirus nucleocapsids. In the absence of pUS3, primary enveloped virions accumulate in the perinuclear space (PNS) in large invaginations of the inner nuclear membrane (INM), pointing to a modulatory function for pUS3 during deenvelopment. The HSV and pseudorabies virus (PrV) US3 genes are transcribed into two mRNAs encoding two pUS3 isoforms, which have different aminoterminal sequences and abundances. To test whether the two isoforms in PrV serve different functions, we constructed mutant viruses expressing exclusively either the larger minor or the smaller major isoform, a mutant virus with decreased expression of the smaller isoform, or a mutant with impaired kinase function. Respective virus mutants were investigated in several cell lines. Our results show that absence of the larger pUS3 isoform has no detectable effect on viral replication in cell culture, while full expression of the smaller isoform and intact kinase activity is required for efficient nuclear egress. Absence of pUS3 resulted in only minor titer reduction in most cell lines tested but disclosed a more severe defect in Madin-Darby bovine kidney cells. However, accumulations of primary virions in the PNS do not account for the observed titer reduction in PrV.IMPORTANCE A plethora of substrates and functions have been assigned to the alphaherpesviral pUS3 kinase, including a role in nuclear egress. In PrV, two different pUS3 isoforms are expressed, which differ in size, abundance, and intracellular localization. Their respective role in replication is unknown, however. Here, we show that efficient nuclear egress of PrV requires the smaller isoform and intact kinase activity, whereas absence of the larger isoform has no significant effect on viral replication. Thus, there is a clear distinction in function between the two US3 gene products of PrV.

Modulation of Voltage-Gated Sodium Channel Activity in Human Dorsal Root Ganglion Neurons by Herpesvirus Quiescent Infection.

The molecular mechanisms of pain associated with alphaherpesvirus latency are not clear. We hypothesize that the voltage-gated sodium channels (VGSC) on the dorsal root ganglion (DRG) neurons controlling electrical impulses may have abnormal activity during latent viral infection and reactivation. We used herpes simplex virus 1 (HSV-1) to infect the human DRG-derived neuronal cell line HD10.6 in order to study the establishment and maintenance of viral latency, viral reactivation, and changes in the functional expression of VGSCs. Differentiated cells exhibited robust tetrodotoxin (TTX)-sensitive sodium currents, and acute infection significantly reduced the functional expression of VGSCs within 24 h and completely abolished VGSC activity within 3 days. A quiescent state of infection mimicking latency can be achieved in the presence of acyclovir (ACV) for 7 days followed by 5 days of ACV washout, and then the viruses can remain dormant for another 3 weeks. It was noted that during the establishment of HSV-1 latency, the loss of VGSC activity caused by HSV-1 infection could not be blocked by ACV treatment. However, neurons with continued ACV treatment for another 4 days showed a gradual recovery of VGSC functional expression. Furthermore, the latently infected neurons exhibited higher VGSC activity than controls. The overall regulation of VGSCs by HSV-1 during quiescent infection was proved by increased transcription and possible translation of Nav1.7. Together, these observations demonstrated a very complex pattern of electrophysiological changes during HSV infection of DRG neurons, which may have implications for understanding of the mechanisms of virus-mediated pain linked to latency and reactivation.IMPORTANCE The reactivation of herpesviruses, most commonly varicella-zoster virus (VZV) and pseudorabies virus (PRV), may cause cranial nerve disorder and unbearable pain. Clinical studies have also reported that HSV-1 causes postherpetic neuralgia and chronic occipital neuralgia in humans. The current work meticulously studies the functional expression profile changes of VGSCs during the processes of HSV-1 latency establishment and reactivation using human dorsal root ganglion-derived neuronal HD10.6 cells as an in vitro model. Our results indicated that VGSC activity was eliminated upon infection but steadily recovered during latency establishment and that latent neurons exhibited even higher VGSC activity. This finding advances our knowledge of how ganglion neurons generate uncharacteristic electrical impulses due to abnormal VGSC functional expression influenced by the latent virus.